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Roberto MARASCA

Professore Associato
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

2023 - Characteristics and clinical behavior of acute myeloid leukemia harboring rare non-A/B/D nucleophosmin (NPM1) gene mutation subtypes: a single-center experience and review of the literature [Articolo su rivista]
Mutti, M.; Cordella, S.; Parisotto, A.; Bettelli, F.; Morselli, M.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Giusti, D.; Colaci, E.; Cassanelli, L.; Paolini, A.; Martinelli, S.; Maffei, R.; Riva, G.; Nasillo, V.; Sarti, M.; Trenti, T.; Comoli, P.; Tagliafico, E.; Manfredini, R.; Eccher, A.; Lagreca, I.; Barozzi, P.; Potenza, L.; Marasca, R.; Candoni, A.; Luppi, M.; Forghieri, F.
abstract

2023 - COVID-19 omicron variant outbreak in a hematopoietic stem cell transplant unit [Articolo su rivista]
Gilioli, A.; Bresciani, P.; Franceschini, E.; Messerotti, A.; Pioli, V.; Colasante, C.; Bettelli, F.; Giusti, D.; Forghieri, F.; Morselli, M.; Colaci, E.; Potenza, L.; Gennari, W.; Pecorari, M.; Marasca, R.; Candoni, A.; Mussini, C.; Trenti, T.; Comoli, P.; Luppi, M.; Cuoghi, A.
abstract

Recommendations and guidelines for management of SARS-COV-2 infection in hematologic patients were developed in the very difficult context of dealing with novel viral variants from one pandemic wave to another, with different susceptibility to available drugs and vaccines. Moreover, the largest SARS-COV-2 case series in patients treated for hematologic malignancies, including stem cell transplant recipients, was published before the Omicron surge, and refers mainly to Alpha and Delta viral variants. These infections had very high mortality, in a period when antivirals and monoclonal antibodies were mostly unavailable. Here, we report for the first time a SARS-COV-2 Omicron variant outbreak inside a Bone Marrow Transplant (BMT) Unit, describing the characteristics, clinical course, and infection outcomes shortly before and shortly after myeloablative transplantation. We detail how infections were treated off-label and managed inside the BMT ward, to guarantee the best possible outcomes while avoiding risks for non-infected inpatients. The positive outcomes observed suggest that it may not be absolutely necessary to obtain SARS-CoV-2 PCR negativity before BMT in hematologic patients after treated infection, in cases with long-term PCR positivity and high-risk hematologic disease.

2023 - Do GWAS-Identified Risk Variants for Chronic Lymphocytic Leukemia Influence Overall Patient Survival and Disease Progression? [Articolo su rivista]
Cabrera-Serrano, Antonio José; Sánchez-Maldonado, José Manuel; Ter Horst, Rob; Macauda, Angelica; García-Martín, Paloma; Benavente, Yolanda; Landi, Stefano; Clay-Gilmour, Alyssa; Niazi, Yasmeen; Espinet, Blanca; Rodríguez-Sevilla, Juan José; Pérez, Eva María; Maffei, Rossana; Blanco, Gonzalo; Giaccherini, Matteo; Cerhan, James R; Marasca, Roberto; López-Nevot, Miguel Ángel; Chen-Liang, Tzu; Thomsen, Hauke; Gámez, Irene; Campa, Daniele; Moreno, Víctor; de Sanjosé, Silvia; Marcos-Gragera, Rafael; García-Álvarez, María; Dierssen-Sotos, Trinidad; Jerez, Andrés; Butrym, Aleksandra; Norman, Aaron D; Luppi, Mario; Slager, Susan L; Hemminki, Kari; Li, Yang; Berndt, Sonja I; Casabonne, Delphine; Alcoceba, Miguel; Puiggros, Anna; Netea, Mihai G; Försti, Asta; Canzian, Federico; Sainz, Juan
abstract

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults worldwide. Although genome-wide association studies (GWAS) have uncovered the germline genetic component underlying CLL susceptibility, the potential use of GWAS-identified risk variants to predict disease progression and patient survival remains unexplored. Here, we evaluated whether 41 GWAS-identified risk variants for CLL could influence overall survival (OS) and disease progression, defined as time to first treatment (TTFT) in a cohort of 1039 CLL cases ascertained through the CRuCIAL consortium. Although this is the largest study assessing the effect of GWAS-identified susceptibility variants for CLL on OS, we only found a weak association of ten single nucleotide polymorphisms (SNPs) with OS (p < 0.05) that did not remain significant after correction for multiple testing. In line with these results, polygenic risk scores (PRSs) built with these SNPs in the CRuCIAL cohort showed a modest association with OS and a low capacity to predict patient survival, with an area under the receiver operating characteristic curve (AUROC) of 0.57. Similarly, seven SNPs were associated with TTFT (p < 0.05); however, these did not reach the multiple testing significance threshold, and the meta-analysis with previous published data did not confirm any of the associations. As expected, PRSs built with these SNPs showed reduced accuracy in prediction of disease progression (AUROC = 0.62). These results suggest that susceptibility variants for CLL do not impact overall survival and disease progression in CLL patients.

2023 - Early palliative care versus usual haematological care in multiple myeloma: retrospective cohort study [Articolo su rivista]
Giusti, D.; Colaci, E.; Pioli, V.; Banchelli, F.; Maccaferri, M.; Leonardi, G.; Marasca, R.; Morselli, M.; Forghieri, F.; Bettelli, F.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Candoni, A.; Cassanelli, L.; Sbadili, E.; Bassoli, I.; Longo, G.; Gilioli, F.; Borelli, E.; Bigi, S.; D'Amico, R.; Porro, C. A.; Odejide, O.; Zimmermann, C.; Efficace, F.; Bruera, E.; Luppi, M.; Bandieri, E.; Potenza, L.
abstract

Objectives Although early palliative care (EPC) is beneficial in acute myeloid leukaemia, little is known about EPC value in multiple myeloma (MM). We compared quality indicators for palliative and end of life (EOL) care in patients with MM receiving EPC with those of patients who received usual haematological care (UHC).Methods This observational, retrospective study was based on 290 consecutive patients with MM. The following indicators were abstracted: providing psychological support, assessing/managing pain, discussing goals of care, promoting advance care plan, accessing home care services; no anti MM treatment within 14 and 30 days and hospice length of stay >7 days before death; no cardiopulmonary resuscitation, no intubation, <2 hospitalisations and emergency department visits within 30 days before death. Comparisons were performed using unadjusted and confounder adjusted regression models.Results 55 patients received EPC and 231 UHC. Compared with UHC patients, EPC patients had a significantly higher number of quality indicators of care (mean 2.62 +/- 1.25 vs 1.12 +/- 0.95; p<0.0001)); a significant reduction of pain intensity over time (p<0.01) and a trend towards reduced aggressiveness at EOL, with the same survival (5.3 vs 5.46 years; p=0.74)).Conclusions Our data support the value of integrating EPC into MM routine practice and lay the groundwork for future prospective comparative studies.

2023 - Effects of the BTN162b2 mRNA COVID-19 vaccine in humoral and cellular immunity in patients with chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, S.; Atene, C. G.; Maffei, R.; Mesini, N.; Debbia, G.; Colasante, C.; Pozzi, S.; Barbieri, E.; Maccaferri, M.; Leonardi, G.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

Chronic lymphocytic leukemia (CLL), the most common leukemia in the western countries, is characterized by immunosuppression due to disease itself and cytotoxic treatments. Since the beginning of COVID-19 pandemic, patients with CLL appear to be a vulnerable population. In addition, phase III mRNA vaccine trials did not provide information about the efficacy in immunocomprised population. In CLL, the antibody-mediated response to SARS-CoV-2 vaccine is impaired. The goal of this study was to evaluate the effects of SARS-CoV-2 vaccination on humoral immune response and on cellular immunity in CLL patients. Humoral immune response to BNT162b2 messenger RNA COVID-19 vaccine was evaluated in 44 CLL patients comprising 20 treatment-naïve, 14 under treatment with ibrutinib and 10 in follow-up after completion of therapy. A positive serological response to SARS-CoV-2 vaccination with IgG titers higher than 13 UA/ml was detected in 54.6% of CLL patients with a higher response in patients who obtained remission after treatment. Reduced antibody response was detected in patients under ibrutinib treatment. T-cell response to overlapping pool of peptides representing the spike region was assessed in paired CLL samples collected before and after 1 month from the second dose of COVID-19 vaccine in treatment-naïve and ibrutinib-treated CLL patients using cytokine secretion assay. Both CD3+ CD4+ and CD3+ CD8+ T cells are able to mount a cellular response to spike peptides with secretion of IFNγ and TNFα before and after vaccination in both treatment naïve and ibrutinib-treated patients and this cellular immune response is independent by COVID-19 vaccination. Collectively, T cell response to spike peptides appeared more blunted in CLL patients under treatment with ibrutinib compared to untreated ones. Our study supports the need for optimization of vaccination strategy to achieve an adequate immune response keeping strict preventive measures by CLL patients against COVID-19.

2023 - PEG-Asparaginase Single-Agent Rescue in an Advanced Case of Monomorphic Epitheliotropic Intestinal T Cell Lymphoma [Articolo su rivista]
Barbieri, E.; Pozzi, S.; Gelmini, R.; Roncati, L.; Maccaferri, M.; Potenza, L.; Marasca, R.; Luppi, M.; Leonardi, G.
abstract

Purpose: MEITL is a very rare and highly aggressive peripheral T cell lymphoma with poor prognosis and for which there is no standard treatment. Treatment options for patients patients with relapsed/refractory disease are scarce and the choice of an appropriate rescue still represents an unmet need. Methods: Here, we report the case of a 65-year-old woman affected by MEITL, progressing after initial treatment with an anthracycline-based chemotherapy and surgery, who received single-agent PEG-asparaginase salvage therapy at our institution. Results: PEG-asparaginase single-agent rescue proved to be rapidly effective in controlling the disease and its associated paraneoplastic features. Nevertheless, toxicity was high and the patient died due to a treatment-related complication. Conclusion: The case we described brings new evidences on the effectiveness of PEG-asparaginase therapy in MEITL patients. Whether PEG-asparaginase should be included in the treatment course of MEITL patients could be the subject of future studies.

2023 - Prognostic Relevance of Multi-Antigenic Myeloma-Specific T-Cell Assay in Patients with Monoclonal Gammopathies [Articolo su rivista]
Lagreca, Ivana; Nasillo, Vincenzo; Barozzi, Patrizia; Castelli, Ilaria; Basso, Sabrina; Castellano, Sara; Paolini, Ambra; Maccaferri, Monica; Colaci, Elisabetta; Vallerini, Daniela; Natali, Patrizia; Debbia, Daria; Pirotti, Tommaso; Ottomano, Anna Maria; Maffei, Rossana; Bettelli, Francesca; Giusti, Davide; Messerotti, Andrea; Gilioli, Andrea; Pioli, Valeria; Leonardi, Giovanna; Forghieri, Fabio; Bresciani, Paola; Cuoghi, Angela; Morselli, Monica; Manfredini, Rossella; Longo, Giuseppe; Candoni, Anna; Marasca, Roberto; Potenza, Leonardo; Tagliafico, Enrico; Trenti, Tommaso; Comoli, Patrizia; Luppi, Mario; Riva, Giovanni
abstract

: Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease (p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.

2023 - Role of chemotherapy in the treatment of chronic lymphocytic leukemia in the era of targeted therapies in Italy. A Campus CLL network report [Articolo su rivista]
Ballotta, L.; Maccaferri, M.; De Paoli, L.; Orsucci, L.; Gottardi, D.; Chiurazzi, F.; Reda, G.; Moia, R.; Cuneo, A.; Foa, R.; Marasca, R.
abstract

2023 - Role of Notch2 pathway in mature B cell malignancies [Articolo su rivista]
Mesini, N.; Fiorcari, S.; Atene, C. G.; Maffei, R.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

In recent decades, the Notch pathway has been characterized as a key regulatory signaling of cell-fate decisions evolutionarily conserved in many organisms and different tissues during lifespan. At the same time, many studies suggest a link between alterations of this signaling and tumor genesis or progression. In lymphopoiesis, the Notch pathway plays a fundamental role in the correct differentiation of T and B cells, but its deregulated activity leads to leukemic onset and evolution. Notch and its ligands Delta/Jagged exhibit a pivotal role in the crosstalk between leukemic cells and their environment. This review is focused in particular on Notch2 receptor activity. Members of Notch2 pathway have been reported to be mutated in Chronic Lymphocytic Leukemia (CLL), Splenic Marginal Zone Lymphoma (SMZL) and Nodal Marginal Zone Lymphoma (NMZL). CLL is a B cell malignancy in which leukemic clones establish supportive crosstalk with non-malignant cells of the tumor microenvironment to grow, survive, and resist even the new generation of drugs. SMZL and NMZL are indolent B cell neoplasms distinguished by a distinct pattern of dissemination. In SMZL leukemic cells affect mainly the spleen, bone marrow, and peripheral blood, while NMZL has a leading nodal distribution. Since Notch2 is involved in the commitment of leukemic cells to the marginal zone as a major regulator of B cell physiological differentiation, it is predominantly affected by the molecular lesions found in both SMZL and NMZL. In light of these findings, a better understanding of the Notch receptor family pathogenic role, in particular Notch2, is desirable because it is still incomplete, not only in the physiological development of B lymphocytes but also in leukemia progression and resistance. Several therapeutic strategies capable of interfering with Notch signaling, such as monoclonal antibodies, enzyme or complex inhibitors, are being analyzed. To avoid the unwanted multiple “on target” toxicity encountered during the systemic inhibition of Notch signaling, the study of an appropriate pharmaceutical formulation is a pressing need. This is why, to date, there are still no Notch-targeted therapies approved. An accurate analysis of the Notch pathway could be useful to drive the discovery of new therapeutic targets and the development of more effective therapies.

2022 - Adverse outcome associated with daratumumab-based treatments in relapsed/refractory multiple myeloma patients with amplification of chromosome arm 1q21: a single-center retrospective experience [Articolo su rivista]
Barbieri, Emiliano; Maccaferri, Monica; Leonardi, Giovanna; Giacobbi, Francesca; Corradini, Giorgia; Lagreca, Ivana; Barozzi, Patrizia; Potenza, Leonardo; Marasca, Roberto; Luppi, Mario
abstract

2022 - An 81-Year-Old Man with a 6-Year History of Chronic Lymphocytic Leukemia Presenting with Disease Flare Following Ibrutinib Discontinuation [Articolo su rivista]
Pozzi, S.; Potenza, L.; Giusti, D.; Colaci, E.; Pioli, V.; Leonardi, G.; Maccaferri, M.; Luppi, M.; Marasca, R.
abstract

Patient: Male, 81-year-old Final Diagnosis: Chronic lymphocytic leukemia Symptoms: Fever Medication: — Clinical Procedure: — Specialty: Hematology Objective: Background: Case Report: Conclusions: Unusual clinical course Chronic lymphocytic leukemia (CLL) is a mature B-cell neoplasm and the most common leukemia in adults in Western countries. Novel agents, including BTK inhibitors and the BCL2 inhibitor venetoclax, have dramati-cally changed the treatment landscape. Moreover, a disease flare, characterized by sudden worsening of clinical symptoms, radiographic findings of rapidly worsening splenomegaly or lymphadenopathy, and laboratory changes (increased absolute lymphocyte count or lactate dehydrogenase), is a phenomenon described in up to 25% of patients with CLL after ibrutinib discontinuation. We describe a patient with CLL with disease flare after ibrutinib discontinuation due to disease progression and describe the subsequent management of vene-toclax initial treatment in the course of the disease flare. We describe the case of an 81-year-old man with a 6-year history of CLL who was treated with multiple lines of therapy and developed worsening of disease-related signs and symptoms with fever, marked increase of lym-phocyte count, acute worsening of renal function, and increase in lymph nodes and spleen size following ces-sation of targeted therapy with ibrutinib at the time of disease progression. There was subsequent overlap-ping of ibrutinib during the venetoclax dose escalation period to prevent disease flare recurrence. Our report highlights the problem of disease flare after ibrutinib discontinuation in order to avoid associated patient morbidity, underscoring the importance of awareness of this phenomenon and focusing on the addition of venetoclax at time of progression in ibrutinib-treated patients, as a temporary overlap strategy, to prevent disease flare.

2022 - BTK Inhibitors Impair Platelet-Mediated Antifungal Activity [Articolo su rivista]
Nasillo, V.; Lagreca, I.; Vallerini, D.; Barozzi, P.; Riva, G.; Maccaferri, M.; Paolini, A.; Forghieri, F.; Fiorcari, S.; Maffei, R.; Martinelli, S.; Atene, C. G.; Castelli, I.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract

In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

2022 - Efficacy of Front-Line Ibrutinib and Rituximab Combination and the Impact of Treatment Discontinuation in Unfit Patients with Chronic Lymphocytic Leukemia: Results of the Gimema LLC1114 Study [Articolo su rivista]
Mauro, F. R.; Paoloni, F.; Molica, S.; Reda, G.; Trentin, L.; Sportoletti, P.; Marchetti, M.; Pietrasanta, D.; Marasca, R.; Gaidano, G.; Coscia, M.; Stelitano, C.; Mannina, D.; Di Renzo, N.; Ilariucci, F.; Liberati, A. M.; Orsucci, L.; Re, F.; Tani, M.; Musuraca, G.; Gottardi, D.; Zinzani, P. L.; Gozzetti, A.; Molinari, A.; Gentile, M.; Chiarenza, A.; Laurenti, L.; Varettoni, M.; Ibatici, A.; Murru, R.; Ruocco, V.; Del Giudice, I.; De Propris, M. S.; Starza, I. D.; Raponi, S.; Nanni, M.; Fazi, P.; Neri, A.; Guarini, A.; Rigolin, G. M.; Piciocchi, A.; Cuneo, A.; Foa, R.
abstract

The GIMEMA group investigated the efficacy, safety, and rates of discontinuations of the ibrutinib and rituximab regimen in previously untreated and unfit patients with chronic lymphocytic leukemia (CLL). Treatment consisted of ibrutinib, 420 mg daily, and until disease progression, and rituximab (375 mg/sqm, given weekly on week 1-4 of month 1 and day 1 of months 2-6). This study included 146 patients with a median age of 73 years, with IGHV unmutated in 56.9% and TP53 disrupted in 22.2%. The OR, CR, and 48-month PFS rates were 87%, 22.6%, and 77%, respectively. Responses with undetectable MRD were observed in 6.2% of all patients and 27% of CR patients. TP53 disruption (HR 2.47; p = 0.03) and B-symptoms (HR 2.91; p = 0.02) showed a significant and independent impact on PFS. The 48-month cumulative rates of treatment discontinuations due to disease progression (DP) or adverse events (AEs) were 5.6% and 29.1%, respectively. AEs leading more frequently to treatment discontinuation were atrial fibrillation in 8% of patients, infections in 8%, and non-skin cancers in 6%. Discontinuation rates due to AEs were higher in male patients (HR: 0.46; p = 0.05), patients aged ≥70 years (HR 5.43, p = 0.0017), and were managed at centers that enrolled <5 patients (HR 5.1, p = 0.04). Patients who discontinued ibrutinib due to an AE showed a 24-month next treatment-free survival rate of 63%. In conclusion, ibrutinib and rituximab combination was an effective front-line treatment with sustained disease control in more than half of unfit patients with CLL. Careful monitoring is recommended to prevent and manage AEs in this patient population.

2022 - Genetic and phenotypic attributes of splenic marginal zone lymphoma [Articolo su rivista]
Bonfiglio, F.; Bruscaggin, A.; Guidetti, F.; Terzi di Bergamo, L.; Faderl, M.; Spina, V.; Condoluci, A.; Bonomini, L.; Forestieri, G.; Koch, R.; Piffaretti, D.; Pini, K.; Pirosa, M. C.; Cittone, M. G.; Arribas, A.; Lucioni, M.; Ghilardi, G.; Wu, W.; Arcaini, L.; Baptista, M. J.; Bastidas, G.; Bea, S.; Boldorini, R.; Broccoli, A.; Buehler, M. M.; Canzonieri, V.; Cascione, L.; Ceriani, L.; Cogliatti, S.; Corradini, P.; Derenzini, E.; Devizzi, L.; Dietrich, S.; Elia, A. R.; Facchetti, F.; Gaidano, G.; Garcia, J. F.; Gerber, B.; Ghia, P.; Gomes da Silva, M.; Gritti, G.; Guidetti, A.; Hitz, F.; Inghirami, G.; Ladetto, M.; Lopez-Guillermo, A.; Lucchini, E.; Maiorana, A.; Marasca, R.; Matutes, E.; Meignin, V.; Merli, M.; Moccia, A.; Mollejo, M.; Montalban, C.; Novak, U.; Oscier, D. G.; Passamonti, F.; Piazza, F.; Pizzolitto, S.; Rambaldi, A.; Sabattini, E.; Salles, G.; Santambrogio, E.; Scarfo, L.; Stathis, A.; Stussi, G.; Geyer, J. T.; Tapia, G.; Tarella, C.; Thieblemont, C.; Tousseyn, T.; Tucci, A.; Vanini, G.; Visco, C.; Vitolo, U.; Walewska, R.; Zaja, F.; Zenz, T.; Zinzani, P. L.; Khiabanian, H.; Calcinotto, A.; Bertoni, F.; Bhagat, G.; Campo, E.; De Leval, L.; Dirnhofer, S.; Pileri, S. A.; Piris, M. A.; Traverse-Glehen, A.; Tzankov, A.; Paulli, M.; Ponzoni, M.; Mazzucchelli, L.; Cavalli, F.; Zucca, E.; Rossi, D.
abstract

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and in genetically modified mouse models, and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

2022 - Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation [Articolo su rivista]
Atene, C. G.; Fiorcari, S.; Mesini, N.; Alboni, S.; Martinelli, S.; Maccaferri, M.; Leonardi, G.; Potenza, L.; Luppi, M.; Maffei, R.; Marasca, R.
abstract

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

2022 - Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1 [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Atene, C. G.; Mesini, N.; Maccaferri, M.; Leonardi, G.; Martinelli, S.; Paolini, A.; Nasillo, V.; Debbia, G.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

2022 - Relative dose intensity of obinutuzumab-chlorambucil in chronic lymphocytic leukemia: A multicenter Italian study [Articolo su rivista]
Fresa, A.; Autore, F.; Piciocchi, A.; Catania, G.; Visentin, A.; Tomasso, A.; Moretti, M.; Vitale, C.; Chiarenza, A.; Morelli, F.; Sportoletti, P.; Marasca, R.; Sapienza, G.; Cuccaro, A.; Murru, R.; Sanna, A.; Patti, C.; Angeletti, I.; Coscia, M.; Trentin, L.; Pietrasanta, D.; Innocenti, I.; Laurenti, L.
abstract

2022 - The Role of T Cell Immunity in Monoclonal Gammopathy and Multiple Myeloma: From Immunopathogenesis to Novel Therapeutic Approaches [Articolo su rivista]
Lagreca, I.; Riva, G.; Nasillo, V.; Barozzi, P.; Castelli, I.; Basso, S.; Bettelli, F.; Giusti, D.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Colasante, C.; Vallerini, D.; Paolini, A.; Maccaferri, M.; Donatelli, F.; Forghieri, F.; Morselli, M.; Colaci, E.; Leonardi, G.; Marasca, R.; Potenza, L.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Comoli, P.; Luppi, M.
abstract

Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cyto-kine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the re-lapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell im-pairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunother-apeutic approaches in these settings.

2022 - Thrombotic and bleeding complications in patients with chronic lymphocytic leukemia and severe COVID-19: a study of ERIC, the European Research Initiative on CLL [Articolo su rivista]
Antic, D.; Milic, N.; Chatzikonstantinou, T.; Scarfo, L.; Otasevic, V.; Rajovic, N.; Allsup, D.; Alonso Cabrero, A.; Andres, M.; Baile Gonzales, M.; Capasso, A.; Collado, R.; Cordoba, R.; Cuellar-Garcia, C.; Correa, J. G.; De Paoli, L.; De Paolis, M. R.; Del Poeta, G.; Dimou, M.; Doubek, M.; Efstathopoulou, M.; El-Ashwah, S.; Enrico, A.; Espinet, B.; Farina, L.; Ferrari, A.; Foglietta, M.; Lopez-Garcia, A.; Garcia-Marco, J. A.; Garcia-Serra, R.; Gentile, M.; Gimeno, E.; da Silva, M. G.; Gutwein, O.; Hakobyan, Y. K.; Herishanu, Y.; Hernandez-Rivas, J. A.; Herold, T.; Itchaki, G.; Jaksic, O.; Janssens, A.; Kalashnikova, O. B.; Kalicinska, E.; Kater, A. P.; Kersting, S.; Koren-Michowitz, M.; Labrador, J.; Lad, D.; Laurenti, L.; Fresa, A.; Levin, M. -D.; Mayor Bastida, C.; Malerba, L.; Marasca, R.; Marchetti, M.; Marquet, J.; Mihaljevic, B.; Milosevic, I.; Miras, F.; Morawska, M.; Motta, M.; Munir, T.; Murru, R.; Nunes, R.; Olivieri, J.; Pavlovsky, M. A.; Piskunova, I.; Popov, V. M.; Quaglia, F. M.; Quaresmini, G.; Reda, G.; Rigolin, G. M.; Shrestha, A.; Simkovic, M.; Smirnova, S.; Spacek, M.; Sportoletti, P.; Stanca, O.; Stavroyianni, N.; Te Raa, D.; Tomic, K.; Tonino, S.; Trentin, L.; Van Der Spek, E.; van Gelder, M.; Varettoni, M.; Visentin, A.; Vitale, C.; Vukovic, V.; Wasik-Szczepanek, E.; Wrobel, T.; Segundo, L. Y. S.; Yassin, M.; Coscia, M.; Rambaldi, A.; Montserrat, E.; Foa, R.; Cuneo, A.; Carrier, M.; Ghia, P.; Stamatopoulos, K.
abstract

Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to COVID-19 related poor outcomes, including thrombosis and death, due to the advanced age, the presence of comorbidities, and the disease and treatment-related immune deficiency. The aim of this study was to assess the risk of thrombosis and bleeding in patients with CLL affected by severe COVID-19.

2022 - Validation and functional characterization of GWAS-identified variants for chronic lymphocytic leukemia: a CRuCIAL study [Articolo su rivista]
Garcia-Martin, P.; Diez, A. M.; Maldonado, J. M. S.; Serrano, A. J. C.; ter Horst, R.; Benavente, Y.; Landi, S.; Macauda, A.; Clay-Gilmour, A.; Hernandez-Mohedo, F.; Niazi, Y.; Gonzalez-Sierra, P.; Espinet, B.; Rodriguez-Sevilla, J. J.; Maffei, R.; Blanco, G.; Giaccherini, M.; Puiggros, A.; Cerhan, J.; Marasca, R.; Canadas-Garre, M.; Lopez-Nevot, M. A.; Chen-Liang, T.; Thomsen, H.; Gamez, I.; Moreno, V.; Marcos-Gragera, R.; Garcia-Alvarez, M.; Llorca, J.; Jerez, A.; Berndt, S.; Butrym, A.; Norman, A. D.; Casabonne, D.; Luppi, M.; Slager, S. L.; Hemminki, K.; Li, Y.; Alcoceba, M.; Campa, D.; Canzian, F.; de Sanjose, S.; Forsti, A.; Netea, M. G.; Jurado, M.; Sainz, J.
abstract

2021 - A single-tube multiplex method for monitoring mutations in cysteine 481 of Bruton Tyrosine Kinase (BTK) gene in chronic lymphocytic leukemia patients treated with ibrutinib [Articolo su rivista]
Maffei, R.; Fiorcari, S.; Atene, C. G.; Martinelli, S.; Scarfo, L.; Bonfiglio, S.; Maccaferri, M.; Ljungstrom, V.; Zucchini, P.; Forghieri, F.; Potenza, L.; Ghia, P.; Marasca, R.; Trenti, T.; Tagliafico, E.; Luppi, M.
abstract

2021 - COVID-19 severity and mortality in patients with CLL: an update of the international ERIC and Campus CLL study [Articolo su rivista]
Chatzikonstantinou, T.; Kapetanakis, A.; Scarfo, L.; Karakatsoulis, G.; Allsup, D.; Cabrero, A. A.; Andres, M.; Antic, D.; Baile, M.; Baliakas, P.; Bron, D.; Capasso, A.; Chatzileontiadou, S.; Cordoba, R.; Correa, J. -G.; Cuellar-Garcia, C.; De Paoli, L.; De Paolis, M. R.; Del Poeta, G.; Demosthenous, C.; Dimou, M.; Donaldson, D.; Doubek, M.; Efstathopoulou, M.; Eichhorst, B.; El-Ashwah, S.; Enrico, A.; Espinet, B.; Farina, L.; Ferrari, A.; Foglietta, M.; Frederiksen, H.; Furstenau, M.; Garcia-Marco, J. A.; Garcia-Serra, R.; Gentile, M.; Gimeno, E.; Glenthoj, A.; Gomes da Silva, M.; Gutwein, O.; Hakobyan, Y. K.; Herishanu, Y.; Hernandez-Rivas, J. A.; Herold, T.; Innocenti, I.; Itchaki, G.; Jaksic, O.; Janssens, A.; Kalashnikova, Оb.; Kalicinska, E.; Karlsson, L. K.; Kater, A. P.; Kersting, S.; Labrador, J.; Lad, D.; Laurenti, L.; Levin, M. -D.; Lista, E.; Lopez-Garcia, A.; Malerba, L.; Marasca, R.; Marchetti, M.; Marquet, J.; Mattsson, M.; Mauro, F. R.; Milosevic, I.; Miras, F.; Morawska, M.; Motta, M.; Munir, T.; Murru, R.; Niemann, C. U.; Rodrigues, R. N.; Olivieri, J.; Orsucci, L.; Papaioannou, M.; Pavlovsky, M. A.; Piskunova, I.; Popov, V. M.; Quaglia, F. M.; Quaresmini, G.; Qvist, K.; Reda, G.; Rigolin, G. M.; Ruchlemer, R.; Saghumyan, G.; Shrestha, A.; Simkovic, M.; Spacek, M.; Sportoletti, P.; Stanca, O.; Stavroyianni, N.; Tadmor, T.; Te Raa, D.; Tonino, S. H.; Trentin, L.; Van Der Spek, E.; van Gelder, M.; van Kampen, R.; Varettoni, M.; Visentin, A.; Vitale, C.; Wasik-Szczepanek, E.; Wrobel, T.; San Segundo, L. Y.; Yassin, M.; Coscia, M.; Rambaldi, A.; Montserrat, E.; Foa, R.; Cuneo, A.; Stamatopoulos, K.; Ghia, P.
abstract

Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to Coronavirus disease 2019 (COVID-19) due to age, disease, and treatment-related immunosuppression. We aimed to assess risk factors of outcome and elucidate the impact of CLL-directed treatments on the course of COVID-19. We conducted a retrospective, international study, collectively including 941 patients with CLL and confirmed COVID-19. Data from the beginning of the pandemic until March 16, 2021, were collected from 91 centers. The risk factors of case fatality rate (CFR), disease severity, and overall survival (OS) were investigated. OS analysis was restricted to patients with severe COVID-19 (definition: hospitalization with need of oxygen or admission into an intensive care unit). CFR in patients with severe COVID-19 was 38.4%. OS was inferior for patients in all treatment categories compared to untreated (p < 0.001). Untreated patients had a lower risk of death (HR = 0.54, 95% CI:0.41–0.72). The risk of death was higher for older patients and those suffering from cardiac failure (HR = 1.03, 95% CI:1.02–1.04; HR = 1.79, 95% CI:1.04–3.07, respectively). Age, CLL-directed treatment, and cardiac failure were significant risk factors of OS. Untreated patients had a better chance of survival than those on treatment or recently treated.

2021 - Cytomegalovirus reactivation after hematopoietic stem cell transplant with CMV-IG prophylaxis: A monocentric retrospective analysis [Articolo su rivista]
Gilioli, A.; Messerotti, A.; Bresciani, P.; Cuoghi, A.; Pioli, V.; Colasante, C.; Bettelli, F.; Giusti, D.; Forghieri, F.; Potenza, L.; Donatelli, F.; Giubbolini, R.; Galassi, L.; Marasca, R.; Banchelli, F.; D'Amico, R.; Pecorari, M.; Gennari, W.; Trenti, T.; Comoli, P.; Luppi, M.; Narni, F.
abstract

Human cytomegalovirus (CMV) represents the most common viral infection after hematopoietic stem cell transplant (HSCT), mainly occurring as reactivation from latency in seropositive patients, with a different prevalence based on the extent and timing of seroconversion in a specific population. Here, we retrospectively analyzed a cohort of patients who underwent HSCT at our Institution between 2013 and 2018, all of whom were prophylactically treated with CMV-IG (Megalotect Biotest®), to define the incidence and clinical outcomes of CMV reactivation and clinically significant infection. CMV infection occurred in 69% of our patient series, mainly resulting from reactivation, and CMV clinically significant infection (CS-CMVi) occurred in 48% of prophylactically treated patients. CMV infection and CS-CMVi impacted neither on relapse incidence nor on overall survival nor on relapse-free survival. Moreover, a very low incidence of CMV end-organ disease was documented. CMV-IG used alone as prophylactic therapy after HSCT does not effectively prevent CMV reactivation.

2021 - Dose/schedule-adjusted Rd-R vs continuous Rd for elderly, intermediate-fit patients with newly diagnosed multiple myeloma [Articolo su rivista]
Larocca, A.; Bonello, F.; Gaidano, G.; D'Agostino, M.; Offidani, M.; Cascavilla, N.; Capra, A.; Benevolo, G.; Tosi, P.; Galli, M.; Marasca, R.; Giuliani, N.; Bernardini, A.; Antonioli, E.; Rota-Scalabrini, D.; Cellini, C.; Pompa, A.; Monaco, F.; Patriarca, F.; Caravita di Toritto, T.; Corradini, P.; Tacchetti, P.; Boccadoro, M.; Bringhen, S.
abstract

Lenalidomide-dexamethasone (Rd) is standard treatment for elderly patients with multiple myeloma (MM). In this randomized phase 3 study, we investigated efficacy and feasibility of dose/schedule-adjusted Rd followed by maintenance at 10 mg per day without dexamethasone (Rd-R) vs continuous Rd in elderly, intermediate-fit newly diagnosed patients with MM. Primary end point was event-free survival (EFS), defined as progression/death from any cause, lenalidomide discontinuation, or hematologic grade 4 or nonhematologic grade 3 to 4 adverse event (AE). Of 199 evaluable patients, 101 received Rd-R and 98 continuous Rd. Median follow-up was 37 months. EFS was 10.4 vs 6.9 months (hazard ratio [HR], 0.70; 95% confidence interval [CI], 0.51-0.95; P = .02); median progression-free survival, 20.2 vs 18.3 months (HR, 0.78; 95% CI, 0.55-1.10; P = .16); and 3-year overall survival, 74% vs 63% (HR, 0.62; 95% CI, 0.37-1.03; P = .06) with Rd-R vs Rd, respectively. Rate of ≥1 nonhematologic grade ≥3 AE was 33% vs 43% (P = .14) in Rd-R vs Rd groups, with neutropenia (21% vs 18%), infections (10% vs 12%), and skin disorders (7% vs 3%) the most frequent; constitutional and central nervous system AEs mainly related to dexamethasone were more frequent with Rd. Lenalidomide was discontinued for AEs in 24% vs 30% and reduced in 45% vs 62% of patients receiving Rd-R vs Rd, respectively. In intermediate-fit patients, switching to reduced-dose lenalidomide maintenance without dexamethasone after 9 Rd cycles was feasible, with similar outcomes to standard continuous Rd. This trial was registered at www.clinicaltrials.gov as #NCT02215980.

2021 - Early palliative/supportive care in acute myeloid leukaemia allows low aggression end-of-life interventions: Observational outpatient study [Articolo su rivista]
Potenza, L.; Scaravaglio, M.; Fortuna, D.; Giusti, D.; Colaci, E.; Pioli, V.; Morselli, M.; Forghieri, F.; Bettelli, F.; Messerotti, A.; Catellani, H.; Gilioli, A.; Marasca, R.; Borelli, E.; Bigi, S.; Longo, G.; Banchelli, F.; D'Amico, R.; L Back, A.; Efficace, F.; Bruera, E.; Luppi, M.; Bandieri, E.
abstract

Objectives: Early palliative supportive care has been associated with many advantages in patients with advanced cancer. However, this model is underutilised in patients with haematological malignancies. We investigated the presence and described the frequency of quality indicators for palliative care and end-of-life care in a cohort of patients with acute myeloid leukaemia receiving early palliative supportive care. Methods: This is an observational, retrospective study based on 215 patients consecutively enrolled at a haematology early palliative supportive care clinic in Modena, Italy. Comprehensive hospital chart reviews were performed to abstract the presence of well-established quality indicators for palliative care and for aggressiveness of care near the end of life. Results: 131 patients received a full early palliative supportive care intervention. All patients had at least one and 67 (51%) patients had four or more quality indicators for palliative care. Only 2.7% of them received chemotherapy in the last 14 days of life. None underwent intubation or cardiopulmonary resuscitation and was admitted to intensive care unit during the last month of life. Only 4% had either multiple hospitalisations or two or more emergency department access. Approximately half of them died at home or in a hospice. More than 40% did not receive transfusions within 7 days of death. The remaining 84 patients, considered late referrals to palliative care, demonstrated sensibly lower frequencies of the same indicators. Conclusions: Patients with acute myeloid leukaemia receiving early palliative supportive care demonstrated high frequency of quality indicators for palliative care and low rates of treatment aggressiveness at the end of life.

2021 - Efficacy of idelalisib and rituximab in relapsed/refractory chronic lymphocytic leukemia treated outside of clinical trials. A report of the Gimema Working Group [Articolo su rivista]
Rigolin, G. M.; Cavazzini, F.; Piciocchi, A.; Arena, V.; Visentin, A.; Reda, G.; Zamprogna, G.; Cibien, F.; Vitagliano, O.; Coscia, M.; Farina, L.; Gaidano, G.; Murru, R.; Varettoni, M.; Paolini, R.; Sportoletti, P.; Pietrasanta, D.; Molinari, A. L.; Quaglia, F. M.; Laurenti, L.; Marasca, R.; Marchetti, M.; Mauro, F. R.; Crea, E.; Vignetti, M.; Gentile, M.; Montillo, M.; Foa, R.; Cuneo, A.; Chiarenza, A.; Perbellini, O.; Mannina, D.; Sancetta, R.; Olivieri, A.; Molica, S.; Pane, F.; Patti, C.; Iliariucci, F.; Gozzetti, A.; Califano, C.; Galieni, P.; Augello, A. F.; Vallisa, D.; Cura, F.; Frustaci, A. M.; Fazi, P.; Trentin, L.; Ferrara, F.
abstract

Because the efficacy of new drugs reported in trials may not translate into similar results when used in the real-life, we analyzed the efficacy of idelalisib and rituximab (IR) in 149 patients with relapsed/refractory chronic lymphocytic leukemia treated at 34 GIMEMA centers. Median progression-free survival (PFS) and overall survival were 22.9 and 44.5 months, respectively; performance status (PS) ≥2 and ≥3 previous lines of therapy were associated with shorter PFS and overall survival (OS). 48% of patients were on treatment at 12 months; the experience of the centers (≥5 treated patients) and PS 0–1 were associated with a significantly longer treatment duration (p = 0.015 and p = 0.002, respectively). TP53 disruption had no prognostic significance. The overall response rate to subsequent treatment was 49.2%, with median OS of 15.5 months and not reached in patients who discontinued, respectively, for progression and for toxicity (p < 0.01). Treatment breaks ≥14 days were recorded in 96% of patients and adverse events mirrored those reported in trials. In conclusion, this real-life analysis showed that IR treatment duration was longer at experienced centers, that the ECOG PS and ≥3 lines of previous therapy are strong prognostic factor and that the overall outcome with this regimen was superimposable to that reported in a randomized trial.

2021 - How to improve prognostication in acute myeloid leukemia with cbfb‐myh11 fusion transcript: Focus on the role of molecular measurable residual disease (mrd) monitoring [Articolo su rivista]
Talami, A.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Galassi, L.; Giubbolini, R.; Catellani, H.; Donatelli, F.; Maffei, R.; Martinelli, S.; Barozzi, P.; Potenza, L.; Marasca, R.; Trenti, T.; Tagliafico, E.; Comoli, P.; Luppi, M.; Forghieri, F.
abstract

Acute myeloid leukemia (AML) carrying inv(16)/t(16;16), resulting in fusion transcript CBFB‐MYH11, belongs to the favorable‐risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well‐established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real‐time reverse transcriptase polymerase chain reaction (qRT‐PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB‐MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low‐level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize postremission monitoring and to define effective MRD‐based therapeutic strategies.

2021 - Ibrutinib interferes with innate immunity in chronic lymphocytic leukemia patients during COVID-19 infection [Articolo su rivista]
Fiorcari, S.; Atene, C. G.; Maffei, R.; Debbia, G.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

2021 - Inflammatory microenvironment and specific t cells in myeloproliferative neoplasms: Immunopathogenesis and novel immunotherapies [Articolo su rivista]
Nasillo, V.; Riva, G.; Paolini, A.; Forghieri, F.; Roncati, L.; Lusenti, B.; Maccaferri, M.; Messerotti, A.; Pioli, V.; Gilioli, A.; Bettelli, F.; Giusti, D.; Barozzi, P.; Lagreca, I.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Maiorana, A.; Tagliafico, E.; Luppi, M.; Trenti, T.
abstract

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hemato-logical malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as in-triguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

2021 - IRF4 L116R mutation promotes proliferation of chronic lymphocytic leukemia B cells inducing MYC [Articolo su rivista]
Benatti, S.; Atene, C. G.; Fiorcari, S.; Mesini, N.; Martinelli, S.; Zucchini, P.; Bacchelli, F.; Maccaferri, M.; Debbia, G.; Potenza, L.; Rossi, D.; Vallisa, D.; Trentin, L.; Gaidano, G.; Luppi, M.; Marasca, R.; Maffei, R.
abstract

2021 - IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK [Articolo su rivista]
Maffei, R.; Fiorcari, S.; Benatti, S.; Atene, C. G.; Martinelli, S.; Zucchini, P.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

Interferon regulatory factor 4 (IRF4) is a transcriptional regulator of immune system development and function. Here, we investigated the role of IRF4 in controlling responsiveness to B-cell receptor (BCR) stimulation in chronic lymphocytic leukemia (CLL). We modulated IRF4 levels by transfecting CLL cells with an IRF4 vector or by silencing using small-interfering RNAs. Higher IRF4 levels attenuated BCR signaling by reducing AKT and ERK phosphorylation and calcium release. Conversely, IRF4 reduction improved the strength of the intracellular cascade activated by BCR engagement. Our results also indicated that IRF4 negatively regulates the expression of the spleen tyrosine kinase SYK, a crucial protein for propagation of BCR signaling, and the zinc finger DNA-binding protein IKAROS. We modulated IKAROS protein levels both by genetic manipulation and pharmacologically by treating CLL cells with lenalidomide and avadomide (IMIDs). IKAROS promoted BCR signaling by reducing the expression of inositol 5-phosphatase SHIP1. Lastly, IMIDs induced IRF4 expression, while down-regulating IKAROS and interfered with survival advantage mediated by BCR triggering, also in combination with ibrutinib. Overall, our findings elucidate the mechanism by which IRF4 tunes BCR signaling in CLL cells. Low IRF4 levels allow an efficient transmission of BCR signal throughout the accumulation of SYK and IKAROS.

2021 - Management of chronic lymphocytic leukemia in Italy during a one year of the COVID-19 pandemic and at the start of the vaccination program. A Campus CLL report [Articolo su rivista]
Cuneo, A.; Rigolin, G. M.; Coscia, M.; Quaresmini, G.; Scarfo, L.; Mauro, F. R.; Motta, M.; Quaglia, F. M.; Trentin, L.; Ferrario, A.; Laurenti, L.; Reda, G.; Ferrari, A.; Pietrasanta, D.; Sportoletti, P.; Re, F.; De Paoli, L.; Foglietta, M.; Giordano, A.; Marchetti, M.; Farina, L.; Del Poeta, G.; Varettoni, M.; Chiurazzi, F.; Marasca, R.; Malerba, L.; Ibatici, A.; Tisi, M. C.; Stefoni, V.; Leone, M.; Barate, C.; Olivieri, J.; Murru, R.; Gentile, M.; Sanna, A.; Gozzetti, A.; Gattei, V.; Gottardi, D.; Derenzini, E.; Levato, L.; Orsucci, L.; Penna, G.; Chiarenza, A.; Foa, R.
abstract

2021 - Multiparametric flow cytometry for MRD monitoring in hematologic malignancies: Clinical applications and new challenges [Articolo su rivista]
Riva, G.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Forghieri, F.; Lusenti, B.; Barozzi, P.; Lagreca, I.; Fiorcari, S.; Martinelli, S.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract

In hematologic cancers, Minimal Residual Disease (MRD) monitoring, using either molecular (PCR) or immunophenotypic (MFC) diagnostics, allows the identification of rare cancer cells, readily detectable either in the bone marrow or in the peripheral blood at very low levels, far below the limit of classic microscopy. In this paper, we outlined the state-of-the-art of MFC-based MRD detection in different hematologic settings, highlighting main recommendations and new challenges for using such a method in patients with acute leukemias or chronic hematologic neoplasms. The combination of new molecular technologies with advanced flow cytometry is progressively allowing clinicians to design a personalized therapeutic path, proportionate to the biological aggressiveness of the disease, in particular by using novel immunotherapies, in view of a modern decision-making process, based on precision medicine. Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCRbased techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL), to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings.

2021 - Neoantigen-specific T-cell immune responses: The paradigm of NPM1-mutated acute myeloid leukemia [Articolo su rivista]
Forghieri, F.; Riva, G.; Lagreca, I.; Barozzi, P.; Bettelli, F.; Paolini, A.; Nasillo, V.; Lusenti, B.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Galassi, L.; Catellani, H.; Donatelli, F.; Talami, A.; Maffei, R.; Martinelli, S.; Potenza, L.; Marasca, R.; Tagliafico, E.; Manfredini, R.; Trenti, T.; Comoli, P.; Luppi, M.
abstract

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicat-ing persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.

2021 - Nurse-Like Cells and Chronic Lymphocytic Leukemia B Cells: A Mutualistic Crosstalk inside Tissue Microenvironments [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Atene, C. G.; Potenza, L.; Luppi, M.; Marasca, R.
abstract

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is an example of hematological disease where cooperation between genetic defects and tumor microenvironmental interaction is involved in pathogenesis. CLL is a disease that is considered as "addicted to the host"; indeed, the crosstalk between leukemic cells and the tumor microenvironment is essential for leukemic clone maintenance supporting CLL cells' survival, proliferation, and protection from drug-induced apoptosis. CLL cells are not innocent bystanders but actively model and manipulate the surrounding microenvironment to their own advantage. Besides the different players involved in this crosstalk, nurse-like cells (NLC) resemble features related to leukemia-associated macrophages with an important function in preserving CLL cell survival and supporting an immunosuppressive microenvironment. This review provides a comprehensive overview of the role played by NLC in creating a nurturing and permissive milieu for CLL cells, illustrating the therapeutic possibilities in order to specifically target and re-educate them.

2021 - Pre-existing cytopenia heralding de novo acute myeloid leukemia: Uncommon presentation of NPM1-mutated AML in a single-center study [Articolo su rivista]
Galassi, L.; Colasante, C.; Bettelli, F.; Gilioli, A.; Pioli, V.; Giusti, D.; Morselli, M.; Paolini, A.; Nasillo, V.; Lusenti, B.; Colaci, E.; Donatelli, F.; Catellani, H.; Pozzi, S.; Barbieri, E.; del Rosso, M. N.; Barozzi, P.; Lagreca, I.; Martinelli, S.; Maffei, R.; Riva, G.; Tenedini, E.; Roncati, L.; Marasca, R.; Potenza, L.; Comoli, P.; Trenti, T.; Manfredini, R.; Tagliafico, E.; Luppi, M.; Forghieri, F.
abstract

2020 - Acute myeloid leukemia in patients living with HIV infection: Several questions, fewer answers [Articolo su rivista]
Forghieri, F.; Nasillo, V.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Mussini, C.; Tagliafico, E.; Trenti, T.; Cossarizza, A.; Maffei, R.; Barozzi, P.; Potenza, L.; Marasca, R.; Narni, F.; Luppi, M.
abstract

Both human immunodeficiency virus (HIV) infection and acute myeloid leukemia (AML) may be considered relatively uncommon disorders in the general population, but the precise incidence of AML in people living with HIV infection (PLWH) is uncertain. However, life expectancy of newly infected HIV-positive patients receiving anti-retroviral therapy (ART) is gradually increasing, rivaling that of age-matched HIV-negative individuals, so that the occurrence of AML is also expected to progressively increase. Even if HIV is not reported to be directly mutagenic, several indirect leukemogenic mechanisms, mainly based on bone marrow microenvironment disruption, have been proposed. Despite a well-controlled HIV infection under ART should no longer be considered per se a contraindication to intensive chemotherapeutic approaches, including allogeneic hematopoietic stem cell transplantation, in selected fit patients with AML, survival outcomes are still generally unsatisfactory. We discussed several controversial issues about pathogenesis and clinical management of AML in PLWH, but few evidence-based answers may currently be provided, due to the limited number of cases reported in the literature, mainly as case reports or small retrospective case series. Prospective multicenter clinical trials are warranted to more precisely investigate epidemiology and cytogenetic/molecular features of AML in PLWH, but also to standardize and further improve its therapeutic management.

2020 - Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia [Articolo su rivista]
Diop, Fary; Moia, Riccardo; Favini, Chiara; Spaccarotella, Elisa; De Paoli, Lorenzo; Bruscaggin, Alessio; Spina, Valeria; Terzi-di-Bergamo, Lodovico; Arruga, Francesca; Tarantelli, Chiara; Deambrogi, Clara; Rasi, Silvia; Adhinaveni, Ramesh; Patriarca, Andrea; Favini, Simone; Sagiraju, Sruthi; Jabangwe, Clive; Kodipad, Ahad A; Peroni, Denise; Mauro, Francesca R; Del Giudice, Ilaria; Forconi, Francesco; Cortelezzi, Agostino; Zaja, Francesco; Bomben, Riccardo; Rossi, Francesca Maria; Visco, Carlo; Chiarenza, Annalisa; Rigolin, Gian Matteo; Marasca, Roberto; Coscia, Marta; Perbellini, Omar; Tedeschi, Alessandra; Laurenti, Luca; Motta, Marina; Donaldson, David; Weir, Phil; Mills, Ken; Thornton, Patrick; Lawless, Sarah; Bertoni, Francesco; Del Poeta, Giovanni; Cuneo, Antonio; Follenzi, Antonia; Gattei, Valter; Boldorini, Renzo Luciano; Catherwood, Mark; Deaglio, Silvia; Foá, Robin; Gaidano, Gianluca; Rossi, Davide
abstract

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Also, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first line fludarabine, cyclophosphamide, and rituximab (FCR). Immunoblotting analysis showed that the non-canonical NF-kB pathway is active in BIRC3 mutated cell lines and primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3 mutated primary CLL cells are less sensitive to fludarabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated CLL receiving first-line FCR was analyzed by targeted next generation sequencing (NGS) of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a very poor prognostic subgroup of patients failing FCR [(median progression free survival (PFS): 2.2 years, p < 0.001] similar to cases harboring TP53 mutations (median PFS: 2.6 years, p < 0.0001]. BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio (HR) of 2.8 (95% C.I. 1.4-5.6, p = 0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

2020 - BTK Inhibition Impairs the Innate Response Against Fungal Infection in Patients With Chronic Lymphocytic Leukemia [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Vallerini, D.; Scarfo, L.; Barozzi, P.; Maccaferri, M.; Potenza, L.; Ghia, P.; Luppi, M.; Marasca, R.
abstract

Infections represent a cause of morbidity and mortality in patients affected by chronic lymphocytic leukemia (CLL). Introduction of new drugs in CLL clinical practice has showed impressive efficacy, in particular those targeting BTK. Among the consistent clinical data, an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported during treatment with ibrutinib in the first 6 months of treatment. The reason underlying manifestations of invasive fungal infections in patients treated with ibrutinib is still under investigation. Our study aimed to understand the impact of BTK inhibition on immune response to fungal infection mediated by macrophages and CD14+ monocytic population obtained from CLL patients. Exposure to ibrutinib and acalabrutinib reduced signaling pathways activated by Aspergillus fumigatus determining an exacerbation of an immunosuppressive signature, a reduction of phagocytosis and a significant deficit in the secretion of inflammatory cytokines either in macrophages and monocytes isolated from CLL patients and healthy donors. These effects lead to a failure in completely counteracting conidia germination. In addition we investigated the biological effects of ibrutinib on monocyte counterpart in patients who were undergoing therapy. A significant impairment in cytokine secretion and a deficit of phagocytosis in circulating monocytes were detected after 3 months of treatment. Thus, our results uncover modifications in the innate response in CLL patients induced by ibrutinib that may impair the immunological response to fungal infection. BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection. Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy.

2020 - Chronic lymphocytic leukemia management in Italy during the COVID-19 pandemic: A Campus CLL report [Articolo su rivista]
Cuneo, A.; Scarfo, L.; Reda, G.; Varettoni, M.; Quaglia, F. M.; Marchetti, M.; De Paoli, L.; Re, F.; Pietrasanta, D.; Rigolin, G. M.; Orsucci, L.; Ibatici, A.; Gattei, V.; Mauro, F. R.; Trentin, L.; Laurenti, L.; Marasca, R.; Foa, R.; Angeletti, I.; Chiurazzi, F.; Del Poeta, G.; Rosaria De Paolis, M.; Farina, L.; Ferrari, A.; Gentile, M.; Gottardi, D.; Gozzetti, A.; Leone, M.; Levato, L.; Maccaferri, M.; Malerba, L.; Motta, M.; Murru, R.; Nocilli, L.; Olivieri, J.; Stefoni, V.
abstract

2020 - Clinical characteristics and outcome of west nile virus infection in patients with lymphoid neoplasms: An italian multicentre study [Articolo su rivista]
Visentin, A.; Nasillo, V.; Marchetti, M.; Ferrarini, I.; Paolini, R.; Sancetta, R.; Rigolin, G. M.; Cibien, F.; Riva, M.; Briani, C.; Marinello, S.; Piazza, F.; Gherlinzoni, F.; Krampera, M.; Bassan, R.; Cuneo, A.; Luppi, M.; Semenzato, G.; Marasca, R.; Trentin, L.
abstract

2020 - COVID-19 severity and mortality in patients with chronic lymphocytic leukemia: a joint study by ERIC, the European Research Initiative on CLL, and CLL Campus [Articolo su rivista]
Scarfo, L.; Chatzikonstantinou, T.; Rigolin, G. M.; Quaresmini, G.; Motta, M.; Vitale, C.; Garcia-Marco, J. A.; Hernandez-Rivas, J. A.; Miras, F.; Baile, M.; Marquet, J.; Niemann, C. U.; Reda, G.; Munir, T.; Gimeno, E.; Marchetti, M.; Quaglia, F. M.; Varettoni, M.; Delgado, J.; Iyengar, S.; Janssens, A.; Marasca, R.; Ferrari, A.; Cuellar-Garcia, C.; Itchaki, G.; Spacek, M.; De Paoli, L.; Laurenti, L.; Levin, M. -D.; Lista, E.; Mauro, F. R.; Simkovic, M.; Van Der Spek, E.; Vandenberghe, E.; Trentin, L.; Wasik-Szczepanek, E.; Ruchlemer, R.; Bron, D.; De Paolis, M. R.; Del Poeta, G.; Farina, L.; Foglietta, M.; Gentile, M.; Herishanu, Y.; Herold, T.; Jaksic, O.; Kater, A. P.; Kersting, S.; Malerba, L.; Orsucci, L.; Popov, V. M.; Sportoletti, P.; Yassin, M.; Pocali, B.; Barna, G.; Chiarenza, A.; dos Santos, G.; Nikitin, E.; Andres, M.; Dimou, M.; Doubek, M.; Enrico, A.; Hakobyan, Y.; Kalashnikova, O.; Ortiz Pareja, M.; Papaioannou, M.; Rossi, D.; Shah, N.; Shrestha, A.; Stanca, O.; Stavroyianni, N.; Strugov, V.; Tam, C.; Zdrenghea, M.; Coscia, M.; Stamatopoulos, K.; Rossi, G.; Rambaldi, A.; Montserrat, E.; Foa, R.; Cuneo, A.; Ghia, P.
abstract

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, characterized by immunodeficiency. Hence, patients with CLL might be considered more susceptible to severe complications from COVID-19. We undertook this retrospective international multicenter study to characterize the course of COVID-19 in patients with CLL and identify potential predictors of outcome. Of 190 patients with CLL and confirmed COVID-19 diagnosed between 28/03/2020 and 22/05/2020, 151 (79%) presented with severe COVID-19 (need of oxygen and/or intensive care admission). Severe COVID-19 was associated with more advanced age (≥65 years) (odds ratio 3.72 [95% CI 1.79–7.71]). Only 60 patients (39.7%) with severe COVID-19 were receiving or had recent (≤12 months) treatment for CLL at the time of COVID-19 versus 30/39 (76.9%) patients with mild disease. Hospitalization rate for severe COVID-19 was lower (p < 0.05) for patients on ibrutinib versus those on other regimens or off treatment. Of 151 patients with severe disease, 55 (36.4%) succumbed versus only 1/38 (2.6%) with mild disease; age and comorbidities did not impact on mortality. In CLL, (1) COVID-19 severity increases with age; (2) antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect; (3) age and comorbidities did not impact on mortality, alluding to a relevant role of CLL and immunodeficiency.

2020 - Epidemiology and clinical outcomes of latent tuberculosis infection in adults affected with acute leukemia or aplastic anemia: a retrospective single-center study [Articolo su rivista]
Bettelli, F.; Giusti, D.; Morselli, M.; Colaci, E.; Nasillo, V.; Pioli, V.; Gilioli, A.; Iotti, S.; Galassi, L.; Giubbolini, R.; Colasante, C.; Catellani, H.; Barozzi, P.; Lagreca, I.; Vallerini, D.; Maffei, R.; Franceschini, E.; Mussini, C.; Banchelli, F.; D'Amico, R.; Marasca, R.; Narni, F.; Potenza, L.; Comoli, P.; Luppi, M.; Forghieri, F.
abstract

2020 - Immunomodulatory effect of ibrutinib: Reducing the barrier against fungal infections [Articolo su rivista]
Maffei, R.; Maccaferri, M.; Arletti, L.; Fiorcari, S.; Benatti, S.; Potenza, L.; Luppi, M.; Marasca, Roberto
abstract

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is increasingly used in the treatment of chronic lymphocytic leukemia (CLL). Moreover, very promising results have been reported in other B-cell malignancies, including primary central nervous system lymphoma (PCNSL). Although well-tolerated in the majority of patients, ibrutinib demonstrates in some cases troublesome toxicities, including invasive fungal infections (IFIs). In the present review, we summarize clinical manifestations of IFIs in patients treated with ibrutinib, generally characterized by an early onset, mild clinical manifestations, asymptomatic/low symptomatic pulmonary localization and high incidence of central nervous system (CNS) involvement. IFI risk appears particularly increased in patients receiving ibrutinib associated with other immune modulator agents, especially with steroids or immune-chemotherapy. Moreover, the immunomodulatory effect of ibrutinib is described, pointing the attention on the involvement of specific molecules targeted by ibrutinib in innate and adaptive response to fungal infection. Overall, the findings indicate the ibrutinib may rapidly impair innate immune cell functions, while concomitantly restoring an effective protective potential of adaptive immune compartment. A correct awareness, especially when other predisposing factors are present, is warranted about the potential risk of IFIs in ibrutinib-treated patients.

2020 - Investigating the association between physicians self-efficacy regarding communication skills and risk of “burnout” [Articolo su rivista]
Messerotti, Andrea; Banchelli, Federico; Ferrari, Silvia; Barbieri, Emiliano; Bettelli, Francesca; Bandieri, Elena; Giusti, Davide; Catellani, Hillary; Borelli, Eleonora; Colaci, Elisabetta; Pioli, Valeria; Morselli, Monica; Forghieri, Fabio; Galeazzi, Gian Maria; Marasca, Roberto; Bigi, Sarah; D’Amico, Roberto; Martin, Peter; Efficace, Fabio; Luppi, Mario; Potenza, Leonardo
abstract

2020 - Npm1‐mutated myeloid neoplasms with <20% blasts: A really distinct clinico‐pathologic entity? [Articolo su rivista]
Forghieri, F.; Nasillo, V.; Paolini, A.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Acquaviva, G.; Riva, G.; Barozzi, P.; Maffei, R.; Potenza, L.; Marasca, R.; Fozza, C.; Tagliafico, E.; Trenti, T.; Comoli, P.; Longo, G.; Luppi, M.
abstract

Nucleophosmin (NPM1) gene mutations rarely occur in non‐acute myeloid neoplasms (MNs) with &lt;20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico‐pathologic entities. Furthermore, fit patients with NPM1‐mutated MNs with &lt;20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS‐directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1‐mutated MNs with blast count &lt;20%, since NPM1‐mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1‐mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.

2020 - Selective inhibition of PI3Kγ affects survival and proliferation of chronic lymphocytic leukemia B cells [Articolo su rivista]
Maffei, R.; Benatti, S.; Atene, C. G.; Debbia, G.; Zucchini, P.; Potenza, L.; Luppi, M.; Fiorcari, S.; Marasca, R.
abstract

the catalytic p110gamma of PI3K is implicated in the survival and proliferation of CLL cells. Our findings support the idea that CLL cells are peculiar in the attitude to sense microenvironmental signals throughout the engagement of multiple PI3Ks. PI3Kgamma inhibition by IPI-549 may directly promote CLL apoptosis, but may also interfere with signals derived from several accessory cells of stromal and immune system. Together with the reported ability of PI3Kgamma inhibition in prevention of CLL migration and adhesion, our data provide knowledge to justify further clinical development of PI3Kc inhibition in CLL cells.

2019 - Characterization and dynamics of specific T cells against nucleophosmin-1 (NPM1)-mutated peptides in patients with NPM1-mutated acute myeloid leukemia [Articolo su rivista]
Forghieri, Fabio; Riva, Giovanni; Lagreca, Ivana; Barozzi, Patrizia; Vallerini, Daniela; Morselli, Monica; Paolini, Ambra; Bresciani, Paola; Colaci, Elisabetta; Maccaferri, Monica; Gilioli, Andrea; Nasillo, Vincenzo; Messerotti, Andrea; Pioli, Valeria; Arletti, Laura; Giusti, Davide; Bettelli, Francesca; Celli, Melania; Donatelli, Francesca; Corradini, Giorgia; Basso, Sabrina; Gurrado, Antonella; Cellini, Monica; Trenti, Tommaso; Marasca, Roberto; Narni, Franco; Martelli, Maria Paola; Falini, Brunangelo; Potenza, Leonardo; Luppi, Mario; Comoli, Patrizia
abstract

Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1-mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9-14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9-14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ-producing and IL2-producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1-mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1-mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1-mutated AML patients.

2019 - Lenalidomide Combination Therapy in Relapsed/Refractory Diffuse Large B Cell Lymphoma: The Italian Real-Life Experience [Articolo su rivista]
Marangon, M.; Stefoni, V.; Castellino, A.; Visco, C.; Tani, M.; Cox, M. C.; Marasca, R.; Tecchio, C.; Devizzi, L.; Monaco, F.; Romano, A.; Rusconi, C.; Rigacci, L.; Castellino, C.; Gaudio, F.; Argnani, L.; Zinzani, P. L.
abstract

2019 - Lenalidomide in Pretreated Patients with Diffuse Large B-Cell Lymphoma: An Italian Observational Multicenter Retrospective Study in Daily Clinical Practice [Articolo su rivista]
Broccoli, A.; Casadei, B.; Chiappella, A.; Visco, C.; Tani, M.; Cascavilla, N.; Conconi, A.; Balzarotti, M.; Cox, M. C.; Marino, D.; Goldaniga, M. C.; Marasca, R.; Tecchio, C.; Patti, C.; Musuraca, G.; Devizzi, L.; Monaco, F.; Romano, A.; Fama, A.; Zancanella, M.; Paolini, R.; Rigacci, L.; Castellino, C.; Gaudio, F.; Argnani, L.; Zinzani, P. L.
abstract

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma subtype, and approximately 50% of the patients are &gt;60 years of age. Patients with relapsed/refractory (rr) disease have a poor prognosis with currently available treatments. Lenalidomide is available in Italy for patients with rrDLBCL based on a local disposition of the Italian Drug Agency. Subjects, Materials, and Methods: An observational retrospective study was conducted in 24 Italian hematology centers with the aim to improve information on effectiveness and safety of lenalidomide use for rrDLBCL in real practice. Results: One hundred fifty-three patients received lenalidomide for 21/28 days with a median of four cycles. At the end of therapy, there were 36 complete responses (23.5%) and 9 partial responses with an overall response rate (ORR) of 29.4%. In the elderly (&gt;65 years) subset, the ORR was 33.6%. With a median follow-up of 36 months, median overall survival was reached at 12 months and median disease-free survival was not reached at 62 months. At the latest available follow-up, 29 patients are still in response out of therapy. Median progression-free survivals differ significantly according to age (2.5 months vs. 9.5 in the younger vs. elderly group, respectively) and to disease status at the latest previous therapy (15 months for relapsed patients vs. 3.5 for refractory subjects). Toxicities were manageable, even if 30 of them led to an early drug discontinuation. Conclusion: Lenalidomide therapy for patients with rrDLBCL is effective and tolerable even in a real-life context, especially for elderly patients. Implications for Practice: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, and approximately 50% of the patients are &gt;60 years of age. Patients with relapsed/refractory (rr) disease have a poor prognosis, reflected by the remarkably short life expectancy of 12 months with currently available treatments. The rrDLBCL therapeutic algorithm is not so well established because data in the everyday clinical practice are still poor. Lenalidomide for patients with rrDLBCL is effective and tolerable even in a real-life context, especially for elderly patients.

2019 - Lenalidomide-based induction and maintenance in elderly newly diagnosed multiple myeloma patients: updated results of the EMN01 randomized trial [Articolo su rivista]
Bringhen, Sara; D'Agostino, Mattia; Paris, Laura; Ballanti, Stelvio; Pescosta, Norbert; Spada, Stefano; Pezzatti, Sara; Grasso, Mariella; Rota-Scalabrini, Delia; De Rosa, Luca; Pavone, Vincenzo; Gazzera, Giulia; Aquino, Sara; Poggiu, Marco; Santoro, Armando; Gentile, Massimo; Baldini, Luca; Petrucci, Maria Teresa; Tosi, Patrizia; Marasca, Roberto; Cellini, Claudia; Palumbo, Antonio; Falco, Patrizia; Hájek, Roman; Boccadoro, Mario; Larocca, Alessandra
abstract

In the EMN01 trial, the addition of an alkylator (melphalan or cyclophosphamide) to lenalidomide-steroid induction has been prospectively evaluated in transplant-ineligible multiple myeloma patients. After induction, patients were randomly assigned to maintenance treatment with lenalidomide alone or with prednisone continuously. This analysis (median follow-up of 71 months) focused on maintenance treatment and on subgroup analyses according to the International Myeloma Working Group Frailty Score. 217 patients in lenalidomide-dexamethasone, 217 in melphalan-prednisone-lenalidomide and 220 in cyclophosphamide-prednisone-lenalidomide arms were evaluable. 284 (43%) patients were fit, 205 (31%) intermediate-fit and 165 (25%) frail. After induction, 402 patients were eligible for maintenance, (lenalidomide arm: 204; lenalidomide-prednisone: 198). After a median duration of maintenance of 22.0 months, progression-free survival from start of maintenance was 22.2 months with lenalidomide-prednisone vs 18.6 months with lenalidomide (HR 0.85,p=0.14), with no differences across frailty subgroups. The most frequent grade ≥3 toxicity was neutropenia (10% of lenalidomide-prednisone and 21% of lenalidomide patients; p=0.001). Grade ≥3 non-hematologic adverse events were rare (&lt;15%). In fit patients, melphalan-prednisone-lenalidomide significantly prolonged progression-free survival compared to cyclophosphamide-prednisone-lenalidomide (HR 0.72,p=0.05) and lenalidomide-dexamethasone (HR 0.72, p=0.04). Likewise, a trend towards a better overall survival was noted for melphalan-prednisone-lenalidomide and cyclophosphamide-prednisone-lenalidomide, as compared to lenalidomide-dexamethasone. No differences were observed in intermediate-fit and frail patients. This analysis showed positive outcomes of maintenance with lenalidomide-based regimens, with a good safety profile. For the first time, we showed that fit patients benefit from a triplet full-dose regimen, while intermediate-fit and frail patients from gentler regimens. ClinicalTrials.gov registration number: NCT01093196.

2019 - Managing chronic myeloid leukemia for treatment-free remission: a proposal from the GIMEMA CML WP [Articolo su rivista]
Baccarani, Michele; Abruzzese, Elisabetta; Accurso, Vincenzo; Albano, Francesco; Annunziata, Mario; Barulli, Sara; Beltrami, Germana; Bergamaschi, Micaela; Binotto, Gianni; Bocchia, Monica; Caocci, Giovanni; Capodanno, Isabella; Cavazzini, Francesco; Cedrone, Michele; Cerrano, Marco; Crugnola, Monica; D'Adda, Mariella; Elena, Chiara; Fava, Carmen; Fazi, Paola; Fozza, Claudio; Galimberti, Sara; Giai, Valentina; Gozzini, Antonella; Gugliotta, Gabriele; Iurlo, Alessandra; La Barba, Gaetano; Levato, Luciano; Lucchesi, Alessandro; Luciano, Luigia; Lunghi, Francesca; Lunghi, Monia; Malagola, Michele; Marasca, Roberto; Martino, Bruno; Melpignano, Angela; Miggiano, Maria Cristina; Montefusco, Enrico; Musolino, Caterina; Palmieri, Fausto; Pregno, Patrizia; Rapezzi, Davide; Rege-Cambrin, Giovanna; Rupoli, Serena; Salvucci, Marzia; Sancetta, Rosaria; Sica, Simona; Spadano, Raffaele; Stagno, Fabio; Tiribelli, Mario; Tomassetti, Simona; Trabacchi, Elena; Bonifacio, Massimiliano; Breccia, Massimo; Castagnetti, Fausto; Pane, Fabrizio; Russo, Domenico; Saglio, Giuseppe; Soverini, Simona; Vigneri, Paolo; Rosti, Gianantonio
abstract

Several papers authored by international experts have proposed recommendations on the management of BCR-ABL1+ chronic myeloid leukemia (CML). Following these recommendations, survival of CML patients has become very close to normal. The next, ambitious, step is to bring as many patients as possible into a condition of treatment-free remission (TFR). The Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA; Italian Group for Hematologic Diseases of the Adult) CML Working Party (WP) has developed a project aimed at selecting the treatment policies that may increase the probability of TFR, taking into account 4 variables: the need for TFR, the tyrosine kinase inhibitors (TKIs), the characteristics of leukemia, and the patient. A Delphi-like method was used to reach a consensus among the representatives of 50 centers of the CML WP. A consensus was reached on the assessment of disease risk (EUTOS Long Term Survival [ELTS] score), on the definition of the most appropriate age boundaries for the choice of first-line treatment, on the choice of the TKI for first-line treatment, and on the definition of the responses that do not require a change of the TKI (BCR-ABL1 ≤10% at 3 months, ≤1% at 6 months, ≤0.1% at 12 months, ≤0.01% at 24 months), and of the responses that require a change of the TKI, when the goal is TFR (BCR-ABL1 &gt;10% at 3 and 6 months, &gt;1% at 12 months, and &gt;0.1% at 24 months). These suggestions may help optimize the treatment strategy for TFR.

2019 - Overexpression of CD49d in trisomy 12 chronic lymphocytic leukemia patients is mediated by IRF4 through induction of IKAROS [Articolo su rivista]
Fiorcari, Stefania; Benatti, Stefania; Zucchetto, Antonella; Zucchini, Patrizia; Gattei, Valter; Luppi, Mario; Marasca, Roberto; Maffei, Rossana
abstract

2019 - Peripheral lymphadenopathy: role of excisional biopsy in differential diagnosis based on a five-year experience. [Articolo su rivista]
Campanelli, M; Cabry, F; Marasca, R; Gelmini, R.
abstract

BACKGROUND: Peripheral lymphadenopathy can be caused by benign disease, or it could be a manifestation of underlying haematological disease or metastasis of a yet undiagnosed malignant condition. Fine-needle aspiration cytology (FNAC) and image- guided core biopsy usually make up the first line of investigation. There are several disadvantages to these techniques: FNAC is an acellular aspirate that may provide non- diagnostic specimens, while core biopsy may fail in the presence of composite lymphoma, nodal necrosis, and insufficiency or fragmentation of the specimens. Our aim was to evaluate the safety and effectiveness of excisional biopsy (EB) in a large case series. METHODS: 220 consecutive patients underwent lymph node EB under local anaesthesia. All patients underwent complete and systematic physical examination. Any palpable lymph node was evaluated for its location, size, consistency, fixation, and tenderness. All specimens were sent to the pathologist as fresh tissue. RESULTS: The EB materials demonstrated 89 (40.5%) benign lesions, 130 (59%) malignant diagnoses, and one (0.5%) unclear diagnosis. Mean operative time was 42.9 minutes (range 10-120 minutes). Harvested lymph nodes had a mean diameter of 3.3 x 2.3 cm. All patients were discharged within 8 hours. No major complications were reported, with a mean of 1.16 post-operative outpatient visits. Temporary seroma and/or minor lymph leak at the site of the incision occurred in 14 cases (6.4%), haematoma in 7 (3.2%), and dehiscence of the surgical incision in 4 (1.8%), and in 3 cases (1.4%) pain was reported up to 7 days post-operatively. CONCLUSIONS: Excisional biopsy is a diagnostic method that can be applied safely with minimal morbidity and mortality.

2018 - Angiopoietin-2 acts as a survival factor for chronic lymphocytic leukemia B cells throughout Tie-2 receptor engagement [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Guarnotta, Carla; Benatti, Stefania; Belmonte, Beatrice; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

we demonstrated that: (i) CLL cells expressed Tie-2 receptor both in peripheral blood and in lymph nodes; (ii) Ang2 may interact with Tie-2 in CLL mediating a survival signal throughout PI3K-AKT signalling, and (iii) the interruption of Ang2/Tie-2 signalling may be effective in CLL.

2018 - Casein kinase 1: A new tale of chronic lymphocytic leukemia (CLL) microenvironment [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Marasca, R.
abstract

2018 - Casein kinase 1: A new tale of chronic lymphocytic leukemia (CLL) microenvironment [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Marasca, R.
abstract

2018 - Effectiveness of originator (Neupogen) and biosimilar (Zarzio) filgrastim in autologous peripheral blood stem cell mobilization in adults with acute myeloid leukemia: a single-center retrospective study [Articolo su rivista]
Nasillo, Vincenzo; Paolini, Ambra; Riva, Giovanni; Morselli, Monica; Potenza, Leonardo; Coluccio, Valeria; Maccaferri, Monica; Colaci, Elisabetta; Fantuzzi, Valeria; Messerotti, Andrea; Arletti, Laura; Pioli, Valeria; Lugli, Elisabetta; Gilioli, Andrea; Quadrelli, Chiara; Zucchini, Patrizia; Vallerini, Daniela; Lagreca, Ivana; Barozzi, Patrizia; Cuoghi, Angela; Bresciani, Paola; Marasca, Roberto; Mariano, Maria Teresa; Ceccherelli, Giovanni; Comoli, Patrizia; Campioli, Daniele; Trenti, Tommaso; Narni, Franco; Luppi, Mario; Forghieri, Fabio
abstract

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2018 - Efficacy of bendamustine and rituximab as first salvage treatment in chronic lymphocytic leukemia and indirect comparison with ibrutinib: A GIMEMA, ERIC and UK CLL FORUM study [Articolo su rivista]
Cuneo, Antonio; Follows, George; Rigolin, Gian Matteo; Piciocchi, Alfonso; Tedeschi, Alessandra; Trentin, Livio; Perez, Angeles Medina; Coscia, Marta; Laurenti, Luca; Musuraca, Gerardo; Farina, Lucia; Delgado, Alfredo Rivas; Orlandi, Ester Maria; Galieni, Piero; Mauro, Francesca Romana; Visco, Carlo; Amendola, Angela; Billio, Atto; Marasca, Roberto; Chiarenza, Annalisa; Meneghini, Vittorio; Ilariucci, Fiorella; Marchetti, Monia; Molica, Stefano; Re, Francesca; Gaidano, Gianluca; Gonzalez, Marcos; Forconi, Francesco; Ciolli, Stefania; Cortelezzi, Agostino; Montillo, Marco; Smolej, Lukas; Schuh, Anna; Eyre, Toby A.; Kennedy, Ben; Bowles, Kris M.; Vignetti, Marco; De La Serna, Javier; Moreno, Carol; Foà, Robin; Ghia, Paolo
abstract

We performed an observational study on the efficacy of bendamustine and rituximab (BR) as first salvage regimen in chronic lymphocytic leukemia (CLL). In an intention-to-treat analysis including 237 patients, the median progression-free survival (PFS) was 25 months. The presence of del (17p), unmutated IGHV and advanced stage were associated with a shorter PFS at multivariate analysis. The median time-to-next treatment was 31.3 months. Front-line treatment with a chemoimmunotherapy regimen was the only predictive factor for a shorter time to next treatment at multivariate analysis. The median overall survival (OS) was 74.5 months. Advanced disease stage (i.e. Rai stage III-IV or Binet stage C) and resistant disease were the only parameters significantly associated with a shorter OS. Grade 3-5 infections were recorded in 6.3% of patients. A matched-adjusted indirect comparison with ibrutinib given second-line within Named Patient Programs in the United Kingdom and in Italy was carried out with OS as objective end point. When restricting the analysis to patients with intact 17p who had received chemoimmunotherapy in first line, there was no difference in OS between patients treated with ibrutinib (63% alive at 36 months) and patients treated with BR (74.4% alive at 36 months). BR is an efficacious first salvage regimen in CLL in a real-life population, including the elderly and unfit patients. BR and ibrutinib may be equally effective in terms of OS when used as first salvage treatment in patients without 17p deletion. (Registered at clinicaltrials.gov identifier: 02491398).

2018 - Idelalisib impairs T-cell-mediated immunity in chronic lymphocytic leukemia [Articolo su rivista]
Martinelli, Silvia; Maffei, Rossana; Fiorcari, Stefania; Quadrelli, Chiara; Zucchini, Patrizia; Benatti, Stefania; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

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2018 - Increased SHISA3 expression characterizes chronic lymphocytic leukemia patients sensitive to lenalidomide [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Benatti, Stefania; Bulgarelli, Jenny; Rizzotto, Lara; Debbia, Giulia; Santachiara, Rita; Rigolin, Gian Matteo; Forconi, Francesco; Rossi, Davide; Laurenti, Luca; Palumbo, Giuseppe A.; Vallisa, Daniele; Cuneo, Antonio; Gaidano, Gianluca; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change =  +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.

2018 - Italian real life experience with ibrutinib: Results of a large observational study on 77 relapsed/refractory mantle cell lymphoma [Articolo su rivista]
Broccoli, Alessandro; Casadei, Beatrice; Morigi, Alice; Sottotetti, Federico; Gotti, Manuel; Spina, Michele; Volpetti, Stefano; Ferrero, Simone; Spina, Francesco; Pisani, Francesco; Merli, Michele; Visco, Carlo; Paolini, Rossella; Zilioli, Vittorio Ruggero; Baldini, Luca; Di Renzo, Nicola; Tosi, Patrizia; Cascavilla, Nicola; Molica, Stefano; Ilariucci, Fiorella; Rigolin, Gian Matteo; D'Alò, Francesco; Vanazzi, Anna; Santambrogio, Elisa; Marasca, Roberto; Mastrullo, Lucia; Castellino, Claudia; Desabbata, Giovanni; Scortechini, Ilaria; Trentin, Livio; Morello, Lucia; Argnani, Lisa; Zinzani, Pier Luigi
abstract

Although sometimes presenting as an indolent lymphoma, mantle cell lymphoma (MCL) is an aggressive disease, hardly curable with standard chemo-immunotherapy. Current approaches have greatly improved patients' outcomes, nevertheless the disease is still characterized by high relapse rates. Before approval by EMA, Italian patients with relapsed/refractory MCL were granted ibrutinib early access through a Named Patient Program (NPP). An observational, retrospective, multicenter study was conducted. Seventyseven heavily pretreated patients were enrolled. At the end of therapy there were 14 complete responses and 14 partial responses, leading to an overall response rate of 36.4%. At 40 months overall survival was 37.8% and progression free survival was 30%; disease free survival was 78.6% at 4 years: 11/14 patients are in continuous complete response with a median of 36 months of follow up. Hematological toxicities were manageable, and main extra-hematological toxicities were diarrhea (9.4%) and lung infections (9.0%). Overall, 4 (5.2%) atrial fibrillations and 3 (3.9%) hemorrhagic syndromes occurred. In conclusions, thrombocytopenia, diarrhea and lung infections are the relevant adverse events to be clinically focused on; regarding effectiveness, ibrutinib is confirmed to be a valid option for refractory/relapsed MCL also in a clinical setting mimicking the real world.

2018 - Lenalidomide in Pretreated Mantle Cell Lymphoma Patients: An Italian Observational Multicenter Retrospective Study in Daily Clinical Practice (the Lenamant Study) [Articolo su rivista]
Stefoni, Vittorio; Pellegrini, Cinzia; Broccoli, Alessandro; Baldini, Luca; Tani, Monica; Cencini, Emanuele; Figuera, Amalia; Ansuinelli, Michela; Bernocco, Elisa; Cantonetti, Maria; Cox, Maria Christina; Ballerini, Filippo; Rusconi, Chiara; Visco, Carlo; Arcaini, Luca; Fama, Angelo; Marasca, Roberto; Volpetti, Stefano; Castellino, Alessia; Califano, Catello; Cavaliere, Marina; Gini, Guido; Liberati, Anna Marina; Musuraca, Gerardo; Lucania, Anna; Ricciuti, Giuseppina; Argnani, Lisa; Zinzani, Pier Luigi
abstract

Background: Mantle cell lymphoma (MCL) has the worst prognosis of B-cell subtypes owing to its aggressive clinical disease course and incurability with standard chemo-immunotherapy. Options for relapsed MCL are limited, although several single agents have been studied. Lenalidomide is available in Italy for patients with MCL based on a local disposition of the Italian Drug Agency. Subjects, Materials, and Methods: An observational retrospective study was conducted in 24 Italian hematology centers with the aim to improve information on effectiveness and safety of lenalidomide use in real practice. Results: Seventy patients received lenalidomide for 21/28 days with a median of eight cycles. At the end of therapy, there were 22 complete responses (31.4%), 11 partial responses, 6 stable diseases, and 31 progressions, with an overall response rate of 47.1%. Eighteen patients (22.9%) received lenalidomide in combination with either dexamethasone (n = 13) or rituximab (n = 5). Median overall survival (OS) was reached at 33 months and median disease-free survival (DFS) at 20 months: 14/22 patients are in continuous complete response with a median of 26 months. Patients who received lenalidomide alone were compared with patients who received lenalidomide in combination: OS and DFS did not differ. Progression-free survivals are significantly different: at 56 months, 36% in the combination group versus 13% in patients who received lenalidomide alone. Toxicities were manageable, even if 17 of them led to an early drug discontinuation. Conclusion: Lenalidomide therapy for relapsed MCL patients is effective and tolerable even in a real-life context. Implication for Practice: Several factors influence treatment choice in relapsed/refractory mantle cell lymphoma (rrMCL), and the therapeutic scenario is continuously evolving. In fact, rrMCL became the first lymphoma for which four novel agents have been approved: temsirolimus, lenalidomide, ibrutinib, and bortezomib. The rrMCL therapeutic algorithm is not so well established because data in the everyday clinical practice are still poor. Lenalidomide for rrMCL patients is effective and tolerable even in a real-life context.

2018 - Lenalidomide treatment of myelodysplastic syndromes with chromosome 5q deletion: Results from the National Registry of the Italian Drug Agency [Articolo su rivista]
Arcioni, F.; Roncadori, A.; Di Battista, V.; Tura, S.; Covezzoli, A.; Cundari, S.; Mecucci, C.; Abbadessa, A.; Alterini, R.; Santini, V.; Cantonetti, M.; Buccisano, F.; Bacigalupo, A.; Sessarego, M.; Tonso, A.; Ferrero, D.; D'Ardia, S.; Tarella, C.; Cascavilla, N.; Bassan, R.; Sancetta, R.; Cortelezzi, A.; Reda, G.; Maria D'Arco, A.; De Fabritiis, P.; Di Renzo, N.; Falini, B.; Alimena, G.; Avanzini, P.; Ilariucci, F.; Iuliano, F.; La Nasa, G.; Caocci, G.; Defina, M.; Latte, G.; Palmas, A.; Levis, A.; Leone, G.; Teresa Voso, M.; Leoni, P.; Poloni, A.; Fozza, C.; Crugnola, M.; Montanaro, M.; Spedini, P.; Lanza, F.; Pizzuti, M.; Pane, F.; Paolini, R.; Borin, L.; Rambaldi, A.; Rossi, G.; Maria Pelizzari, A.; Russo, D.; D'Emilio, A.; Ruggeri, M.; Semenzato, G.; Specchia, G.; Tagariello, G.; Sartori, R.; Testore, F.; Ciravegna, G.; Marasca, R.; Cimarosto, L.; Fontanive, O.; Visani, G.
abstract

Objective: The most typical cytogenetic aberration in myelodysplastic syndromes is del(5q), which, when isolated, is associated with refractory anaemia and good prognosis. Based on high rates of erythroid response and transfusion independence, Lenalidomide (LEN) became the standard treatment. This multi-centre study was designed to supplement Italian Registry data on LEN by addressing prescription, administration appropriateness, haematological and cytogenetic responses and disease evolution. Methods: MORE study was an observational, non-interventional, multi-centre, retrospective and prospective study. Cases were recruited from 45 Haematological Centres throughout Italy. Data were collected from the Italian National Registry for Lenalidomide administration and supplemented by a MORE data form. Results: Data from 190/213 patients were analysed. In all, 149 had been diagnosed by conventional cytogenetics (GROUP A) and 41 only by FISH (GROUP B). Overall erythroid response was obtained in 92.8% of cases. Overall cytogenetic remission was achieved in 22.6% of cases. Disease progression occurred in 15.6% of cases. Clonal cytogenetic evolution characterised progression to AML but not to higher risk MDS. Conclusions: Erythroid response to Lenalidomide was similar in MDS with isolated del(5q) and with del(5q) plus one anomaly. Progression to AML or higher risk MDS showed different cytogenetic features.

2018 - Minimal/Measurable Residual Disease Monitoring in NPM1-Mutated Acute Myeloid Leukemia: A Clinical Viewpoint and Perspectives [Articolo su rivista]
Forghieri, Fabio; Comoli, Patrizia; Marasca, Roberto; Potenza, Leonardo; Luppi, Mario
abstract

Acute myeloid leukemia (AML) with NPM1 gene mutations is currently recognized as a distinct entity, due to its unique biological and clinical features. We summarize here the results of published studies investigating the clinical application of minimal/measurable residual disease (MRD) in patients with NPM1-mutated AML, receiving either intensive chemotherapy or hematopoietic stem cell transplantation. Several clinical trials have so far demonstrated a significant independent prognostic impact of molecular MRD monitoring in NPM1-mutated AML and, accordingly, the Consensus Document from the European Leukemia Net MRD Working Party has recently recommended that NPM1-mutated AML patients have MRD assessment at informative clinical timepoints during treatment and follow-up. However, several controversies remain, mainly with regard to the most clinically significant timepoints and the MRD thresholds to be considered, but also with respect to the optimal source to be analyzed, namely bone marrow or peripheral blood samples, and the correlation of MRD with other known prognostic indicators. Moreover, we discuss potential advantages, as well as drawbacks, of newer molecular technologies such as digital droplet PCR and next-generation sequencing in comparison to conventional RQ-PCR to quantify NPM1-mutated MRD. In conclusion, further prospective clinical trials are warranted to standardize MRD monitoring strategies and to optimize MRD-guided therapeutic interventions in NPM1-mutated AML patients.

2018 - Practical management of ibrutinib in the real life: Focus on atrial fibrillation and bleeding [Articolo su rivista]
Boriani, Giuseppe; Corradini, Paolo; Cuneo, Antonio; Falanga, Anna; Foà, Robin; Gaidano, Gianluca; Ghia, Paolo Prospero; Martelli, Maurizio; Marasca, Roberto; Massaia, Massimo; Mauro, Francesca Romana; Minotti, Giorgio; Molica, Stefano; Montillo, Marco; Pinto, Antonio; Tedeschi, Alessandra; Vitolo, Umberto; Zinzani, Pier Luigi
abstract

The Bruton tyrosine kinase inhibitor ibrutinib (IB) has attained an important role in the treatment of patients with chronic lymphocytic leukaemia, mantle cell lymphoma, and Waldenström macroglobulinemia, significantly improving clinical outcomes. However, IB therapy has been associated with an increased risk of atrial fibrillation (AF) and bleeding. We report on the expert opinion that a group of Italian haematologists, cardiologists, and pharmacologists jointly released to improve the practical management of patients at risk for AF and bleeding during treatment with IB. A proper pretreatment assessment to identify patients who are at a higher risk, careful choice of concomitant drugs, regular monitoring, and multispecialist approach were characterized as the main principles of clinical management of these patients. For patients developing AF, anticoagulant and antiarrhythmic therapy must be guided by considerations about efficacy, safety, and risk of pharmacokinetic interactions with IB. For patients experiencing bleeding or requiring procedures that increase the risk of bleeding, considerations about platelet turnover, IB-related platelet dysfunctions, and bleeding worsening by concomitant anticoagulants or antiplatelet agents provide clues to manage bleeding. Overall, AF and bleeding are manageable clinical events in patients receiving IB, not requiring drug interruption in most cases. Preexisting AF should not represent an absolute contraindication to IB therapy. For each patient candidate for IB, strategies of risk assessment and mitigation may allow to exploit the life-saving effects of in chronic lymphocytic leukaemia and mantle cell lymphoma.

2017 - A population-based study of chronic myeloid leukemia patients treated with imatinib in first line [Articolo su rivista]
Castagnetti, Fausto; Di Raimondo, Francesco; De Vivo, Antonio; Spitaleri, Antonio; Gugliotta, Gabriele; Fabbiano, Francesco; Capodanno, Isabella; Mannina, Donato; Salvucci, Marzia; Antolino, Agostino; Marasca, Roberto; Musso, Maurizio; Crugnola, Monica; Impera, Stefana; Trabacchi, Elena; Musolino, Caterina; Cavazzini, Francesco; Mineo, Giuseppe; Tosi, Patrizia; Tomaselli, Carmela; Rizzo, Michele; Siragusa, Sergio; Fogli, Miriam; Ragionieri, Riccardo; Zironi, Alessandro; Soverini, Simona; Martinelli, Giovanni; Cavo, Michele; Vigneri, Paolo; Stagno, Fabio; Rosti, Gianantonio; Baccarani, Michele
abstract

Chronic myeloid leukemia (CML) treatment is based on company-sponsored and academic trials testing different tyrosine kinase inhibitors (TKIs) as first-line therapy. These studies included patients selected according to many inclusion–exclusion criteria, particularly age and comorbidities, with specific treatment obligations. In daily clinical practice (real-life), inclusion–exclusion criteria do not exist, and the treatment outcome does not only depend on the choice of first-line TKI but also on second- and third-line TKIs. To investigate in a real-life setting the response and the outcome on first-line imatinib, with switch to second generation TKIs in case of unsatisfying response or intolerance, we analyzed all newly diagnosed patients (N = 236), living in two Italian regions, registered in a prospective study according to population-based criteria and treated front-line with imatinib. A switch from imatinib to second-generation TKIs was reported in 14% of patients for side effects and in 24% for failure or suboptimal response, with an improvement of molecular response in 57% of them. The 5-year overall survival (OS) and leukemia-related survival (LRS) were 85% and 93%, respectively; the 4-year rates of MR3.0 and MR4.0 were 75% and 48%, respectively. Cardiovascular complications were reported in 4% of patients treated with imatinib alone and in 6% of patients receiving nilotinib as second-line. Older age (≥70 years) affected OS, but not LRS. These data provide an unbiased reference on the CML management and on the results of TKI treatment in real-life, according to ELN recommendations, using imatinib as first-line treatment and second-generation TKIs as second-line therapy. Am. J. Hematol. 92:82–87, 2017. © 2016 Wiley Periodicals, Inc.

2017 - BCR-ABL-specific T-cell therapy in Ph+ ALL patients on tyrosine-kinase inhibitors [Articolo su rivista]
Comoli, Patrizia; Basso, Sabrina; Riva, Giovanni; Barozzi, Patrizia; Guido, Ilaria; Gurrado, Antonella; Quartuccio, Giuseppe; Rubert, Laura; Lagreca, Ivana; Vallerini, Daniela; Forghieri, Fabio; Morselli, Monica; Bresciani, Paola; Cuoghi, Angela; Paolini, Ambra; Colaci, Elisabetta; Marasca, Roberto; Cuneo, Antonio; Iughetti, Lorenzo; Trenti, Tommaso; Narni, Franco; Foà, Robin; Zecca, Marco; Luppi, Mario; Potenza, Leonardo
abstract

Although the emergence of bone marrow (BM)-resident (p190)BCR-ABL-specific T lymphocytes has been correlated with hematologic and cytogenetic remissions in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) undergoing maintenance tyrosine-kinase inhibitor treatment, little is known about the possibility of culturing these cells ex vivo and using them in T-cell therapy strategies. We investigated the feasibility of expanding/priming (p190)BCR-ABL-specific T cells in vitro by stimulation with dendritic cells pulsed with (p190)BCR-ABL peptides derived from the BCR-ABL junctional region and alternative splicing, and of adoptively administering them to patients with relapsed disease. We report on the feasibility of producing clinical-grade BCR-ABL-specific cytotoxic T lymphocytes (CTLs), endowed with antileukemia activity, from Ph(+) ALL patients and healthy donors. We treated 3 patients with Ph(+) ALL with autologous or allogeneic (p190)BCR-ABL-specific CTLs. No postinfusion toxicity was observed, except for a grade II skin graft-versus-host disease in the patient treated for hematologic relapse. All patients achieved a molecular or hematologic complete remission (CR) after T-cell therapy, upon emergence of (p190)BCR-ABL-specific T cells in the BM. Our results show that (p190)BCR-ABL-specific CTLs are capable of controlling treatment-refractory Ph(+) ALL in vivo, and support the development of adoptive immunotherapeutic approaches with BCR-ABL CTLs in Ph(+) ALL.

2017 - Chlorambucil plus rituximab as front-line therapy for elderly and/or unfit chronic lymphocytic leukemia patients: Correlation with biologically-based risk stratification [Articolo su rivista]
Laurenti, Luca; Innocenti, Idanna; Autore, Francesco; Ciolli, Stefania; Mauro, Francesca Romana; Mannina, Donato; Del Poeta, Giovanni; D’Arena, Giovanni; Massaia, Massimo; Coscia, Marta; Molica, Stefano; Pozzato, Gabriele; Efremov, Dimitar G.; Vannata, Barbara; Marasca, Roberto; Galieni, Pietro; Cuneo, Antonio; Orlando, Sonia; Piciocchi, Alfonso; Boncompagni, Riccardo; Vincelli, Donatella; Liberati, Anna Marina; Russo, Filomena; Foa, Robin
abstract

Chlorambucil plus rituximab as front-line therapy for elderly and/or unfit chronic lymphocytic leukemia patients: correlation with biologically-based risk stratification. A Real life experience.

2017 - Detection of Fusarium-specific T cells in hematologic patients with invasive fusariosis [Articolo su rivista]
Vallerini, Daniela; Forghieri, Fabio; Lagreca, Ivana; Riva, Giovanni; Barozzi, Patrizia; Quadrelli, Chiara; Morselli, Monica; Bresciani, Paola; Cuoghi, Angela; Coluccio, Valeria; Maccaferri, Monica; Paolini, Ambra; Colaci, Elisabetta; Marasca, Roberto; Narni, Franco; Cellini, Monica; Beauvais, Anne; Latgè, Jean Paul; Romani, Luigina; Iughetti, Lorenzo; Comoli, Patrizia; Campioli, Daniele; Trenti, Tommaso; Luppi, Mario; Potenza, Leonardo
abstract

N.A.

2017 - Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Vaisitti, Tiziana; Martinelli, Silvia; Benatti, Stefania; Debbia, Giulia; Rossi, Davide; Zucchini, Patrizia; Potenza, Leonardo; Luppi, Mario; Gaidano, Gianluca; Deaglio, Silvia; Marasca, Roberto
abstract

The crosstalk between chronic lymphocytic leukemia (CLL) cells and tumor microenvironment is essential for leukemic clone maintenance, supporting CLL cells survival, proliferation and protection from drug-induced apoptosis. Over the past years, the role of several soluble factors involved in these processes has been studied. CLL cells express higher levels of endothelin 1 (ET-1) and ETA receptor as compared to normal B cells. Upon ET-1 stimulation, CLL cells improve their survival and proliferation and reduce their sensitivity to the phosphoinositide-3-kinase d inhibitor idelalisib and to fludarabine. Here, we demonstrate that CLL cells express not only ETA receptor but also ETB receptor. ET-1 acts as a homing factor supporting CLL cells migration and adhesion to microenvironmental cells. In addition, ET-1 stimulates a pro-angiogenic profile of CLL cells increasing VEGF expression through hypoxia-inducible factor-1 (HIF-1α) accumulation in CLL cells. Macitentan, a specific dual inhibitor of ETAand ETBreceptors, targets CLL cells affecting leukemic cells migration and adhesion and overcoming the pro-survival and proliferation signals mediated by microenvironment. Furthermore, macitentan cooperates with ibrutinib inhibiting the BCR pathway and with ABT-199 disrupting BCL2 pathway. Our data describe the biological effects of a new drug, macitentan, able to counteract essential processes in CLL pathobiology as survival, migration, trafficking and drug resistance. These findings envision the possibility to interfere with ET receptors activity using macitentan as a possible novel therapeutic strategy for CLL patients.

2017 - New players in the Bruton's tyrosine kinase vs. ibrutinib match [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Marasca, R.
abstract

2017 - The expression of endothelin-1 in chronic lymphocytic leukemia is controlled by epigenetic mechanisms and extracellular stimuli. [Articolo su rivista]
Martinelli, Silvia; Maffei, Rossana; Fiorcari, Stefania; Quadrelli, Chiara; Zucchini, Patrizia; Benatti, Stefania; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Endothelin-1 (ET-1) is a hormone peptide widely expressed and is involved in several biological processes, important not only for normal cell function but also for tumor development, including cell proliferation, invasion, metastasis, angiogenesis and osteogenesis. In accordance, ET-1 was already shown to contribute to the growth and progression of many different solid cancers. We recently demonstrated that ET-1 has a role in the pathogenesis of chronic lymphocytic leukemia (CLL) where it is abnormally expressed. In the context of this malignancy, ET-1 is able to mediate survival, drug-resistance and growth signals in leukemic cells. Previous studies, not conducted in CLL, have shown that ET-1 regulatory mechanisms are numerous and cell specific. Here, we valued the expression of ET-1 in CLL, in relation to DNA methylation but also in response to stimulation of some important pathways for the dialogue between CLL and microenvironment. We found that a high methylation of ET-1 first intron affects the basal expression of ET-1 in CLL. Moreover, we showed that the activation of CD40 or Toll-like receptor (TLR) by extracellular stimuli produces an augment of ET-1 level in CLL cells. Finally, we demonstrated the fundamental role of NF-kB signalling pathway in promoting and maintaining ET-1 expression in CLL cells, both in basal conditions and after CD40 activation.

2017 - The importance of cytogenetic and molecular analyses in eosinophilia-associated myeloproliferative neoplasms: an unusual case with normal karyotype and TNIP1- PDGFRB rearrangement and overview of PDGFRB partner genes [Articolo su rivista]
Maccaferri, Monica; Pierini, V.; Di Giacomo, D.; Zucchini, Patrizia; Forghieri, Fabio; Bonacorsi, G.; Paolini, Ambra; Quadrelli, Chiara; Giacobbi, F.; Fontana, Francesco; Cappelli, Gianni; Potenza, Leonardo; Marasca, Roberto; Luppi, Mario; Mecucci, C.
abstract

Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of eosinophilia-associated myeloproliferative neoplasms (Eo-MPN). Here, we would like to report a case of Eo-MPN with normal karyotype and rearrangement of TNIP1-PDGFRB, with unusual clinical presentation, which delayed the diagnosis and initiation of an appropriate therapy. We would also like to provide a review of the so far reported PDGFRB fusion partners in myeloid neoplasms, either myeloproliferative or myelodysplastic, summarizing clinical features and response to imatinib

2016 - All-trans retinoic acid (ATRA) in non-promyelocytic acute myeloid leukemia (AML): results of combination of ATRA with low-dose Ara-C in three elderly patients with NPM1-mutated AML unfit for intensive chemotherapy and review of the literature [Articolo su rivista]
Forghieri, Fabio; Bigliardi, Sara; Quadrelli, Chiara; Morselli, Monica; Potenza, Leonardo; Paolini, Ambra; Colaci, Elisabetta; Barozzi, Patrizia; Zucchini, Patrizia; Riva, Giovanni; Vallerini, Daniela; Lagreca, Ivana; Marasca, Roberto; Narni, Franco; Venditti, Adriano; Martelli, Maria Paola; Falini, Brunangelo; Lo Coco, Francesco; Amadori, Sergio; Luppi, Mario
abstract

Based upon the clinical behavior of three patients, we suggest that the combination of low-dose Ara-C and all-trans retinoic acid may potentially be effective in some elderly patients, unfit for intensive chemotherapy, affected with NPM1-mutated acute myeloid leukemia without FLT3 mutations, warranting perspective clinical studies in these selected patients.

2016 - Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation [Articolo su rivista]
Forghieri, Fabio; Luppi, Mario; Barozzi, Patrizia; Riva, Giovanni; Monica, Morselli; Bigliardi, Sara; Quadrelli, Chiara; Vallerini, Daniela; Maccaferri, Monica; Coluccio, Valeria; Paolini, Ambra; Colaci, Elisabetta; Goretta, Bonacorsi; Maiorana, Antonino; Sara, Tagliazucchi; Fabio, Rumpianesi; Mattioli, Francesco; Presutti, Livio; Gelmini, Roberta; Cermelli, Claudio; Giulio, Rossi; Patrizia, Comoli; Marasca, Roberto; Narni, Franco; Potenza, Leonardo
abstract

Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. We retrospectively anal- ysed a consecutive series of 486 human immunodeficiency virus-negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61 5%) and 187 (38 5%) cases, respectively. Of note, seven of the 111 (6 3%) patients with benign lymphadenopathy without well-defined aetiology, showed chronic/ recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reac- tive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)-6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV-6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well-known aetiology and without recurrences, positivity for HHV-6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter-follicular regions. Immunohistochemistry for HHV-6A and HHV-6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV- 6B reactivation and chronic/recurrent benign lymphadenopathy.

2016 - Circulating functional T cells specific to human herpes virus 6 (HHV6) antigens in individuals with chromosomally integrated HHV6 [Articolo su rivista]
Barozzi, Patrizia; Riva, Giovanni; Vallerini, Daniela; Quadrelli, Chiara; Lagreca, Ivana; Eccheli, Roberta; Forghieri, Fabio; Coluccio, Valeria; Maccaferri, Monica; Paolini, Ambra; Colaci, Elisabetta; Morselli, Monica; Marasca, Roberto; Narni, Franco; Potenza, Leonardo; Luppi, Mario; Comoli, Patrizia; Campioli, Daniele; Trenti, Tommaso
abstract

Circulating functional T cells specific to human herpes virus 6 (HHV6) antigens in individuals with chromosomally integrated HHV6

2016 - Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, Stefania; Maffei, Rossana; Audrito, Valentina; Martinelli, Silvia; Hacken, Elisa Ten; Zucchini, Patrizia; Grisendi, Giulia; Potenza, Leonardo; Luppi, Mario; Burger, Jan A; Deaglio, Silvia; Marasca, Roberto
abstract

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

2016 - Lenalidomide in chronic lymphocytic leukemia: The present and future in the era of tyrosine kinase inhibitors [Articolo su rivista]
Maffei, Rossana; Colaci, Elisabetta; Fiorcari, Stefania; Martinelli, Silvia; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is an immunomodulatory agent (IMiD) clinically active in chronic lymphocytic leukemia (CLL), both in heavily pre-treated patients and upfront. Lenalidomide has a unique mechanism of action in CLL. Its efficacy relies on a multifactorial mode-of-action (MOA), comprising a plethora of immunomodulatory actions, the disruption of mutualistic interactions inside CLL microenvironment and direct effects against leukemic cells. In the last few years, a number of new and highly effective drugs appeared in the scenario of CLL therapeutic options, i.e. tyrosine kinase inhibitors (TKIs), showing a good safety profile and impressive clinical response, also in high-risk patients. In this review, we describe the data from clinical studies about lenalidomide efficacy in CLL and we critically dissect the different mechanisms of action of this drug. We point the attention on open issues, including drug dosage and administration schedule, prediction of clinical response to lenalidomide, and combination therapeutic strategies. This overview would be useful to envision a possible role of lenalidomide in the treatment flow-chart of CLL, exploiting its peculiar MOA and also exploring the possible synergetic effect with new drugs.

2016 - Mucorales-specific T cells in patients with hematologic malignancies [Articolo su rivista]
Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Gilioli, Andrea; Forghieri, Fabio; Candoni, Anna; Cesaro, Simone; Quadrelli, Chiara; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Codeluppi, Mauro; Mussini, Cristina; Colaci, Elisabetta; Messerotti, Andrea; Paolini, Ambra; Maccaferri, Monica; Fantuzzi, Valeria; DEL GIOVANE, Cinzia; Stefani, Alessandro; Morandi, Uliano; Maffei, Rossana; Marasca, Roberto; Narni, Franco; Fanin, Renato; Comoli, Patrizia; Romani, Luigina; Beauvais, Anne; Viale, Pier Luigi; Latgè, Jean Paul; Lewis, Russell E.; Luppi, Mario
abstract

Background: Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. Methods and Findings: By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during highdose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD):2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Conclusions: Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

2016 - Randomized phase 2 study: Elotuzumab plus bortezomib/dexamethasone vs bortezomib/dexamethasone for relapsed/refractory MM [Articolo su rivista]
Jakubowiak, Andrzej; Offidani, Massimo; Pégourie, Brigitte; De La Rubia, Javier; Garderet, Laurent; Laribi, Kamel; Bosi, Alberto; Marasca, Roberto; Laubach, Jacob; Mohrbacher, Ann; Carella, Angelo Michele; Singhal, Anil K.; Tsao, L. Claire; Lynch, Mark; Bleickardt, Eric; Jou, Ying Ming; Robbins, Michael; Palumbo, Antonio
abstract

In this proof-of-concept, open-label, phase 2 study, patients with relapsed/refractory multiple myeloma (RRMM) received elotuzumab with bortezomib and dexamethasone (EBd) or bortezomib and dexamethasone (Bd) until disease progression/unacceptable toxicity. Primary endpoint was progression-free survival (PFS); secondary/exploratory endpoints included overall response rate (ORR) and overall survival (OS). Two-sided 0.30 significance level was specified (80% power, 103 events) to detect hazard ratio (HR) of 0.69. Efficacy and safety analyses were performed on all randomized patients and all treated patients, respectively. Of 152 randomized patients (77 EBd, 75 Bd), 150 were treated (75 EBd, 75 Bd). PFS was greater with EBd vs Bd (HR, 0.72; 70% confidence interval [CI], 0.59-0.88; stratified log-rank P 5 .09); median PFS was longer with EBd (9.7 months) vs Bd (6.9 months). In an updated analysis, EBd-treated patients homozygous for the high-affinity FcγRIIIa allele had median PFS of 22.3 months vs 9.8 months in EBd-treated patients homozygous for the low-affinity allele. ORR was 66% (EBd) vs 63% (Bd). Very good partial response or better occurred in 36% of patients (EBd) vs 27% (Bd). Early OS results, based on 40 deaths, revealed an HR of 0.61 (70% CI, 0.43-0.85). To date, 60 deaths have occurred (28 EBd, 32 Bd). No additional clinically significant adverse events occurred with EBd vs Bd. Grade 1/2 infusion reaction rate was low (5% EBd) and mitigated with premedication. In patients with RRMM, elotuzumab, an immunostimulatory antibody, appears to provide clinical benefit without added clinically significant toxicity when combined with Bd vs Bd alone. Registered to ClinicalTrials.gov as NCT01478048.

2016 - Safety and efficacy of rituximab plus bendamustine in relapsed or refractory diffuse large B-cell lymphoma patients: an Italian retrospective multicenter study [Articolo su rivista]
Arcari, Annalisa; Chiappella, Annalisa; Spina, Michele; Zanlari, Luca; Bernuzzi, Patrizia; Valenti, Vanessa; Tani, Monica; Marasca, Roberto; Cabras, Maria Giuseppina; Zambello, Renato; Santagostino, Alberto; Ilariucci, Fiorella; Carli, Giuseppe; Musto, Pellegrino; Savini, Paolo; Marino, Dario; Ghio, Francesco; Gentile, Massimo; Cox, Maria Christina; Vallisa, Daniele
abstract

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not suitable for high dose chemotherapy with autologous stem cell transplantation (ASCT) has a dismal prognosis and no standard therapy. We designed an Italian multicenter retrospective study aimed at evaluating the safety and efficacy of rituximab plus bendamustine (R-B) as salvage treatment in patients not eligible for ASCT because of age and/or comorbidity or in patients with post-ASCT recurrence. Fifty-five patients with a median age of 76 years were included. The overall response rate was 50%, including 28% complete remission and 22% partial remission. The median overall survival (OS) was 10.8 months. The median progression free survival (PFS) was 8.8 months. Eleven patients are still alive and in complete remission at last follow-up (12-71 months). Toxicity was moderate, mainly grades 1 and 2. R-B showed promising efficacy results with an acceptable toxicity profile and should be further investigated, possibly in combination with novel drugs.

2016 - The bone marrow represents an enrichment site of specific T lymphocytes against filamentous fungi [Articolo su rivista]
Vallerini, Daniela; Riva, Giovanni; Barozzi, Patrizia; Forghieri, Fabio; Lagreca, Ivana; Quadrelli, Chiara; Morselli, Monica; Bresciani, Paola; Cuoghi, Angela; Coluccio, Valeria; Maccaferri, Monica; Paolini, Ambra; Colaci, Elisabetta; Marasca, Roberto; Narni, Franco; Latgè, Jean Paul; Romani, Luigina; Comoli, Patrizia; Campioli, Daniele; Trenti, Tommaso; Luppi, Mario; Potenza, Leonardo
abstract

Bone marrow has already been described as an enrichment site for several antigen-specific T lymphocytes, but the presence of mould-specific T cells has never been investigated in the bone marrow. We have previously demonstrated that mould-specific T cells emerge in the peripheral blood of patients with invasive fungal infections (IFI) but tend to become undetectable after disease resolution. In seven patients with a history of IFI, we investigated the presence of mould-specific T cells secreting different cytokines in bone marrow and peripheral blood paired samples. The results showed that the frequencies of mould-specific T cells secreting the protective cytokine IFNI3 are significantly higher in bone marrow (BM) and are mainly represented by CD8+ T lymphocytes with effector phenotype. A putative disappearance of such protective BM responses after myeloablative therapy could contribute to the increased risk of IFI in hematologic patients.

2016 - The genetics of nodal marginal zone lymphoma [Articolo su rivista]
Spina, Vincenzo; Khiabanian, H; Messina, M; Monti, S; Cascione, L; Bruscaggin, A; Spaccarotella, E; Holmes, Ab; Arcaini, L; Lucioni, M; Tabbò, F; Zairis, S; Diop, F; Cerri, M; Chiaretti, S; Marasca, Roberto; Ponzoni, Martina; Deaglio, S; Ramponi, A; Tiacci, E; Pasqualucci, L; Paulli, M; Falini, Brunangelo; Inghirami, G; Bertoni, Francesco; Foà, R; Rabadan, R; Gaidano, G; Rossi, D.
abstract

Nodal marginal zone lymphoma (NMZL) is a rare, indolent B-cell tumor that is distinguished from splenic marginal zone lymphoma (SMZL) by the different pattern of dissemination. NMZL still lacks distinct markers and remains orphan of specific cancer gene lesions. By combining whole-exome sequencing, targeted sequencing of tumor-related genes, whole-transcriptome sequencing, and high-resolution single nucleotide polymorphism array analysis, we aimed at disclosing the pathways that are molecularly deregulated in NMZL and we compare the molecular profile of NMZL with that of SMZL. These analyses identified a distinctive pattern of nonsilent somatic lesions in NMZL. In 35 NMZL patients, 41 genes were found recurrently affected in ≥3 (9%) cases, including highly prevalent molecular lesions of MLL2 (also known as KMT2D; 34%), PTPRD (20%), NOTCH2 (20%), and KLF2 (17%). Mutations of PTPRD, a receptor-type protein tyrosine phosphatase regulating cell growth, were enriched in NMZL across mature B-cell tumors, functionally caused the loss of the phosphatase activity of PTPRD, and were associated with cell-cycle transcriptional program deregulation and increased proliferation index in NMZL. Although NMZL shared with SMZL a common mutation profile, NMZL harbored PTPRD lesions that were otherwise absent in SMZL. Collectively, these findings provide new insights into the genetics of NMZL, identify PTPRD lesions as a novel marker for this lymphoma across mature B-cell tumors, and support the distinction of NMZL as an independent clinicopathologic entity within the current lymphoma classification.

2015 - Antineoplastic effects of liposomal siRNA treatment targeting BLIMP1/PRDM1 in primary effusion lymphoma [Articolo su rivista]
Riva, Giovanni; Lagreca, Ivana; Mattiolo, Adriana; Belletti, Daniela; Lignitto, Laura; Barozzi, Patrizia; Ruozi, Barbara; Vallerini, Daniela; Quadrelli, Chiara; Corradini, Giorgia; Forghieri, Fabio; Marasca, Roberto; Narni, Franco; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Amadori, Alberto; Chieco Bianchi, Luigi; Potenza, Leonardo; Calabro', Maria Luisa; Luppi, Mario
abstract

RNA interference (RNAi) has been suggested to represent a promising therapeutic approach in different disease settings. Primary effusion lymphoma (PEL) is a plasmablastic lymphoma consistently expressing B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells. Here we report, for the first time, that transient knockdown of the BLIMP1 gene (also known as PR Domain Containing 1 with ZNF Domain, or PRDM1) using small interfering RNA (siRNA) delivered by liposomes, induced remarkable killing in PEL cell lines. Furthermore, in a murine model of PEL, significantly prolonged survival was achieved by intraperitoneal treatment with such anti-BLIMP1 lipoplexes, while no vector-induced toxicity was observed. This effective and safe RNAi strategy, based on liposomal siRNA targeting a master transcription factor of post-germinal center B cells, may indeed be a potential treatment against plasmablastic lymphoma

2015 - Differences among young adults, adults and elderly chronic myeloid leukemia patients [Articolo su rivista]
Castagnetti, Fausto; Gugliotta, G.; Baccarani, M.; Breccia, M.; Specchia, G.; Levato, L.; Abruzzese, E.; Rossi, G.; Iurlo, A.; Martino, B.; Pregno, P.; Stagno, F.; Cuneo, A.; Bonifacio, M.; Gobbi, M.; Russo, D.; Gozzini, A.; Tiribelli, M.; De Vivo, A.; Alimena, G.; Cavo, M.; Martinelli, G.; Pane, F.; Saglio, G.; Rosti, G.; Salvi, F.; Pini, M.; Leoni, P.; Rupoli, S.; Galieni, P.; Bigazzi, C.; Cantore, N.; Palmieri, F.; Albano, F.; Russo Rossi, A.; Rambaldi, A.; Intermesoli, T.; Palandri, F.; Testoni, N.; Luatti, S.; Soverini, S.; Iacobucci, I.; Bochicchio, M. T.; Apolinari, M.; Fogli, M.; Cervello, I.; Capucci, A.; Malagola, M.; Malpignano, A.; Girasoli, M.; Angelucci, E.; Usala, E.; Storti, S.; De Biasi, E.; Tagariello, G.; Sartori, R.; Di Raimondo, F.; Vigneri, P.; Impera, S.; Molica, S.; Lanza, F.; Viganò, C.; Grasso, M.; Rapezzi, D.; Cavazzini, F.; Bosi, A.; Santini, V.; Capalbo, S. F.; Spinosa, G.; Pierri, I.; Bergamaschi, M.; Carella, A. M.; Bacigalupo, A.; De Blasio, A.; Ciccone, F.; Di Renzo, N.; Musolino, C.; Russo, S.; Cortelezzi, A.; Morra, E.; Pungolino, E. M.; Luppi, Mario; Marasca, Roberto; Pogliani, E. M.; Gambacorti Passerini, C.; Luciano, L.; Ferrara, F.; Annunziata, M.; Latte, G.; Noli, D.; Rege Cambrin, G.; Fava, C.; Semenzato, G.; Binotto, G.; Fabbiano, F.; Turri, D.; Siragusa, S.; Caracciolo, C.; Musso, M.; Porretto, F.; Aversa, F.; Crugnola, M.; Cazzola, M.; Orlandi, E.; Falini, B.; Falzetti, F.; Visani, G.; Isidori, A.; Fioritoni, G.; Di Lorenzo, R.; Vallisa, D.; Trabacchi, E.; Petrini, M.; Galimberti, S.; Pizzuti, M.; Zaccaria, A.; Salvucci, M.; Ronco, F.; Ielo, D.; Merli, F.; Avanzini, P.; Tosi, P.; Merli, A.; Musto, P.; De Stefano, V.; Sica, S.; Latagliata, R.; De Fabritiis, P.; Trawiska, M.; Majolino, I.; Pacilli, L.; Ronci, B.; Cedrone, M.; Petti, M. C.; Pisani, F.; Tafuri, A.; Montefusco, E.; Iuliano, F.; Dore, F.; Pardini, S.; Bocchia, M.; Defina, M.; Liberati, A. M.; Luzzi, D.; Boccadoro, M.; Ferrero, D.; Vitolo, U.; Gherlinzoni, F.; Calistri, E.; Fanin, R.; Pizzolo, G.; Meneghini, V.; Rodighiero, F.; D'Emilio, A.
abstract

Background: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. Patients and methods: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CMLWP over a 40-year period. Results: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P &lt; 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P &lt; 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P &lt; 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). Conclusions: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome.

2015 - DNA methylation-profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features [Articolo su rivista]
Arribas, Aj; Rinaldi, A; Mensah, Aa; Kwee, I; Cascione, L; Robles, Ef; Martinez Climent, Ja; Oscier, D; Arcaini, L; Baldini, L; Marasca, Roberto; Thieblemont, C; Briere, J; Forconi, F; Zamò, A; Bonifacio, M; Mollejo, M; Facchetti, F; Dirnhofer, S; Ponzoni, M; Bhagat, G; Piris, Ma; Gaidano, G; Zucca, E; Rossi, D; Bertoni, F.
abstract

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 gene are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted, therefore identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here, we integrated genome-wide DNA promoter methylation profiling with gene expression profiling, and clinical and biologic variables. An unsupervised clustering analysis of a test series of 98 samples identified two clusters with different degrees of promoter methylation. The cluster comprising samples with higher promoter methylation (High-M) had a poorer overall survival compared to the Low-M cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several pro-survival lymphoma genes were un-methylated and over-expressed. A model based on the methylation of three genes (CACNB2, HTRA1 and KLF4) identified a poorer outcome patient subset. Exposure of SMZL cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

2015 - Epidemiology and clinical outcome of lower respiratory tract infections by respiratory syncytial virus or parainfluenza virus type 3 in adults receiving treatment for either acute leukemia or severe aplastic anemia: a retrospective single center study [Articolo su rivista]
Bigliardi, Sara; Morselli, Monica; Potenza, Leonardo; Riva, Giovanni; Coluccio, Valeria; Maccaferri, Monica; Paolini, Ambra; Colaci, Elisabetta; Fantuzzi, Valeria; Soci, Francesco; Nasillo, Vincenzo; Messerotti, Andrea; Arletti, Laura; Pioli, Valeria; Lugli, Elisabetta; Gilioli, Andrea; Quadrelli, Chiara; Vallerini, Daniela; Barozzi, Patrizia; Lagreca, Ivana; Marasca, Roberto; Narni, Franco; Franceschini, Erica; Codeluppi, Mauro; Mussini, Cristina; Luppi, Mario; Forghieri, Fabio
abstract

Epidemiology and clinical outcome of lower respiratory tract infections by respiratory syncytial virus or parainfluenza virus type 3 in adults receiving treatment for either acute leukemia or severe aplastic anemia: a retrospective single center study

2015 - First report of FIP1L1-PDGFRα-positive eosinophilic granulomatosis with polyangiitis [Articolo su rivista]
Emmi, Giacomo; Silvestri, Elena; Marconi, Roberto; Carrai, Valentina; Fanelli, Tiziana; Zucchini, Patrizia; Marasca, Roberto; Vannucchi, ALESSANDRO MARIA; Emmi, Lorenzo; Prisco, Domenico; Vaglio, Augusto
abstract

First report of FIP1L1-PDGFRα-positive eosinophilic granulomatosis with polyangiitis

2015 - Lenalidomide interferes with tumor-promoting properties of nurse-like cells in chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, Stefania; Martinelli, Silvia; Bulgarelli, Jenny; Audrito, V; Zucchini, Patrizia; Colaci, Elisabetta; Potenza, Leonardo; Narni, Franco; Luppi, Mario; Deaglio, S; Marasca, Roberto; Maffei, Rossana
abstract

Lenalidomide is an immunomodulatory agent clinically active in chronic lymphocytic leukemia patients. The specific mechanism of action is still undefined, but includes modulation of the microenvironment. In chronic lymphocytic leukemia patients, nurse-like cells differentiate from CD14(+) mononuclear cells and protect chronic lymphocytic leukemia cells from apoptosis. Nurse-like cells resemble M2 macrophages with potent immunosuppressive functions. Here, we examined the effect of lenalidomide on the monocyte/macrophage population in chronic lymphocytic leukemia patients. We found that lenalidomide induces high actin polymerization on CD14(+) monocytes through activation of small GTPases, RhoA, Rac1 and Rap1 that correlated with increased adhesion and impaired monocyte migration in response to CCL2, CCL3 and CXCL12. We observed that lenalidomide increases the number of nurse-like cells that lost the ability to nurture chronic lymphocytic leukemia cells, acquired properties of phagocytosis and promoted T-cell proliferation. Gene expression signature, induced by lenalidomide in nurse-like cells, indicated a reduction of pivotal pro-survival signals for chronic lymphocytic leukemia, such as CCL2, IGF1, CXCL12, HGF1, and supported a modulation towards M1 phenotype with high IL2 and low IL10, IL8 and CD163. Our data provide new insights into the mechanism of action of lenalidomide that mediates a pro-inflammatory switch of nurse-like cells affecting the protective microenvironment generated by chronic lymphocytic leukemia into tissues.

2015 - Molecular prediction of durable remission after first-line fludarabinecyclophosphamide-rituximab in chronic lymphocytic leukemia [Articolo su rivista]
Rossi, Davide; Terzi Di Bergamo, Lodovico; De Paoli, Lorenzo; Cerri, Michaela; Ghilardi, Guido; Chiarenza, Annalisa; Bulian, Pietro; Visco, Carlo; Mauro, Francesca R.; Morabito, Fortunato; Cortelezzi, Agostino; Zaja, Francesco; Forconi, Francesco; Laurenti, Luca; Del Giudice, Ilaria; Gentile, Massimo; Vincelli, Iolanda; Motta, Marina; Coscia, Marta; Rigolin, Gian Matteo; Tedeschi, Alessandra; Neri, Antonino; Marasca, Roberto; Perbellini, Omar; Moreno, Carol; Del Poeta, Giovanni; Massaia, Massimo; Zinzani, Pier Luigi; Montillo, Marco; Cuneo, Antonio; Gattei, Valter; Foà, Robin; Gaidano, Gianluca
abstract

Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant treatment advancement in chronic lymphocytic leukemia (CLL). In the new scenario of targeted agents, there is an increasing interest in identifying patients who gain the maximum benefit from FCR. In this observational multicenter retrospective analysis of 404 CLL patients receiving frontline FCR, the combination of three biomarkers that are widely tested before treatment (IGHV mutation status, 11q deletion and 17p deletion; available in 80% of the study cohort) allowed to identify a very low-risk category of patients carrying mutated IGHV genes but neither 11q or 17p deletion that accounted for 28% of all cases. The majority of very low-risk patients (71%) remained free of progression after treatment and their hazard of relapse decreased after 4 years from FCR. The life expectancy of very low-risk patients (91% at 5 years) was superimposable to that observed in the matched normal general population, indicating that neither the disease nor complications of its treatment affected survival in this favorable CLL group. These findings need a prospective validation and may be helpful for the design of clinical trials aimed at comparing FCR to new targeted treatments of CLL, and, possibly, for optimized disease management

2015 - NPM1 mutations may reveal acute myeloid leukemia in cases otherwise morphologically diagnosed as myelodysplastic syndromes or myelodysplastic/myeloproliferative neoplasms [Articolo su rivista]
Forghieri, Fabio; Paolini, Ambra; Morselli, Monica; Bigliardi, Sara; Bonacorsi, Goretta; Leonardi, Giovanna; Coluccio, Valeria; Maccaferri, Monica; Fantuzzi, Valeria; Faglioni, Laura; Colaci, Elisabetta; Soci, Francesco; Nasillo, Vincenzo; Messerotti, Andrea; Arletti, Laura; Pioli, Valeria; Zucchini, Patrizia; Quadrelli, Chiara; Corradini, Giorgia; Giacobbi, Francesca; Vallerini, Daniela; Riva, Giovanni; Barozzi, Patrizia; Lagreca, Ivana; Marasca, Roberto; Narni, Franco; Mecucci, Cristina; Ottaviani, Emanuela; Martinelli, Giovanni; Falini, Brunangelo; Luppi, Mario; Potenza, Leonardo
abstract

NPM1 mutations may reveal acute myeloid leukemia in cases otherwise morphologically diagnosed as myelodysplastic syndromes or myelodysplastic/myeloproliferative neoplasms.

2015 - Ofatumumab in poor-prognosis chronic lymphocytic leukemia: a Phase 4, non--interventional, observational study from the European Research Initiative on Chronic Lymphocytic Leukemia [Articolo su rivista]
Moreno, C; Montillo, M; Panayiotidis, P; Dimou, M; Bloor, A; Dupuis, J; Schuh, A; Norin, S; Geisler, C; Hillmen, P; Doubek, M; Trněný, M; Obrtlikova, P; Laurenti, L; Stilgenbauer, S; Smolej, L; Ghia, P; Cymbalista, F; Jaeger, U; Stamatopoulos, K; Stavroyianni, N; Carrington, P; Zouabi, H; Leblond, V; Gomez Garcia, Jc; Rubio, M; Marasca, Roberto; Musuraca, G; Rigacci, L; Farina, L; Paolini, R; Pospisilova, S; Kimby, E; Bradley, C; Montserrat, E.
abstract

We report the largest retrospective, phase IV non-interventional, observational study of ofatumumab therapy in heavily pre-treated patients with poor-prognosis chronic lymphocytic leukemia. The total number of patients was 103, with a median age of 65 years (range, 39-85). The median number of prior lines of therapy was 4 (range, 1-13), including in most cases rituximab-, fludarabine- and alemtuzumab- based regimens; thirteen patients had been allografted. Of 113 adverse events, 28 (29%) were considered to be directly related to ofatumumab. Grade 3-4 toxicities included neutropenia (10%), thrombocytopenia (5%), anemia (3%), pneumonia (17%), and fever (3%). Two heavily pre-treated patients developed progressive multifocal leukoencephalopathy. On an intention-to-treat analysis, the overall response rate was 22% (3 complete response, 1 incomplete complete response). Median progression-free and overall survival times were 5 and 11 months, respectively. This study confirms in a daily-life setting the feasibility and acceptable toxicity of ofatumumab treatment in advanced chronic lymphocytic leukemia. The complete response rate, however, was low. Therefore, treatment with ofatumumab should be moved to earlier phases of the disease. Ideally, this should be done in combination with other agents, as recently approved for ofatumumab plus chlorambucil as front-line treatment for patients unfit for fludarabine. The study herein reported is registered as ClinicalTrial.gov National Cancer Institute 01453062.

2015 - Targeting neoplastic B cells and harnessing microenvironment: the “double face” of ibrutinib and idelalisib [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not suitable for high dose chemotherapy with autologous stem cell transplantation (ASCT) has a dismal prognosis and no standard therapy. We designed an Italian multicenter retrospective study aimed at evaluating the safety and efficacy of rituximab plus bendamustine (R–B) as salvage treatment in patients not eligible for ASCT because of age and/or comorbidity or in patients with post-ASCT recurrence. Fifty-five patients with a median age of 76 years were included. The overall response rate was 50%, including 28% complete remission and 22% partial remission. The median overall survival (OS) was 10.8 months. The median progression free survival (PFS) was 8.8 months. Eleven patients are still alive and in complete remission at last follow-up (12–71 months). Toxicity was moderate, mainly grades 1 and 2. R–B showed promising efficacy results with an acceptable toxicity profile and should be further investigated, possibly in combination with novel drugs.

2015 - The Krüppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma [Articolo su rivista]
Piva, R; Deaglio, S; Famà, R; Buonincontri, R; Scarfò, I; Bruscaggin, A; Mereu, E; Serra, S; Spina, V; Brusa, D; Garaffo, G; Monti, S; Dal Bo, M; Marasca, Roberto; Arcaini, L; Neri, A; Gattei, V; Paulli, M; Tiacci, E; Bertoni, F; Pileri, Sa; Foà, R; Inghirami, G; Gaidano, G; Rossi, D.
abstract

Splenic marginal zone lymphoma (SMZL) is an indolent B-cell tumor involving the spleen, and is characterized by recurrent deletion of chromosome 7q and biased usage of the immunoglobulin heavy variable (IGHV) allele 1-2*04.1 Genomic studies have partially unraveled the typical SMZL-coding genome, which is characterized by lesions affecting genes involved in the physiological homeostasis of marginal zone (MZ) B cells, including mutations of NOTCH2.2, 3, 4, 5 However, the full spectrum of lesions that contribute to the malignant transformation of SMZL remains unknown.

2014 - An unusual case of B-ALL occurring in a patient with acute promyelocytic leukemia in remission after two hematopoietic SCTs: whose are the leukemic cells? [Articolo su rivista]
Bigliardi, S; Morselli, M; Potenza, Leonardo; Bresciani, P; Cuoghi, A; Coluccio, Valeria; Riva, Giovanni; Paolini, Ambra; Fantuzzi, Valeria; Faglioni, Laura; Nasillo, Vincenzo; Messerotti, Andrea; Marasca, Roberto; Narni, Franco; Luppi, Mario; Forghieri, Fabio
abstract

We read with interest the recent article by Shiozaki H, et al. (Bone Marrow Transplant 2014; 49: 102–109) reporting a case of donor cell leukemia (DCL) occurring in a patient who previously underwent cord blood (CB) transplantation for myelodysplastic syndrome and describing the clinical and biological differences between DCL arising after hematopoietic SCT (HSCT) from either CB or BM sources. We would like to comment on these issues, reporting an unusual case of DCL in a patient with a long history of acute promyelocytic leukemia (APL).

2014 - Bendamustine in combination with ofatumumab in relapsed or refractory chronic lymphocytic leukemia: a GIMEMA Multicenter Phase II Trial [Articolo su rivista]
Cortelezzi, A; Sciumè, M; Liberati, A. M; Vincenti, D; Cuneo, A; Reda, G; Laurenti, L; Zaja, F; Marasca, Roberto; Chiarenza, A; Gritti, G; Orsucci, L; Storti, S; Angelucci, E; Cascavilla, N; Gobbi, M; Mauro, F. R; Morabito, F; Fabris, S; Piciocchi, A; Vignetti, M; Neri, A; Rossi, D; Giannarelli, D; Guarini, A; Foà, R.
abstract

We conducted a phase II, noncomparative, open-label, multicenter GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) study (CLL0809) to assess the efficacy and safety of bendamustine in combination with ofatumumab (BendOfa) in relapsed/refractory chronic lymphocytic leukemia (CLL). Forty-seven patients from 14 centers were evaluated. Therapy consisted of bendamustine (70 mg/m(2)) for 2 consecutive days every 28 days, and ofatumumab 300 mg on day 1 and 1000 mg on day 8 during the first cycle, and 1000 mg on day 1 subsequently. Treatment was administered up to six cycles. The overall response rate (ORR), as per intention-to-treat analysis, was 72.3% (95% confidence of interval (CI), 57-84%), with 17% complete responses. After a median follow-up of 24.2 months, the overall survival was 83.6% (95% CI, 73.0-95.7%) and the progression-free survival (PFS) was 49.6% (95% CI, 35.9-68.6%). The median PFS was 23.6 months. Univariate and multivariate analyses were used to identify clinical and biological characteristics associated with ORR and PFS. Myelosuppression was the most common toxicity; grade ≥3 neutropenia was observed in 61.7% of patients; however, grade ≥3 infections occurred in 6% of patients. BendOfa is feasible and effective in relapsed/refractory CLL patients, including patients with high-risk clinical and biological features.

2014 - Bortezomib-melphalan-prednisone-thalidomide followed by maintenance with bortezomib-thalidomide compared with bortezomib-melphalan-prednisone for initial treatment of multiple myeloma: updated follow-up and improved survival [Articolo su rivista]
Palumbo, A; Bringhen, S; Larocca, A; Rossi, D; Di Raimondo, F; Magarotto, V; Patriarca, F; Levi, A; Benevolo, G; Vincelli, Id; Grasso, M; Franceschini, L; Gottardi, D; Zambello, R; Montefusco, V; Falcone, Ap; Omedé, P; Marasca, Roberto; Morabito, F; Mina, R; Guglielmelli, T; Nozzoli, C; Passera, R; Gaidano, G; Offidani, M; Ria, R; Petrucci, Mt; Musto, P; Boccadoro, M; Cavo, M.
abstract

PURPOSE: Bortezomib-melphalan-prednisone (VMP) has improved overall survival in multiple myeloma. This randomized trial compared VMP plus thalidomide (VMPT) induction followed by bortezomib-thalidomide maintenance (VMPT-VT) with VMP in patients with newly diagnosed multiple myeloma. PATIENTS AND METHODS: We randomly assigned 511 patients who were not eligible for transplantation to receive VMPT-VT (nine 5-week cycles of VMPT followed by 2 years of VT maintenance) or VMP (nine 5-week cycles without maintenance). RESULTS: In the initial analysis with a median follow-up of 23 months, VMPT-VT improved complete response rate from 24% to 38% and 3-year progression-free-survival (PFS) from 41% to 56% compared with VMP. In this analysis, median follow-up was 54 months. The median PFS was significantly longer with VMPT-VT (35.3 months) than with VMP (24.8 months; hazard ratio [HR], 0.58; P < .001). The time to next therapy was 46.6 months in the VMPT-VT group and 27.8 months in the VMP group (HR, 0.52; P < .001). The 5-year overall survival (OS) was greater with VMPT-VT (61%) than with VMP (51%; HR, 0.70; P = .01). Survival from relapse was identical in both groups (HR, 0.92; P = .63). In the VMPT-VT group, the most frequent grade 3 to 4 adverse events included neutropenia (38%), thrombocytopenia (22%), peripheral neuropathy (11%), and cardiologic events (11%). All of these, except for thrombocytopenia, were significantly more frequent in the VMPT-VT patients. CONCLUSION: Bortezomib and thalidomide significantly improved OS in multiple myeloma patients not eligible for transplantation.

2014 - Endothelin-1 promotes survival and chemoresistance in chronic lymphocytic leukemia B cells through eta receptor [Articolo su rivista]
Maffei, Rossana; Bulgarelli, Jenny; Fiorcari, Stefania; Martinelli, Silvia; Castelli, Ilaria; Valenti, Vanessa; Rossi, Davide; Bonacorsi, Goretta; Zucchini, Patrizia; Potenza, Leonardo; Vallisa, Daniele; Gattei, Valter; Del Poeta, Giovanni; Forconi, Francesco; Gaidano, Gianluca; Narni, Franco; Luppi, Mario; Marasca, Roberto
abstract

The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ET(A)R and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ET(A)R axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ET(A)R triggering. Moreover, the blockade of ET(A)R by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ET(A)R via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ET(A)R blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ET(A)R signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ET(A)R inhibition.

2014 - Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Rizzotto, Lara; Martinelli, Silvia; Rigolin, Gian Matteo; Debbia, Giulia; Castelli, Ilaria; Bonacorsi, Goretta; Santachiara, Rita; Forconi, Francesco; Rossi, Davide; Laurenti, Luca; Palumbo, Giuseppe A.; Vallisa, Daniele; Cuneo, Antonio; Gaidano, Gianluca; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.

2014 - Fludarabine plus alemtuzumab (FA) front-line treatment in young patients with chronic lymphocytic leukemia (CLL) and an adverse biologic profile [Articolo su rivista]
Mauro, Fr; Molica, S; Laurenti, L; Cortelezzi, A; Carella, Am; Zaja, F; Chiarenza, A; Angrilli, F; Nobile, F; Marasca, Roberto; Musolino, C; Brugiatelli, M; Piciocchi, A; Vignetti, M; Fazi, P; Gentile, G; De Propris, Ms; Della Starza, I; Marinelli, M; Chiaretti, S; Del Giudice, I; Nanni, M; Albano, F; Cuneo, A; Guarini, A; Foà, R.
abstract

In 45, ≤ 60 years old patients with CLL and an adverse biologic profile, a front-line treatment with Fludarabine and Campath (Alemtuzumab(®)) was given. The overall response rate was 75.5%, the complete response rate (CR) 24.4% with the lowest CR rates, 16.7% and 8.3%, in 11q and 17p deleted cases. The 3-year progression-free survival (PFS) and overall survival were 42.5% and 79.9%, respectively. PFS was significantly influenced by CLL duration, beta2-microglobulin, and improved by post-remissional stem cell transplantation. Front-line fludarabine and alemtuzumab showed a manageable safety profile and evidence of a benefit in a small series of CLL patients with adverse biologic features.

2014 - Long-term molecular remission with persistence of BCR-ABL1-specific cytotoxic T cells following imatinib withdrawal in an elderly patient with Philadelphia-positive ALL [Articolo su rivista]
Riva, Giovanni; Luppi, Mario; Lagreca, Ivana; Barozzi, Patrizia; Quadrelli, Chiara; Vallerini, Daniela; Zanetti, Eleonora; Basso, Sabrina; Forghieri, Fabio; Morselli, Monica; Maccaferri, Monica; Paolini, Ambra; Fantuzzi, Valeria; Messerotti, Andrea; Maffei, Rossana; Iacobucci, Ilaria; Martinelli, Giovanni; Marasca, Roberto; Narni, Franco; Comoli, Patrizia; Potenza, Leonardo
abstract

Long-term molecular remission with persistence of BCR-ABL1-specific cytotoxic T cells following imatinib withdrawal in an elderly patient with Philadelphia-positive ALL

2014 - NOX-A12: Mobilizing CLL away from home [Articolo su rivista]
Marasca, R.; Maffei, R.
abstract

Comment on "The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization". Hoellenriegel J, Zboralski D, Maasch C, Rosin NY, Wierda WG, Keating MJ, Kruschinski A, Burger JA. Blood. 2014 Feb 13;123(7):1032-9. doi: 10.1182/blood-2013-03-493924. Epub 2013 Nov 25.

2014 - Ruxolitinib for pulmonary extramedullary hematopoiesis in myelofibrosis [Articolo su rivista]
Maccaferri, Monica; Leonardi, Giovanna; Marasca, Roberto; Colaci, Elisabetta; Paolini, Ambra; Soci, Francesco; Forghieri, Fabio; Potenza, Leonardo; Narni, Franco; Luppi, Mario
abstract

Here we report an uncommon case of a patient with MF and pulmonary EMH treated with ruxolitinib

2014 - Severe anemia in a patient with multiple sclerosis treated with natalizumab [Articolo su rivista]
Simone, ANNA MARIA; Ferraro, Diana; Vitetta, Francesca; Marasca, Roberto; Bonacorsi, Goretta; Pinelli, Giovanni; Federzoni, Lucia; Nichelli, Paolo Frigio; Sola, Patrizia
abstract

The paper is a case report of a 51-year-old woman with a 16-year history of relapsing-remitting multiple sclerosis, who developed a severe anemia following a treatment with natalizumab

2014 - Tumor evolutionary directed graphs and the history of chronic lymphocytic leukemia [Articolo su rivista]
Wang, Jiguang; Khiabanian, Hossein; Rossi, Davide; Fabbri, Giulia; Gattei, Valter; Forconi, Francesco; Laurenti, Luca; Marasca, Roberto; Del Poeta, Giovanni; Foà, Robin; Pasqualucci, Laura; Gaidano, Gianluca; Rabadan, Raul
abstract

Cancer is a clonal evolutionary process, caused by successive accumulation of genetic alterations providing milestones of tumor initiation, progression, dissemination, and/or resistance to certain therapeutic regimes. To unravel these milestones we propose a framework, tumor evolutionary directed graphs (TEDG), which is able to characterize the history of genetic alterations by integrating longitudinal and cross-sectional genomic data. We applied TEDG to a chronic lymphocytic leukemia (CLL) cohort of 70 patients spanning 12 years and show that: (a) the evolution of CLL follows a time-ordered process represented as a global flow in TEDG that proceeds from initiating events to late events; (b) there are two distinct and mutually exclusive evolutionary paths of CLL evolution; (c) higher fitness clones are present in later stages of the disease, indicating a progressive clonal replacement with more aggressive clones. Our results suggest that TEDG may constitute an effective framework to recapitulate the evolutionary history of tumors.

2013 - Age and organ damage correlate with poor survival in myeloma patients: meta-analysis of 1435 individual patient data from 4 randomized trials [Articolo su rivista]
Bringhen, Sara; Mateos, Maria Victoria; Zweegman, Sonja; Larocca, Alessandra; Falcone, Antonietta Pia; Oriol, Albert; Rossi, Davide; Cavalli, Maide; Wijermans, Pierre; Ria, Roberto; Offidani, Massimo; Lahuerta, Juan Jose; Liberati, Anna Marina; Mina, Roberto; Callea, Vincenzo; Schaafsma, Martijn; Cerrato, Chiara; Marasca, Roberto; Franceschini, Luca; Evangelista, Andrea; Teruel, Ana Isabel; van der Holt, Bronno; Montefusco, Vittorio; Ciccone, Giovannino; Boccadoro, Mario; San Miguel, Jesus; Sonneveld, Pieter; Palumbo, Antonio
abstract

Thalidomide and bortezomib are extensively used to treat elderly myeloma patients. In these patients, treatment-related side effects are frequent and full drug doses difficult to tolerate. We retrospectively analyzed data from 1435 elderly patients enrolled in 4 European phase III trials including thalidomide and/or bortezomib. After a median follow up of 33 months (95%CI: 10-56 months), 513 of 1435 patients (36%) died; median overall survival was 50 months (95%CI: 46-60 months). The risk of death was increased in patients aged 75 years or over (HR 1.44, 95%CI: 1.20-1.72; P&lt;0.001), in patients with renal failure (HR 2.02, 95%CI: 1.51-2.70; P&lt;0.001), in those who experienced grade 3-4 infections, cardiac or gastrointestinal adverse events during treatment (HR 2.53, 95%CI: 1.75-3.64; P&lt;0.001) and in those who required drug discontinuation due to adverse events (HR 1.67, 95%CI; 1.12-2.51; P=0.01). This increased risk was restricted to the first six months after occurrence of adverse events or drug discontinuation and declined over time. More intensive approaches, such as the combination of bortezomib-thalidomide, negatively affected outcome. Bortezomib-based combinations may overcome the negative impact of renal failure. Age 75 years or over or renal failure at presentation, occurrence of infections, cardiac or gastrointestinal adverse events negatively affected survival. A detailed geriatric assessment, organ evaluation and less intense individualized approaches are suggested in elderly unfit subjects.

2013 - ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia [Articolo su rivista]
Martinelli, Silvia; Kanduri, M; Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Marasca, Roberto; Rosenquist, R.
abstract

Increasing evidence suggests a key role for angiopoietin-2 (ANGPT2) in influencing the aggressiveness of chronic lymphocytic leukemia (CLL). In the presence of vascular endothelial growth factor (VEGF), ANGPT2 causes vessel destabilization leading to neoangiogenesis. Accordingly, high expression levels of ANGPT2 and high degree of angiogenesis have consistently been associated with poor prognosis in CLL; however, the molecular mechanisms behind the variability in ANGPT2 expression are still to be discovered. Here, for the first time, we investigated the DNA methylation status of the ANGPT2 promoter in a large CLL cohort (n = 88) using pyrosequencing and correlated methylation data with ANGPT2 expression levels, prognostic factors and outcome. Importantly, methylation levels of the ANGPT2 gene correlated inversely with its mRNA expression levels (p<0.001). Moreover, low ANGPT2 methylation status was highly associated with adverse prognostic markers, shorter time to first treatment and overall survival. Finally, treatment with methyl inhibitors induced re-expression of ANGPT2 in two B-cell lymphoma cell lines, underscoring the importance of DNA methylation in regulating transcriptional silencing of this gene. In conclusion, we believe that the known variability in ANGPT2 expression among CLL patients could be explained by differential promoter DNA methylation and that low methylation levels of the ANGPT2 promoter have an adverse prognostic impact in CLL.

2013 - Association between molecular lesions and specific B-cell receptor subsets in chronic lymphocytic leukemia [Articolo su rivista]
Rossi, D; Spina, Vincenzo; Bomben, R; Rasi, S; Dal Bo, M; Bruscaggin, A; Rossi, Fm; Monti, S; Degan, M; Ciardullo, C; Serra, R; Zucchetto, A; Nomdedeu, J; Bulian, P; Grossi, A; Zaja, F; Pozzato, G; Laurenti, L; Efremov, Dg; Di Raimondo, F; Marasca, Roberto; Forconi, F; Del Poeta, G; Gaidano, G; Gattei, Valter
abstract

Genetic lesions and B-cell receptor (BCR) signaling are both oncogenic drivers in chronic lymphocytic leukemia (CLL). However, scant data are available on preferential associations between specific genetic alterations and stereotyped BCR subsets. By analyzing 1419 cases, 2 CLL subsets (2 and 8) harboring stereotyped BCR are enriched in specific molecular alterations influencing disease course. SF3B1 mutations are the genetic hallmark of IGHV3-21-CLL belonging to subset 2 (52%) but are evenly represented in nonstereotyped IGHV3-21-CLL. Trisomy 12 (87%) and NOTCH1 mutations (62%) characterize IGHV4-39-CLL belonging to subset 8 but occur with the expected frequency in IGHV4-39-CLL with heterogeneous BCR. Clinically, co-occurrence of SF3B1 mutations and subset 2 BCR configuration prompts disease progression in IGHV3-21-CLL, whereas cooperation between NOTCH1 mutations, +12, and subset 8 BCR configuration invariably primes CLL transformation into Richter syndrome. These findings provide a proof of concept that specific stereotyped BCR may promote or select molecular lesions influencing outcome.

2013 - Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients [Articolo su rivista]
Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Forghieri, Fabio; Beauvais, Anne; Beau, Remi; Candoni, Anna; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Zanetti, Eleonora; Quadrelli, Chiara; Codeluppi, Mauro; Guaraldi, Giovanni; Pagano, Livio; Caira, Morena; DEL GIOVANE, Cinzia; Maccaferri, Monica; Stefani, Alessandro; Morandi, Uliano; Tazzioli, Giovanni; Girardis, Massimo; Delia, Mario; Specchia, Giorgina; Longo, Giuseppe; Marasca, Roberto; Narni, Franco; Merli, Francesco; Imovilli, Annalisa; Apolone, Giovanni; Carvalho, Agostinho; Comoli, Patrizia; Romani, Luigina; Latgè, Jean Paul; Luppi, Mario
abstract

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1-3glucan, β1-3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA. © 2013 Potenza et al.

2013 - Clinical heterogeneity of de novo 11q deletion chronic lymphocytic leukaemia: prognostic relevance of extent of 11q deleted nuclei inside leukemic clone. [Articolo su rivista]
Marasca, Roberto; Maffei, Rossana; Martinelli, Silvia; Fiorcari, Stefania; Bulgarelli, Jenny; Debbia, Giulia; Rossi, D; Rossi, Fm; Rigolin, Gm; Martinelli, S; Gattei, V; Del Poeta, G; Laurenti, L; Forconi, F; Montillo, M; Gaidano, G; Luppi, Mario
abstract

Deletion on the long arm of chromosome 11 occurs in 5-20% of chronic lymphocytic leukaemia (CLL) patients. We analysed clinical-biological characteristics of 131 CLL patients carrying 11q deletion documented before therapy (de novo 11q deleted CLL). De novo 11q deleted CLL were characterized by high frequencies of unmutated immunoglobulin variable heavy genes, multiple fluorescence in situ hybridization aberrations and lymph node involvement. Factors significantly associated with shorter time to first treatment (TTFT) were advanced Binet stages, high white blood cell count, increased β(2) -microglobulin levels, 17p in addition, splenomegaly and more extensive lymphadenopathy. We found that patients with <25% 11q deleted nuclei (n = 22) experienced longer TTFT compared with patients with ≥25% 11q deleted nuclei (n = 87; median TTFT, 40 vs. 14 months, p = 0.011) and also showed better response to treatments (complete response, 50% vs. 21%, p = 0.016). The variables identified by multivariate analysis as independently associated with reduced TTFT were advanced Binet stages [hazard ratio (HR) 4.69; p < 0.001] and ≥25% 11q deleted nuclei (HR 4.73; p = 0.004). De novo 11q deleted CLLs exhibit variable clinical outcome. The percentage of deleted nuclei inside leukemic clone should be included in the prognostic definition of therapy-naïve 11q deleted CLL patients.

2013 - Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia [Articolo su rivista]
Rossi, D; Rasi, S; Spina, V; Bruscaggin, A; Monti, S; Ciardullo, C; Deambrogi, C; Khiabanian, H; Serra, R; Bertoni, F; Forconi, F; Laurenti, L; Marasca, Roberto; Dal Bo, M; Rossi, Fm; Bulian, P; Nomdedeu, J; Del Poeta, G; Gattei, V; Pasqualucci, L; Rabadan, R; Foà, R; Dalla Favera, R; Gaidano, G.
abstract

The identification of new genetic lesions in chronic lymphocytic leukemia (CLL) prompts a comprehensive and dynamic prognostic algorithm including gene mutations and chromosomal abnormalities and their changes during clonal evolution. By integrating mutational and cytogenetic analysis in 1274 CLL samples and using both a training-validation and a time-dependent design, 4 CLL subgroups were hierarchically classified: (1) high-risk, harboring TP53 and/or BIRC3 abnormalities (10-year survival: 29%); (2) intermediate-risk, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival: 37%); (3) low-risk, harboring +12 or a normal genetics (10-year survival: 57%); and (4) very low-risk, harboring del13q14 only, whose 10-year survival (69.3%) did not significantly differ from a matched general population. This integrated mutational and cytogenetic model independently predicted survival, improved CLL prognostication accuracy compared with FISH karyotype (P < .0001), and was externally validated in an independent CLL cohort. Clonal evolution from lower to higher risk implicated the emergence of NOTCH1, SF3B1, and BIRC3 abnormalities in addition to TP53 and 11q22-q23 lesions. By taking into account clonal evolution through time-dependent analysis, the genetic model maintained its prognostic relevance at any time from diagnosis. These findings may have relevant implications for the design of clinical trials aimed at assessing the use of mutational profiling to inform therapeutic decisions.

2013 - Large genomic aberrations detected by SNP array are independent prognosticators of a shorter time to first treatment in chronic lymphocytic leukemia patients with normal FISH [Articolo su rivista]
Mian, M; Rinaldi, A; Mensah, A. A; Rossi, D; Ladetto, M; Forconi, F; Marasca, Roberto; Uhr, M; Stussi, G; Kwee, I; Cavalli, F; Gaidano, G; Zucca, E; Bertoni, F.
abstract

Genomic complexity can predict the clinical course of patients affected by chronic lymphocytic leukemia (CLL) with a normal FISH. However, large studies are still lacking. Here, we analyzed a large series of CLL patients and also carried out the so far largest comparison of FISH versus single-nucleotide polymorphism (SNP) array in this disease.

2013 - MGA, a suppressor of MYC, is recurrently inactivated in high risk chronic lymphocytic leukemia [Articolo su rivista]
De Paoli, L; Cerri, M; Monti, S; Rasi, S; Spina, V; Bruscaggin, A; Greco, M; Ciardullo, C; Famà, R; Cresta, S; Maffei, Rossana; Ladetto, M; Martini, M; Laurenti, L; Forconi, F; Marasca, Roberto; Larocca, Lm; Bertoni, F; Gaidano, G; Rossi, D.
abstract

The genetic lesions identified to date in CLL do not fully explain the development of chemorefractoriness and RS transformation. Given the role of the MYC pathway in Richter syndrome, here we investigated high risk CLL for genetic alterations of the MAX gene associated (MGA) gene, a suppressor of MYC transcriptional activation. MGA mutations and deletions were restricted to a single allele in all evaluable cases and were not accompanied by alterations of the second allele. Collectively, by combining mutations and deletions, MGA disruption was observed in 5/48 (10.4%) high risk CLLs, including 3/19 (15.7%) fludarabine-refractory CLLs and 2/29 (6.8%) RS.This observation, along with the notion that MGA is a suppressor of MYC and an inhibitor of MYC-induced cell transformation [4], points to MGA disruption as a new genetic lesion affecting the MYC pathway in high risk CLL and corroborates the notion that activation of MYC is a major contributor to the pathogenesis of RS.

2013 - Monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation [Articolo su rivista]
Maffei, Rossana; Bulgarelli, Jenny; Fiorcari, Stefania; Bertoncelli, L; Martinelli, Silvia; Guarnotta, C; Castelli, Ilaria; Deaglio, S; Debbia, Giulia; DE BIASI, Sara; Bonacorsi, G; Zucchini, Patrizia; Narni, Franco; Tripodo, C; Luppi, Mario; Cossarizza, Andrea; Marasca, Roberto
abstract

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B-cells and the extent of infiltration is associated with adverse prognostic factors. Blood monocyte population was studied by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate T cell proliferation in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes treated with leukemia were also evaluated. Chronic lymphocytic leukemia patients showed an increase in the absolute number of monocytes compared to normal controls (792+/-86 cells/mL vs. 485+/-46 cells/mL, p=0.003). Higher number of nonclassical CD14+CD16++ and Tie-2 expressing monocytes (TEMs) was also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment in phagocytosis. We also detected gene alterations such as the down-regulation of PTGR2, a reductase able to inactivate the prostaglandin E2, indicating an immunosuppressive activity. Accordingly, T cell proliferation was inhibited in contact with monocytes from patients compared to normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated 'education' of immune elements may also include the establishment of a skewed phenotype in monocyte/macrophage population.

2013 - Promoter methylation patterns in Richter syndrome affect stem-cell maintenance and cell cycle regulation and differ from de novo diffuse large B-cell lymphoma [Articolo su rivista]
Rinaldi, A; Mensah, Aa; Kwee, I; Forconi, F; Orlandi, Em; Lucioni, M; Gattei, V; Marasca, Roberto; Berger, F; Cogliatti, S; Cavalli, F; Zucca, E; Gaidano, G; Rossi, D; Bertoni, F.
abstract

In a fraction of patients, chronic lymphocytic leukaemia (CLL) can transform to Richter syndrome (RS), usually a diffuse large B-cell lymphoma (DLBCL). We studied genome-wide promoter DNA methylation in RS and clonally related CLL-phases of transformed patients, alongside de novo DLBCL (of non-germinal centre B type), untransformed-CLL and normal B-cells. The greatest differences in global DNA methylation levels were observed between RS and DLBCL, indicating that these two diseases, although histologically similar, are epigenetically distinct. RS was more highly methylated for genes involved in cell cycle regulation. When RS was compared to the preceding CLL-phase and with untransformed-CLL, RS presented a higher degree of methylation for genes possessing the H3K27me3 mark and PRC2 targets, as well as for gene targets of TP53 and RB1. Comparison of the methylation levels of individual genes revealed that OSM, a stem cell regulatory gene, exhibited significantly higher methylation levels in RS compared to CLL-phases. Its transcriptional repression by DNA methylation was confirmed by 5-aza-2'deoxycytidine treatment of DLBCL cells, determining an increased OSM expression. Our results showed that methylation patterns in RS are largely different from de novo DLBCL. Stem cell-related genes and cell cycle regulation genes are targets of DNA methylation in RS.

2013 - The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells [Articolo su rivista]
Fiorcari, Stefania; Brown, Wells S; Mcintyre, Bradley W; Estrov, Zeev; Maffei, Rossana; O'Brien, Susan; Sivina, Mariela; Hoellenriegel, Julia; Wierda, William G; Keating, Michael J; Ding, Wei; Kay, Neil E; Lannutti, Brian J; Marasca, Roberto; Burger, Jan A.
abstract

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

2013 - Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome [Articolo su rivista]
Chigrinova, Ekaterina; Rinaldi, Andrea; Kwee, Ivo; Rossi, Davide; Rancoita, Paola M. V; Strefford, Jonathan C; Oscier, David; Stamatopoulos, Kostas; Papadaki, Theodora; Berger, Francoise; Young, Ken H; Murray, Fiona; Rosenquist, Richard; Greiner, Timothy C; Chan, Wing C; Orlandi, Ester M; Lucioni, Marco; Marasca, Roberto; Inghirami, Giorgio; Ladetto, Marco; Forconi, Francesco; Cogliatti, Sergio; Votavova, Hana; Swerdlow, Steven H; Stilgenbauer, Stephan; Piris, Miguel A; Matolcsy, Andras; Spagnolo, Dominic; Nikitin, Eugene; Zamò, Alberto; Gattei, Valter; Bhagat, Govind; Ott, German; Zucca, Emanuele; Gaidano, Gianluca; Bertoni, Francesco
abstract

Richter syndrome (RS) occurs in up to 15% of patients with chronic lymphocytic leukemia (CLL). Although RS, usually represented by the histologic transformation to a diffuse large B-cell lymphoma (DLBCL), is associated with a very poor outcome, especially when clonally related to the preexisting CLL, the mechanisms leading to RS have not been clarified. To better understand the pathogenesis of RS, we analyzed a series of cases including 59 RS, 28 CLL phase of RS, 315 CLL, and 127 de novo DLBCL. RS demonstrated a genomic complexity intermediate between CLL and DLBCL. Cell-cycle deregulation via inactivation of TP53 and of CDKN2A was a main mechanism in the histologic transformation from CLL phase, being present in approximately one half of the cases, and affected the outcome of the RS patients. A second major subgroup was characterized by the presence of trisomy 12 and comprised one third of the cases. Although RS shared some of the lesions seen in de novo DLBCL, its genomic profile was clearly separate. The CLL phase preceding RS had not a generalized increase in genomic complexity compared with untransformed CLL, but it presented clear differences in the frequency of specific genetic lesions.

2012 - Atraumatic splenic rupture in patients with myelodysplastic syndromes: Report of a case occurred during treatment with 5-azacitidine and review of the literature. [Articolo su rivista]
Forghieri, Fabio; Morselli, M; Leonardi, G; Potenza, Leonardo; Bonacorsi, G; Coluccio, V; Paolini, Ambra; Maccaferri, Monica; Colaci, Elisabetta; Fantuzzi, Valeria; Bigliardi, Sara; Zaldini, Piera; Riva, Giovanni; Barozzi, Patrizia; Leonardi, L; Rossi, A; Marasca, Roberto; Narni, Franco; Luppi, Mario
abstract

No abstract available

2012 - Chronic/relapsing lymphadenopathy associated with HHV-6B infection: a new benign clinico-pathologic entity occurring in immunocompetent individuals [Abstract in Rivista]
Forghieri, Fabio; Potenza, Leonardo; Barozzi, Patrizia; Vallerini, Daniela; Riva, Giovanni; Zanetti, E; Quadrelli, C; Morselli, M; Leonardi, G; Maccaferri, M; Paolini, Ambra; Coluccio, Valeria; Colaci, Elisabetta; Pedrazzi, Letizia; Fantuzzi, Valeria; Bigliardi, Sara; Soci, Francesco; Bonacorsi, G; Zaldini, P; Rossi, G; Milani, M; Rivasi, Francesco; Gennari, W; Pecorari, M; Grottola, Antonella; Tagliazucchi, S; Rumpianesi, F; Mattioli, F; Presutti, Livio; Franzoni, Chiara; Gelmini, Roberta; Saviano, Massimo; Cermelli, Claudio; Marasca, Roberto; Narni, Franco; Luppi, Mario
abstract

Background. HHV-6 DNA sequences were disclosed in lymph node (LN) tis- sues of several patients with lymphoid malignancies, but a direct major role of HHV-6 in lymphoid malignant transformation has so far not been confirmed. In contrast, active HHV-6 infection has been associated to either infectious mononucleosis-like syndrome or acute lymphadenitis occurring in febrilepatients with systemic symptoms, or to Rosai-Dorfman disease in which viral antigens have been detected by immunohistochimical (IHC) analyses in both histiocytes and follicular dendritic cells (FDCs). Methods. We have retrospec- tively analyzed clinical and pathological data of 365 adult patients, consecutive- ly observed at our Institution over a period of 5 years (2006-2010), because of enlarged superficial lymph nodes and subsequently undergoing lymphadenec- tomy. In the benign/reactive cases in which well-recognized etiologies have been excluded, an involvement of HHV-6 active infection or reactivation was investigated by molecular and immunohistochemical examinations. Results. Malignant disorders, namely malignant lymphoproliferative disorders or solid cancer metastases, were found in 227 cases (62%), whereas in 138 cases (38%) benign/reactive pictures were documented on lymph node examination. Among these latter cases, a well-recognized etiology was demonstrated in 84 patients (61%), while in 54 cases (39%), a well-defined non-malignant reactive/infectious cause could not be documented. Immunohistochemical analyses resulted negative for both HHV-6A and HHV-6B in 38 of these latter lymph nodes (70%). In 7 patients (13%), a scattered, scanty and aspecific pos- itivity for HHV-6B late protein was documented in rare interfollicular plasma cells and histiocytes. Surprisingly, in 9 patients (17%), immunohistochemical analyses showed HHV-6B positive staining of FDCs, together with scattered positivity of interfollicular cells. These 9 HIV-negative adult patients (median age 42 years, range 18-76 years), with either localized or generalized LAP, were observed for a median follow-up of 38 months (range 28-166). Of note, six of them presented with recurrent LAP (one to 3 recurrences), without evolving into lymphoma. A common LN histological pattern at presentation showed florid fol- licular hyperplasia with concurrent mild paracortical expansion. Three cases also showed features consistent with PTGC. Constitutional symptoms were absent in all patients. The IHC reactions for both HHV-6A and HHV-6B, per- formed on further control cases, represented by 131 LN tissues from patients with either benign LAP induced by other known etiologies or lymphoma, were invariably negative. Serology was positive for both IgM and IgG with high avid- ity suggesting viral reactivation/reinfection. However, the molecular analyses failed to detect HHV-6 viremias in cell-free-serum samples of all the 9 patients with positive HHV-6B IHC staining, while positivity for HHV-6B DNA was dis- closed by PCR analyses in 7 out of the 7 LN tissues studied. Conclusions. We show for the first time that local reactivation/infection of HHV-6B should be con- sidered among the possible causes of chronic/relapsing benign LAP in immuno- competent individuals. IHC is the method of choice for investigating the pres- ence of HHV-6 infection in such cases. HHV-6B may indirectly modulate and trigger the proliferation of lymphocytes, by locally affecting FDCs and LN microenvironment. FDCs may indeed be involved in presenting HHV-6B anti- gens to other immune cells, mainly cortical B lymphocytes.

2012 - Del(13q14.3) length matters: an integrated analysis of genomic, fluorescence in situ hybridization and clinical data in 169 chronic lymphocytic leukaemia patients with 13q deletion alone or a normal karyotype.Hematol Oncol. 2011 Jun 13. doi: 10.1002/hon.997 [Articolo su rivista]
Mian, M; Rinaldi, A; Mensah, Aa; Rossi, D; Ladetto, M; Forconi, F; Marasca, Roberto; Gattei, V; Zucca, E; Cavalli, F; Gaidano, G; Kwee, I; Bertoni, F.
abstract

Del(13q14.3) length matters: an integrated analysis of genomic, fluorescence in situ hybridization and clinical data in 169 chronic lymphocytic leukaemia patients with 13q deletion alone or a normal karyotype

2012 - Different impact of NOTCH1 and SF3B1 mutations on the risk of chronic lymphocytic leukemia transformation to Richter syndrome [Articolo su rivista]
Davide, Rossi; Silvia, Rasi; Valeria, Spina; Marco, Fangazio; Sara, Monti; Mariangela, Greco; Carmela, Ciardullo; Rosella, Famà; Stefania, Cresta; Alessio, Bruscaggin; Luca, Laurenti; Maurizio, Martini; Pellegrino, Musto; Francesco, Forconi; Marasca, Roberto; Luigi M., Larocca; Robin, Foà; Gianluca, Gaidano
abstract

Different impact of NOTCH1 and SF3B1 mutations on the risk of chronic lymphocytic leukemia transformation to Richter syndrome

2012 - IGHV gene mutational status and 17p deletion are independent molecular predictors in a comprehensive clinical-biological prognostic model for overall survival prediction in chronic lymphocytic leukemia. [Articolo su rivista]
Bulian, P; Rossi, D; Forconi, F; Del Poeta, G; Bertoni, F; Zucca, E; Montillo, M; Pozzato, G; D'Arena, G; Efremov, D; Marasca, Roberto; Lauria, F; Gaidano, G; Gattei, V; Laurenti, L.
abstract

BACKGROUND: Prognostic index for survival estimation by clinical-demographic variables were previously proposed in chronic lymphocytic leukemia (CLL) patients. Our objective was to test in a large retrospective cohort of CLL patients the prognostic power of biological variables in a comprehensive model including clinical and demographic parameters. A new prognostic index was proposed.METHODS: Overall survival and time to treatment in 620 untreated CLL patients were analyzed retrospectively to evaluate the independent predictive power of mutational status of immunoglobulin heavy chain variable gene segments (IGHV), high-risk chromosomal aberration such as 17p or 11q deletions, CD38 and ZAP-70 expression, age, gender, Binet stage, beta2-microglobulin levels, absolute lymphocyte count and number of lymph node regions.RESULTS: IGHV mutational status and 17p deletion, but not CD38 and ZAP-70 expression, were independent prognostic biological variables in a multivariate model for overall survival, which included demographic (age and gender) and clinical parameters (Binet staging, beta2-microglobulin levels). Analysis of time to treatment in Binet A patients below 70 years of age showed that IGHV was the most important predictor. A novel 6-variable clinical-biological prognostic index was developed and internally validated, which assigned 3 points for Binet C stage, 2 points/each for Binet B stage and for age &gt; 65 years, 1 point/each for male gender, high beta2-microglobulin levels, presence of an unmutated IGHV gene status or 17p deletion. Patients were classified at low-risk (score = 0-1; 21%), intermediate-risk (score 2-5; 63% of cases), high-risk (score 6-9; 16% of cases). Projected 5-year overall survival was 98%, 90% and 58% in low-, intermediate- and high-risk groups, respectively. A nomogram for individual patient survival estimation was also proposed.CONCLUSIONS: Data indicate that IGHV mutational status and 17p deletion may be integrated with clinical-demographic variables in new prognostic tools to estimate overall survival.

2012 - Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes [Articolo su rivista]
Ferrero, S; Capello, D; Svaldi, M; Boi, M; Gatti, D; Drandi, D; Rossi, D; Barbiero, S; Mantoan, B; Mantella, E; Zanni, M; Ghione, P; Larocca, A; Passera, R; Bertoni, F; Gattei, V; Forconi, F; Laurenti, L; Del Poeta, G; Marasca, Roberto; Cortelazzo, S; Gaidano, G; Palumbo, A; Boccadoro, M; Ladetto, M.
abstract

Background. Characterization of the immunoglobulin genes repertoire improved the understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. Design and Methods. To verify this hypothesis we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptors clusters. Results. Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving few genes, demonstrating that myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity-determining-region 3 was 15.5 aminoacids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. Conclusions. Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

2012 - Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia. [Articolo su rivista]
Rossi, D; Rasi, S; Fabbri, G; Spina, V; Fangazio, M; Forconi, F; Marasca, Roberto; Laurenti, L; Bruscaggin, A; Cerri, M; Monti, S; Cresta, S; Famà, R; De Paoli, L; Bulian, P; Gattei, V; Guarini, A; Deaglio, S; Capello, D; Rabadan, R; Pasqualucci, L; Dalla Favera, R; Foà, R; Gaidano, G.
abstract

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

2012 - NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL [Articolo su rivista]
Del Giudice, I; Rossi, D; Chiaretti, S; Marinelli, M; Tavolaro, S; Gabrielli, S; Laurenti, L; Marasca, Roberto; Rasi, S; Fangazio, M; Guarini, A; Gaidano, G; Foa', R.
abstract

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (p=.003) and +12 as a sole cytogenetic abnormality (p=.008). NOTCH1 mutations in +12 CLL associated with a ~2.4 fold increase in the death risk, a significant survival shortening (p&lt;.001) and proved an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biologic contribution of NOTCH1 mutations in determining a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL

2012 - Pathogenetic mechanisms of hepatitis C virus-induced B-cell lymphomagenesis. [Articolo su rivista]
Forghieri, Fabio; Luppi, Mario; Barozzi, Patrizia; Maffei, Rossana; Potenza, Leonardo; Narni, Franco; Marasca, Roberto
abstract

Hepatitis C virus (HCV) infection is probably the most common chronic viral infection and affects an estimated 180 million people worldwide, accounting for 3% of the global population. Although the liver is considered to be the primary target, extrahepatic manifestations are well recognized among patients with chronic HCV infection. Epidemiological studies have clearly demonstrated a correlation between chronic HCV infection and occurrence of B-cell non-Hodgkin's lymphomas (B-NHL). The clinical evidence that antiviral therapy has a significant role in the treatment at least of some HCV-associated lymphoproliferative disorders, especially indolent B-NHL, further supports the existence of an etiopathogenetic link. However, the mechanisms exploited by HCV to induce B-cell lymphoproliferation have so far not completely clarified. It is conceivable that different biological mechanisms, namely, chronic antigen stimulation, high-affinity interaction between HCV-E2 protein and its cellular receptors, direct HCV infection of B-cells, and "hit and run" transforming events, may be combined themselves and cooperate in a multifactorial model of HCV-associated lymphomagenesis.

2012 - Physical contact with endothelial cells through β1- and β2- integrins rescues chronic lymphocytic leukemia from spontaneous and drug-induced apoptosis and induces a peculiar gene expression profile on leukemic cells. [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Martinelli, Silvia; Castelli, Ilaria; Deaglio, S; Debbia, Giulia; Fontana, M; Coluccio, Valeria; Bonacorsi, G; Zucchini, Patrizia; Narni, Franco; Torelli, Giuseppe; Luppi, Mario; Marasca, Roberto
abstract

Background: Chronic lymphocytic leukemia B-cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interaction with endothelial cells in infiltrated tissues and during recirculation may have a pathogenetic role in chronic lymphocytic leukemia.Design and Methods: We evaluated apoptosis of leukemic cells after co-culture on HUVEC monolayer with addition of Fludarabine and blocking adhesion antibodies. Then, we compared microarray-based expression profiles between leukemic cells at baseline and after co-culture.ùResults: We found that endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days co-culture. Moreover, endothelial layer decreased sensitivity of chronic lymphocytic leukemia B-cells to Fludarabine-induced apoptosis. Physical contact with endothelium mediated by both β1- and β2- integrins is essential for survival advantage. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B-cells determined the almost complete abrogation of survival advantage (&gt;70% inhibition of viability). Conversely, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes on chronic lymphocytic leukemia B-cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, increase of genes involved in TGFβ and Wnt signalling pathways, secretion of cytokines recruiting stromal cells and macrophages and increase in anti-apoptotic molecules such as Bcl2 and Survivin. Conclusion: Our study supports the notion that endothelial cells are major players in chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly sustains survival, protects from drug-induced apoptosis and widely modifies gene expression profile of leukemic cells.

2012 - The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development [Articolo su rivista]
D., Rossi; V., Trifonov; M., Fangazio; A., Bruscaggin; S., Rasi; V., Spina; S., Monti; T., Vaisitti; F., Arruga; R., Fama; C., Ciardullo; M., Greco; S., Cresta; D., Piranda; A., Holmes; G., Fabbri; M., Messina; A., Rinaldi; J., Wang; C., Agostinelli; P. P., Piccaluga; M., Lucioni; F., Tabbo; R., Serra; S., Franceschetti; C., Deambrogi; G., Daniele; V., Gattei; Marasca, Roberto; F., Facchetti; L., Arcaini; G., Inghirami; F., Bertoni; S. A., Pileri; S., Deaglio; R., Foa; R., Dalla Favera; L., Pasqualucci; R., Rabadan; G., Gaidano
abstract

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

2011 - A case of JAK2 V617F-positive myelodysplastic/myeloproliferative neoplasm with unusual morphology, resembling acute promyelocytic leukemia-like disorder with a chronic course. [Articolo su rivista]
Forghieri, Fabio; Morselli, M; Potenza, Leonardo; Maccaferri, Monica; Pedrazzi, Letizia; Coluccio, Valeria; Barozzi, Patrizia; Vallerini, Daniela; Riva, Giovanni; Zanetti, Eleonora; Quarelli, C; Bonacorsi, G; Artusi, Tullio; Zaldini, Piera; Zucchini, Patrizia; Marasca, Roberto; Narni, Franco; Falini, B; Torelli, Giuseppe; Luppi, Mario
abstract

An atypical case of myelodysplastic syndrome with morphologic aspect resempling acute promyelocytic leukemia, carring JA2 mutations.

2011 - A variant of the LRP4 gene affects the risk of chronic lymphocytic leukaemia transformation to Richter syndrome [Articolo su rivista]
Rasi, S; Spina, V; Bruscaggin, A; Vaisitti, T; Tripodo, C; Forconi, F; De Paoli, L; Fangazio, M; Sozzi, E; Cencini, E; Laurenti, L; Marasca, Roberto; Visco, C; Xu Monette, Zy; Gattei, V; Young, Kh; Malavasi, F; Deaglio, S; Gaidano, G; Rossi, D.
abstract

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma. Risk factors of CLL transformation to RS are only partly known. We explored the role of the host genetic background as a risk factor for RS occurrence. Forty-five single nucleotide polimorphisms (SNPs) known to be relevant for CLL prognosis were genotyped in a consecutive cohort of 331 CLL, of which 21 had transformed to RS. After correcting for multiple testing and adjusting for previously reported RS risk factors, the LRP4 rs2306029 TT variant genotype was the sole SNP independently associated with a higher risk of RS transformation (Hazard Ratio: 4·17; P = 0·001; q = 0·047). The enrichment of LRP4 TT genotype in RS was confirmed in an independent series (n = 44) used for validation purposes. The LRP4 protein was expressed in CLL (n =66). Bioinformatic analysis scored LRP4 rs2306029 as a variant with possible deleterious and damaging variant of LRP4. LRP4 genotyping may help the recognition of patients with increased risk of RS at the time of CLL diagnosis

2011 - Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma [Articolo su rivista]
Rossi, D; Deaglio, S; Dominguez Sola, D; Rasi, S; Vaisitti, T; Agostinelli, C; Spina, V; Bruscaggin, A; Monti, S; Cerri, M; Cresta, S; Fangazio, M; Arcaini, L; Lucioni, M; Marasca, Roberto; Thieblemont, C; Capello, D; Facchetti, F; Kwee, I; Pileri, Sa; Foà, R; Bertoni, F; Dalla Favera, R; Pasqualucci, L; Gaidano, G.
abstract

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

2011 - Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation [Articolo su rivista]
Fabbri, G; Rasi, S; Rossi, D; Trifonov, V; Khiabanian, H; Ma, J; Grunn, A; Fangazio, M; Capello, D; Monti, S; Cresta, S; Gargiulo, E; Forconi, F; Guarini, A; Arcaini, L; Paulli, M; Laurenti, L; Larocca, Lm; Marasca, Roberto; Gattei, V; Oscier, D; Bertoni, F; Mullighan, Cg; Foá, R; Pasqualucci, L; Rabadan, R; Dalla Favera, R; Gaidano, G.
abstract

The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains <20 clonally represented gene alterations/case, including predominantly nonsilent mutations, and fewer copy number aberrations. These analyses led to the discovery of several genes not previously known to be altered in CLL. Although most of these genes were affected at low frequency in an expanded CLL screening cohort, mutational activation of NOTCH1, observed in 8.3% of CLL at diagnosis, was detected at significantly higher frequency during disease progression toward Richter transformation (31.0%), as well as in chemorefractory CLL (20.8%). Consistent with the association of NOTCH1 mutations with clinically aggressive forms of the disease, NOTCH1 activation at CLL diagnosis emerged as an independent predictor of poor survival. These results provide initial data on the complexity of the CLL coding genome and identify a dysregulated pathway of diagnostic and therapeutic relevance

2011 - Aspirin, Warfarin, or Enoxaparin Thromboprophylaxis in Patients With Multiple Myeloma Treated With Thalidomide: A Phase III, Open-Label, Randomized Trial. [Articolo su rivista]
Palumbo, A; Cavo, M; Bringhen, S; Zamagni, E; Romano, A; Patriarca, F; Rossi, D; Gentilini, F; Crippa, C; Galli, M; Nozzoli, C; Ria, R; Marasca, Roberto; Montefusco, V; Baldini, L; Elice, F; Callea, V; Pulini, S; Carella, Am; Zambello, R; Benevolo, G; Magarotto, V; Tacchetti, P; Pescosta, N; Cellini, C; Polloni, C; Evangelista, A; Caravita, T; Morabito, F; Offidani, M; Tosi, P; Boccadoro, M.
abstract

PurposeIn patients with myeloma, thalidomide significantly improves outcomes but increases the risk ofthromboembolic events. In this randomized, open-label, multicenter trial, we compared aspirin(ASA) or fixed low-dose warfarin (WAR) versus low molecular weight heparin (LMWH) forpreventing thromboembolism in patients with myeloma treated with thalidomide-based regimens.Patients and MethodsA total of 667 patients with previously untreated myeloma who received thalidomide-containingregimens and had no clinical indication or contraindication for a specific antiplatelet or anticoagulanttherapy were randomly assigned to receive ASA (100 mg/d), WAR (1.25 mg/d), or LMWH(enoxaparin 40 mg/d). A composite primary end point included serious thromboembolic events,acute cardiovascular events, or sudden deaths during the first 6 months of treatment.ResultsOf 659 analyzed patients, 43 (6.5%) had serious thromboembolic events, acute cardiovascularevents, or sudden death during the first 6 months (6.4% in the ASA group, 8.2% in the WARgroup, and 5.0% in the LMWH group). Compared with LMWH, the absolute differences were1.3% (95% CI, 3.0% to 5.7%; P .544) in the ASA group and 3.2% (95% CI, 1.5% to7.8%; P .183) in the WAR group. The risk of thromboembolism was 1.38 times higher inpatients treated with thalidomide without bortezomib. Three major (0.5%) and 10 minor (1.5%)bleeding episodes were recorded.ConclusionIn patients with myeloma treated with thalidomide-based regimens, ASA and WAR showed similarefficacy in reducing serious thromboembolic events, acute cardiovascular events, and sudden deathscompared with LMWH, except in elderly patients where WAR showed less efficacy than LMWH.

2011 - B-cell receptor, clinical course and prognosis in chronic lymphocytic leukaemia: the growing saga of the IGHV3 subgroup gene usage. [Articolo su rivista]
Dal Bo, M; Del Giudice, I; Bomben, R; Capello, D; Bertoni, F; Forconi, F; Laurenti, L; Rossi, D; Zucchetto, A; Pozzato, G; Marasca, Roberto; Efremov, Dg; Guarini, A; Del Poeta, G; Foà, R; Gaidano, G; Gattei, V.
abstract

The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement.

2011 - BCR-ABL-specific cytotoxic T cells in the bone marrow of patients with Ph(+) acute lymphoblastic leukemia during second-generation tyrosine-kinase inhibitor therapy. [Articolo su rivista]
Riva, Giovanni; Luppi, Mario; Quadrelli, Chiara; Barozzi, Patrizia; Basso, S; Vallerini, Daniela; Zanetti, E; Morselli, M; Forghieri, Fabio; Maccaferri, M; Paolini, Ambra; DEL GIOVANE, Cinzia; D'Amico, Roberto; Marasca, Roberto; Narni, Franco; Iacobucci, I; Martinelli, G; Baccarani, M; Comoli, P; Potenza, Leonardo
abstract

BCR-ABL-specific cytotoxic T cells in the bone marrow of patients with Ph+ acute lymphoblastic leukemia during second-generation tyrosine-kinase inhibitor therapy

2011 - Bendamustine with or without rituximab in the treatment of relapsed chronic lymphocytic leukaemia: an Italian retrospective study [Articolo su rivista]
Iannitto, E; Morabito, F; Mancuso, S; Gentile, M; Montanini, A; Augello, A; Bongarzoni, V; D'Arco, A; Di Renzo, N; Fazzi, R; Franco, G; Marasca, Roberto; Mulè, A; Musso, M; Musto, P; Pennese, E; Piccin, A; Rota Scalabrini, D; Visani, G; Rigacci, L.
abstract

To retrospectively assess the efficacy of bendamustine alone and with rituximab (R-B), 109 patients with relapsed chronic lymphocytic leukaemia (CLL) were enrolled in 24 Italian centres. The median age was 66 years (range 39-85). Forty-three percent of patients had relapsed and 57% were resistant (median previous therapies = 3; range 1-8). Twenty-two patients received bendamustine alone and 87 patients received R-B (median B dosage: 100 mg/m(2) per day, range 90-130 mg/m(2) per day). The overall response rate was 69·6% (complete response 28·6%; partial response 41%), and was significantly higher in patients treated with R-B (P = 0·014) and in those responsive to the previous treatment (P=0·04). After a median follow-up of 7·9 months (range 1-148), the median progression-free survival was 16 months and the median duration of response was 13 months. Median overall survival (OS) was 16·8 months for the whole cohort; patients not responding to the treatment had a significantly worse outcome than those who attained a response (P = 0·0001). In multivariate analysis, only resistant disease status at start of bendamustine treatment (HR 3·2, 95% CI 1·4-7·3, P = 0·006) had an independent prognostic value for OS. Toxicity was manageable and mostly haematological. In conclusion, in our experience R-B was an effective and well-tolerated treatment for relapsed/refractory CLL patients, producing a remarkable high CR rate and mild toxicity

2011 - Chronic eosinophilic leukaemia with ETV6-NTRK3 fusion transcript in an elderly patient affected with pancreatic carcinoma [Articolo su rivista]
Forghieri, Fabio; Morselli, M; Potenza, Leonardo; Maccaferri, Monica; Pedrazzi, Letizia; Paolini, Ambra; Bonacorsi, G; Artusi, Tullio; Giacobbi, F; Corradini, G; Barozzi, Patrizia; Zucchini, Patrizia; Marasca, Roberto; Narni, Franco; Crescenzi, B; Mecucci, C; Falini, B; Torelli, Giuseppe; Luppi, Mario
abstract

Chronic eosinophilic leukaemia with ETV6-NTRK3 fusion transcript in an elderly patient affected with pancreatic carcinoma.A case report

2011 - Genome-wide DNA profiling better defines the prognosis of chronic lymphocytic leukaemia [Articolo su rivista]
Rinaldi, A; Mian, M; Kwee, I; Rossi, D; Deambrogi, C; Mensah, Aa; Forconi, F; Spina, V; Cencini, E; Drandi, D; Ladetto, M; Santachiara, Rita; Marasca, Roberto; Gattei, V; Cavalli, F; Zucca, E; Gaidano, G; Bertoni, F.
abstract

The integration of molecular and clinical information to tailor treatments remains an important research challenge in chronic lymphocytic leukaemia (CLL). This study aimed to identify genomic lesions associated with a poor outcome and a higher risk of histological transformation. A mono-institutional cohort of 147 cases was used as the test series, and a multi-institutional cohort of 176 cases as a validation series. Genomic profiles were obtained using Affymetrix SNP 6.0. The impact of the recurrent minimal common regions (MCRs) on overall survival was evaluated by univariate analysis followed by multiple-test correction. The independent prognostic significance was assessed by multivariate analysis. Eight MCRs showed a prognostic impact: gains at 2p25.3-p22.3 (MYCN), 2p22.3, 2p16.2-p14 (REL), 8q23.3-q24.3 (MYC), losses at 8p23.1-p21.2, 8p21.2, and of the TP53 locus. Gains at 2p and 8q and TP53 inactivation maintained prognostic significance in multivariate analysis and a hierarchical model confirmed their relevance. Gains at 2p also determined a higher risk of Richter syndrome transformation. The prediction of outcome for CLL patients might be improved by evaluating the presence of gains at 2p and 8q as novel genomic regions besides those included in the 'standard' fluorescence in situ hybridization panel.

2011 - Genome-wide DNA profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome [Articolo su rivista]
Rinaldi, A; Mian, M; Chigrinova, E; Arcaini, L; Bhagat, G; Novak, U; Rancoita, Pm; De Campos, Cp; Forconi, F; Gascoyne, Rd; Facchetti, F; Ponzoni, M; Govi, S; Ferreri, Aj; Mollejo, M; Piris, Ma; Baldini, L; Soulier, J; Thieblemont, C; Canzonieri, V; Gattei, V; Marasca, Roberto; Franceschetti, S; Gaidano, G; Tucci, A; Uccella, S; Tibiletti, Mg; Dirnhofer, S; Tripodo, C; Doglioni, C; Dalla Favera, R; Cavalli, F; Zucca, E; Kwee, I; Bertoni, F.
abstract

Marginal zone B-cell lymphomas (MZLs) have been divided into 3 distinct subtypes (extranodal MZLs of mucosa-associated lymphoid tissue [MALT] type, nodal MZLs, and splenic MZLs). Nevertheless, the relationship between the subtypes is still unclear. We performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question. Samples from 218 MZL patients (25 nodal, 57 MALT, 134 splenic, and 2 not better specified MZLs) were analyzed with the Affymetrix Human Mapping 250K SNP arrays, and the data combined with matched gene expression in 33 of 218 cases. MALT lymphoma presented significantly more frequently gains at 3p, 6p, 18p, and del(6q23) (TNFAIP3/A20), whereas splenic MZLs was associated with del(7q31), del(8p). Nodal MZLs did not show statistically significant differences compared with MALT lymphoma while lacking the splenic MZLs-related 7q losses. Gains of 3q and 18q were common to all 3 subtypes. del(8p) was often present together with del(17p) (TP53). Although del(17p) did not determine a worse outcome and del(8p) was only of borderline significance, the presence of both deletions had a highly significant negative impact on the outcome of splenic MZLs

2011 - Identification and characterization of Aspergillus-specific immune responses to diagnose invasive aspergillosis in high risk patients: a multicenter study [Abstract in Rivista]
Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Maertens, J; Candoni, A; Beauvais, A; Zanetti, Eleonora; Quadrelli, C; Morselli, M; Forghieri, Fabio; Maccaferri, M; Paolini, Ambra; Marasca, Roberto; DEL GIOVANE, Cinzia; D'Amico, Roberto; Ciceri, F; Comoli, P; Cesaro, S; Caira, M; Pagano, L; Romani, L; Narni, Franco; Latgè, Jp; Luppi, Mario
abstract

Background. The mortality of Invasive Aspergillosis (IA) still affects from 27% to 55% of high risk hematologic patients. The reasons of such a poor outcome also rely on several drawbacks limiting the di- agnostic accuracy of non cultural based diagnostic methods (NCBDM) and hampering the opportunities for an early intervention. Studies in mice model of IA and in healthy subjects have shown that Aspergillus-specific T-cells producing interferon-gamma (IFN- gamma-T1) are protective, while Aspergillus-specific T-cells pro- ducing interleukin-10 (IL-10-T2) are non-protective to IA. Aims. We have investigated whether the identification of Aspergillus-specific IFN-gamma-T1 and/or IL-10-T2 through an ex-vivo enzyme linked immunospot (ELISPOT) assay may be effective in the diagnosis of IA in high risk patients. Furthermore, in the proven IA patients, we have functionally and phenotipically characterized such T cells through the cytokine secretion assay (CSA). Methods. 180 patients (168 hemato- logic and 12 solid organ transplant patients) have been enrolled. They were classified, according the revised EORTC/MSG criteria, as fol- lows: 18 proven, 35 probable, 17 possible IA cases and 110 controls. The control patients were divided in two groups: group 1 included 86 (78.2%) patients with hystological and/or cultural verified infec- tious/inflammatory/neoplastic diseases, but other than IA; group 2 in- cluded 24 (21.8%) patients without clinical and/or microbiological features of IA. ELISPOT has been performed, as described [Potenza et al. Leukemia 2007; 21: 578-81], by using as antigens Aspergillus either conidia or recombinant antigens, namely CRF1p, GEL1p, PEP1p, SOD1p, α1-3 glucan, β1-3 glucan and galactomannan (GM). Results. The patient and sample positivity rates were 94.4%/89.5% in proven, 45.7%/35.3% in probable, 35.3%/50% in possible IA cases and 1.8%/4.5% in the controls, respectively. The sensitivity and speci- ficity of ELISPOT for the diagnosis of IA resulted 94.4% (95% CI, 73%-99%) and 98.2% (95% CI, 93%-99%), respectively. The PPV of the test was 89.5% (95% CI, 67%-99%), the NPV was 99.1% (95% CI, 94%-100%) and the efficiency was 97.6% (95% CI, 92.3%- 99.4%). The positive likelihood ratio (LR) resulted 51.89, the negative LR was 0.06 (Table 1A,B). In proven IA patients, CSA demonstrated that Aspergillus-specific IL-10-T2 were predominantly central memory (CM) CD4+ T cells (median frequency 0.37%/0.22%), while Aspergillus-specific IFN-gamma-T1 were either CD4+ or CD8+ cells of either effector memory (EM) or CM phenotype (median frequen- cies 0.24%/0.20%). Also lower frequencies of Aspergillus-specific ei- ther CD4+ or CD8+ T cells producing IL-4 (0.11%/0.19%) of EM phenotype, and EM CD8+ cells producing IL-17 (0.18%), were de- tected. Moreover, although CRF1p, GEL1p, α1-3 glucan and SOD1p resulted the antigens eliciting the highest number of Aspergillus-spe- cific IFN-gamma-T1, only GEL1p and α1-3 glucan were those most constantly targeted by protective immune responses along the entire course of the IA. Conclusions. Our findings demonstrate the potential of ELISPOT in the diagnosis of IA, suggesting that it may comple- ment the other NCBDM, enabling a more consistent diagnosis of IA. Furthermore, this study describes for the first time the Aspergillus- specific immune responses in patients with proven IA, identifying also the antigens predominantly targeted by protective IFN-gamma- T1, with possible consequences in designing strategies of either adoptive cell infusion or vaccine therapies.

2011 - INTERACTION BETWEEN ENDOTHELIUM AND CHRONIC LYMPHOCYTIC LEUKEMIA B-CELLS RESCUES FROM APOPTOSIS AND MODULATES GENE EXPRESSION PROFILE OF LEUKEMIC CELLS [Abstract in Rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Bulgarelli, Jenny; Debbia, Giulia; Fontana, M; Faglioni, Laura; Bigliardi, Sara; Zucchini, Patrizia; Narni, Franco; Torelli, Giuseppe; Luppi, Mario; Marasca, Roberto
abstract

Background. Despite an apparent long life in vivo, CLL cells die rap- idly in vitro. This observation suggests that the apoptotic resistance is not intrinsic to leukemia B cells but extrinsic factors are necessary for CLL prolonged survival. Aims. we investigated the interactions be- tween endothelial cells and CLL cells, highlighting molecular net- works involved in this cellular crosstalk. Methods. we co-cultured CLL cells on HUVEC endothelial monolayer (HC) or in medium alone (CLL only). Then, we detected CLL viability by flow cytometry and we performed whole-genome high density microarrays. Results. we found that endothelial cells protected CLL from spontaneous apop- tosis. After 48h, increased number of alive CLL cells was present in HC condition (59.7 ± 4.2%) compared to CLL alone (22.9 ± 5.1%) (p<0.0001). Moreover, we found that spontaneous in vitro apoptosis was higher in unmutated IGHV CLL (UM-CLL) compared to mutated ones (M-CLL). In HC condition, similar survival was detected be- tween M-CLL and UM-CLL, implying a 2.2-fold increase in relative viability in M-CLL and a 6.1-fold increase in UM-CLL. Moreover, the endothelial cell layer decreased the in vitro sensitivity of CLL cells to Fludarabine-induced apoptotic cell death. The mean viability of CLL cells treated with 10 µM Fludarabine was 19.8% (±4.4%) after 48 hours and 3.8% (±1.3%) after 72 hours. In HC with Fludarabine ad- dition, the mean viability of CLL cells was 37.8% (±9.1%) after 48 hours and 14.3% (±3.2%) after 72 hours. Then, we compared gene expression profiles (GEP) between CLL cultured in contact with EC layer and CLL at baseline to unravel the transcriptional modifications induced by EC cells. Overall 1944 genes were found to be modulated (FC≥2, p<0.05). CLL cells in HC condition showed a 22.6-fold in- crease of CCL2, able to recruit tumor-activated monocytes (p=0.0032) and a 6.5-fold increase of PDGFC, chemoattractant for mesenchymal stromal cells (p=0.0051). Other soluble factors up-reg- ulated by EC/CLL contact were VEGFC (FC=9.4, p=0.0061), ANGTL4 (FC=8.6, p=0.015), EDN1 (FC=9.2, p=0.0061), AMOTL2 (FC=4.3, p=0.019) and THBS1 (FC=45.1, p=0.0004) as well as the metalloproteases MMP2 (FC=8.3, p=0.02) and MMP4 (FC=3.0, p=0.039). The GEP data were confirmed by evaluating the secreted levels of soluble factors in conditioned medium collected after 48h- HC culture. In addition, CLL cells on endothelial layer maintained or increased the expression levels of anti-apoptotic factors Bcl-2, Bcl2A1, BIRC3/c-IAP2 and BIRC5/Survivin compared to CLL cells at baseline. Of interest, the Ang2 tyrosine kinase receptor Tie2 mRNA was found to be increased in CLL cells in co-culture (FC=10.7, p=0.017). We confirmed GEP data by flow cytometry finding a 2-fold and a 4.3-fold increase of percentage of Tie2+CLL cells at 48h and 72h in HC. Conclusion. our results demonstrate a role of endothelial cells in CLL survival advantage and Fludarabine-resistance. The inti- mate contacts with EC seem to determine a microenvironmental- driven angiogenic switch of CLL phenotype, improve the secretion of cytokines involved in regulation of microenvironmental elements such as stromal cells and macrophages and increase the expression of anti-apoptotic molecules.

2011 - May the indirect effects of CIHHV-6 in transplant patients be exerted through the reactivation of the viral replicative machinery? [Articolo su rivista]
Potenza, Leonardo; Barozzi, Patrizia; Rossi, G; Riva, Giovanni; Vallerini, Daniela; Zanetti, Eleonora; Quadrelli, Chiara; Morselli, M; Forghieri, Fabio; Maccaferri, Monica; Paolini, Ambra; Marasca, Roberto; Narni, Franco; Luppi, Mario
abstract

Indirect effects of CIHHV-6 in transplant patients

2011 - Mucorles-specific T cells emerge in the course of invasive mucormucosis and may be used as a surrogate diagnostic marker in high-risk patients [Articolo su rivista]
Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Forghieri, Fabio; Zanetti, Eleonora; Quadrelli, Chiara; Candoni, A; Maertens, J; Rossi, Giulio; Morselli, M; Codeluppi, M; Paolini, Ambra; Maccaferri, Monica; DEL GIOVANE, Cinzia; D'Amico, Roberto; Rumpianesi, F; Pecorari, M; Cavalleri, F; Marasca, Roberto; Narni, Franco; Luppi, Mario
abstract

Mucorales-specific T cells have been investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 infections of known etiologies but other than IM, and 8 never showed fever upon the period of observation. The Mucorales-specific T cells may be detected only in patients with IM, at diagnosis and along the entire course of the IM, but neither before nor long time after the resolution of the infection. Such T cells produced predominantly interleukin-4, interferon-gamma (IFN-γ), interleukin-10, and to a lesser extent also interleukin-17, and belonged to either CD4+ or CD8+ subsets. The specific T cells producing IFN-γ were able to directly induce damage of Mucorales hyphae. None of the 25 patients without IM showed Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

2011 - Multicentre validation of a prognostic index for overall survival in chronic lymphocytic leukaemia. [Articolo su rivista]
Bulian, P; Tarnani, M; Rossi, D; Forconi, F; Del Poeta, G; Bertoni, F; Zucca, E; Montillo, M; Pozzato, G; Deaglio, S; D'Arena, G; Efremov, D; Marasca, Roberto; Lauria, F; Gattei, V; Gaidano, G; Laurenti, L.
abstract

We wish to validate in a multicentric CLL population a nomogram and a risk score recently developed to predict overall survival (OS). Complete records from 1037 CLL patients were retrospectively collected to estimate OS and time to treatment (TTT). Cox models were used to test the independence of age, β-2-microglobulin, absolute lymphocyte count (ALC), sex, Rai stage and number of involved lymph node regions (LNR). Accuracy of prognostic models was tested with the concordance index (c-index). Median follow-up was 5.5 years, with 151 deaths and 475 treated patients. Median OS was not reached (65% survival rate at 13.9 years), median TTT was 6 years. We confirmed the ability of the prognostic score to predict OS and TTT in three risk groups, with results comparable with those reported in the original report. However, ALC and Rai stage were not independent predictors, whereas the Binet staging system, which incorporates LNR variable, showed independent predictive power; furthermore, both 5- and 10-year OS estimates from nomogram were lower compared to real data. When separately analysed, the impact of therapy on OS was not selected as independent predictor of OS in our series. According to these results, we proposed a simpler four-variable model (age, sex, Binet staging, β-2-microglobulin) and a new nomogram. This model had a c-index of 0.78 versus 0.76 of the six-variable model (p = 0.043), showing better predictive accuracy. External validation and refinement are needed on independent data sets, possibly from cancer registry patients' series.

2011 - Safety and efficacy of bortezomib-melphalan-prednisone-thalidomide followed by bortezomib-thalidomide maintenance (VMPT-VT) versus bortezomib-melphalan-prednisone (VMP) in untreated multiple myeloma patients with renal impairment [Articolo su rivista]
Morabito, F; Gentile, M; Mazzone, C; Rossi, D; Di Raimondo, F; Bringhen, S; Ria, R; Offidani, M; Patriarca, F; Nozzoli, C; Petrucci, Mt; Benevolo, G; Vincelli, I; Guglielmelli, T; Grasso, M; Marasca, Roberto; Baldini, L; Montefusco, V; Musto, P; Cascavilla, N; Majolino, I; Musolino, C; Cavo, M; Boccadoro, M; Palumbo, A.
abstract

We assessed efficacy, safety, and reversal of renal impairment (RI) in untreated patients with multiple myeloma given bortezomib-melphalan-prednisone-thalidomide followed by bortezomib-thalidomide (VMPT-VT) maintenance or bortezomib-melphalan-prednisone (VMP). Exclusion criteria included serum creatinine ≥ 2.5 mg/dL. In the VMPT-VT/VMP arms, severe RI (estimated glomerular filtration rate [eGFR] ≤ 30 mL/min), moderate RI (eGFR 31-50 mL/min), and normal renal function (eGFR > 50 mL/min), were 6%/7.9%, 24.1%/24.9%, and 69.8%/67.2%, respectively. Statistically significant improvements in overall response rates and progression-free survival were observed in VMPT-VT versus VMP arms across renal cohorts, except in severe RI patients. In the VMPT group, severe RI reduced overall survival (OS). RI was reversed in 16/63 (25.4%) patients receiving VMPT-VT versus 31/77 (40.3%) receiving VMP. Multivariate analysis showed male sex (P = .022) and moderate RI (P = .003) significantly predicted RI recovery. VMP patients achieving renal response showed longer OS. In both arms, greater rates of severe hematologic adverse events were associated with RI (eGFR < 50 mL/min), however, therapy discontinuation rates were unaffected. VMPT-VT was superior to VMP for cases with normal renal function and moderate RI, whereas VMPT-VT failed to outperform VMP in patients with severe RI, although the relatively low number of cases analyzed preclude drawing definitive conclusions. VMPT-VT had no advantage in terms of RI reversal over VMP. This study is registered at http://www.clinicaltrials.gov as NCT01063179.

2011 - The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation. [Articolo su rivista]
Rossi, D; Spina, V; Deambrogi, C; Rasi, S; Laurenti, L; Stamatopoulos, K; Arcaini, L; Lucioni, M; Rocque, Gb; Xu Monette, Zy; Visco, C; Chang, J; Chigrinova, E; Forconi, F; Marasca, Roberto; Besson, C; Papadaki, T; Paulli, M; Larocca, Lm; Pileri, Sa; Gattei, V; Bertoni, F; Foà, R; Young, Kh; Gaidano, G.
abstract

Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma in the context of chronic lymphocytic leukemia. The scarcity of biologic information about RS has hampered the identification of molecular predictors of RS outcome. We addressed this issue by performing a comprehensive molecular characterization of 86 pathologically proven RS. TP53 disruption (47.1%) and c-MYC abnormalities (26.2%) were the most frequent alterations, whereas common genetic lesions of de novo diffuse large B-cell lymphoma were rare or absent. By multivariate analysis, lack of TP53 disruption (hazard ratio, 0.43; P = .003) translated into significant survival advantage with 57% reduction in risk of death. An algorithm based on TP53 disruption, response to RS treatment, and Eastern Cooperative Oncology Group performance status had 80.9% probability of correctly discriminating RS survival (c-index = .809). RS that were clonally unrelated to the paired chronic lymphocytic leukemia phase were clinically and biologically different from clonally related RS because of significantly longer survival (median, 62.5 months vs 14.2 months; P = .017) and lower prevalence of TP53 disruption (23.1% vs 60.0%; P = .018) and B-cell receptor stereotypy (7.6% vs 50.0%; P = .009). The molecular dissection of RS into biologically distinct categories highlights the genetic heterogeneity of this disorder and provides clinically relevant information for refining the prognostic stratification of patients.

2011 - Tumor-targeted immunoliposomal nanosystems to deliver either Cidofovir or Antineoplastic SiRNA against Primary Effusion Lymphoma (PEL) [Relazione in Atti di Convegno]
Riva, Giovanni; Belletti, Daniela; Ruozi, Barbara; Barozzi, Patrizia; Vallerini, Daniela; Quadrelli, Chiara; Zanetti, Eleonora; Morselli, M; Forghieri, Fabio; Marasca, Roberto; Narni, Franco; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela; Potenza, Leonardo; Luppi, Mario
abstract

Therapeutic applications of siRNA-mediated gene silencing appear to be highly dependent on the use of pharmaco-technologic carrier systems, able to protect siRNAs from rapid degradation upon administration, as well as to specifically deliver them to target cells. Actually, while siRNA-expressing viral vectors are burdened with safety concerns for their clinical use, the development of modified liposomal nanocarriers may represent a feasible option to harness the therapeutic potential of targetedantineoplastic siRNAs. Recently, we have successfully developed and characterized effective immunoliposomal nanosystems (ILNs) for targeted delivery of Cidofovir (an anti-herpesviral nucleotideanalogue, also showing antitumor activity) against PEL cell lines, demonstrating a significant improvement of the antineoplastic activity of the drug, especially at lower doses (less than 1nM). Thus, we tried to adapt such PEL-specific ILNs (PEGilated nanovescicles made of cationic/neutral lipids, engineered with anti-CD138 moAb on their surface) to efficiently encapsulate siRNAs and deliver them into PEL cell lines (highly expressing CD138 membrane protein). Our preliminary data showed thatsingle treatments with anti-PEL ILNs, delivering specific siRNAs against Blimp1 (Prdm1), which is a master transcription factor in PEL (a plasmablast/pre-plasmacell lymphoma, consistently Bcl-6 neg, Blimp1 pos), were able to induce a dose-dependent (50-200nM) inhibition of Blimp1 production (as assessed by Blimp1 mRNA and protein levels using RT-PCR and Western Blot, respectively), and this was strongly associated with enhanced cell death (more than 80%, using Annexin V/PI test). In particular, we observed a massive reduction of PEL viability (mean viable cells 8%, range 3-15%) as soon as 48-72 hours after treatment with 100nM anti-Blimp1 siRNAs. Interestingly, these data may resemble those described in multiple myeloma cell lines, after transduction with lentiviral vector constitutively expressing anti-Blimp1 shRNAs. Further studies on PEL murine models are now warranted to assess the efficacy and toxicity profile of in-vivo treatment with PEL-specific ILNs, loadedwith either Cidofovir or anti-Blimp1 siRNAs.

2011 - 13q14 deletion size and number of deleted cells both influence prognosis in chronic lymphocytic leukemia [Articolo su rivista]
Dal Bo, M; Rossi, Fm; Rossi, D; Deambrogi, C; Bertoni, F; Del Giudice, I; Palumbo, G; Nanni, M; Rinaldi, A; Kwee, I; Tissino, E; Corradini, G; Gozzetti, A; Cencini, E; Ladetto, M; Coletta, Am; Luciano, F; Bulian, P; Pozzato, G; Laurenti, L; Forconi, F; Di Raimondo, F; Marasca, Roberto; Del Poeta, G; Gaidano, G; Foà, R; Guarini, A; Gattei, V.
abstract

Deletion at 13q14 is detected by fluorescence in situ hybridization (FISH) in about 50% of chronic lymphocytic leukemia (CLL). Although CLL with 13q deletion as the sole cytogenetic abnormality (del13q-only) usually have good prognosis, more aggressive clinical courses are documented for del13q-only CLL carrying higher percentages of 13q deleted nuclei. Moreover, deletion at 13q of different sizes have been described, whose prognostic significance is still unknown. In a multi-institutional cohort of 342 del13q-only cases and in a consecutive unselected cohort of 265 CLL, we investigated the prognostic significance of 13q deletion, using the 13q FISH probes locus-specific identifier (LSI)-D13S319 and LSI-RB1 that detect the DLEU2/MIR15A/MIR16-1 and RB1 loci, respectively. Results indicated that both percentage of deleted nuclei and presence of larger deletions involving the RB1 locus cooperated to refine the prognosis of del13q-only cases. In particular, CLL carrying <70% of 13q deleted nuclei with deletions not comprising the RB1 locus were characterized by particularly long time-to-treatment. Conversely, CLL with 13q deletion in <70% of nuclei but involving the RB1 locus, or CLL carrying 13q deletion in ≥70% of nuclei, with or without RB1 deletions, collectively experienced shorter time-to-treatment. A revised flowchart for the prognostic FISH assessment of del13q-only CLL, implying the usage of both 13q probes, is proposed.

2010 - Angiopoietin-2 plasma dosage predicts time to first treatment and overall survival in chronic lymphocytic leukemia. [Articolo su rivista]
Maffei, Rossana; Martinelli, Silvia; Santachiara, R; Rossi, D; Guarnotta, C; Sozzi, E; Zucchetto, A; Rigolin, Gm; Fiorcari, Stefania; Castelli, Ilaria; Fontana, M; Coluccio, Valeria; Leonardi, G; Zucchini, P; Tripodo, C; Cuneo, A; Gattei, V; Del Poeta, G; Forconi, F; Gaidano, G; Torelli, Giuseppe; Marasca, Roberto
abstract

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P &lt; .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P &lt; .001), high beta(2)-microglobulin (P &lt; .001), unmutated variable region of immunoglobulin heavy chain gene status (P &lt; .001), high CD38 and zeta-chain-associated protein kinase 70 expression (P &lt; .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

2010 - Circulating endothelial cells in patients with chronic lymphocytic leukemia: clinical-prognostic and biologic significance. [Articolo su rivista]
Rigolin, Gm; Maffei, Rossana; Rizzotto, L; Ciccone, M; Sofritti, O; Daghia, G; Cibien, F; Cavazzini, F; Marasca, Roberto; Cuneo, A.
abstract

BACKGROUND: In patients with cancer, circulating endothelial cells (CECs) are increased and are correlated with an aggressive disease course. However, the clinical and biologic significance of CECs in chronic lymphocytic leukemia (CLL) remains uncertain.METHODS: In 170 patients with CLL, CEC levels were quantified by flow cytometry and were correlated with clinical and biologic data. In addition, CECs were characterized by immunophenotypic, fluorescence in situ hybridization (FISH), and gene expression profile analyses.RESULTS: In patients with CLL, CECs were increased compared with controls. A higher level of CECs (>20/microL) identified a subset of patients with a more aggressive disease course characterized by a shorter time to first treatment both in univariate and multivariate analyses. In FISH analysis, 7 patients had a significant proportion of CECs and presented with the same cytogenetic lesion of neoplastic lymphocytes and immunophenotypic features of endothelial progenitor cells. The gene expression profile of sorted CECs revealed a molecular pattern, suggesting a derivation from CLL leukemic cells with increased cell survival and proliferation, diminished cell adhesion to extracellular matrix, and enhanced proangiogenic function compared with their normal counterparts.CONCLUSIONS: The current data suggest that, in CLL, CECs may represent a biologic marker of aggressiveness and disease progression to be considered for new, targeted antiangiogenic treatments.

2010 - Circulating endothelial cells in patients with chronic lymphocytic leukemia: Clinical-prognostic and biologic significance (Cancer (2010) 116, (1926-37)) [Articolo su rivista]
Rigolin, G. M.; Maffei, R.; Rizzotto, L.; Ciccone, M.; Sofritti, O.; Daghia, G.; Cibien, F.; Cavazzini, F.; Marasca, R.; Cuneo, A.
abstract

2010 - Expression of mutated IGHV3-23 genes in chronic lymphocytic leukemia identifies a disease subset with peculiar clinical and biological features. [Articolo su rivista]
Bomben, R; Dal Bo, M; Benedetti, D; Capello, D; Forconi, F; Marconi, D; Bertoni, F; Maffei, Rossana; Laurenti, L; Rossi, D; Del Principe, Mi; Luciano, F; Sozzi, E; Cattarossi, I; Zucchetto, A; Rossi, Fm; Bulian, P; Zucca, E; Nicoloso, Ms; Degan, M; Marasca, Roberto; Efremov, Dg; Del Poeta, G; Gaidano, G; Gattei, V.
abstract

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL. Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23–expressing CLL. Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non–IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non–IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non–IGHV3-23 CLL. Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features. Clin Cancer Res; 16(2); 620–8

2010 - IGHD3-3 fails to behave as unfavourable prognostic marker in chronic lymphocytic leukaemia. [Articolo su rivista]
Bomben, R; Dal Bo, M; Capello, D; Forconi, F; Bertoni, F; Maffei, Rossana; Laurenti, L; Rossi, D; Zucca, E; Degan, M; Marasca, Roberto; Efremov, Dg; Del Poeta, G; Gaidano, G; Gattei, V.
abstract

IGHD3-3 fails to behave as unfavourable prognostic marker in chronic lymphocytic leukaemia.

2010 - Immunogenetics features and genomic lesions in splenic marginal zone lymphoma [Articolo su rivista]
Rinaldi, A; Forconi, F; Arcaini, L; Mian, M; Sozzi, E; Zibellini, S; Baldini, L; Franceschetti, S; Gaidano, G; Marasca, Roberto; Mollejo, M; Piris, Ma; Tucci, A; Facchetti, F; Bhagat, G; Favera, Rd; Rancoita, Pm; Zucca, E; Kwee, I; Bertoni, F.
abstract

Splenic marginal zone lymphomas (MZL) express mutated (M)) or unmutated (U)) immunoglobulin heavy chain (IGHV) genes. To investigate the IGHV mutational status impact on genetic lesions, this study combined single nucleotide polymorphism-arrays and IGHV sequencing in 83 cases. Clinical features and outcome were similar between U- and M-IGHV cases. Recurrent lesions frequency was higher in U-IGHV cases, including poor prognosticators. Frequencies differed among cases bearing individual IGHV genes or lambda light chains. In conclusion, SMZL comprises subgroups based on genetic abnormalities and immunogenetic status. Genomic lesion frequency differed and was higher in U-IGHV cases, possibly affecting the outcome.

2010 - Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF. [Articolo su rivista]
Maffei, Rossana; Martinelli, Silvia; Castelli, Ilaria; Santachiara, R; Zucchini, Patrizia; Fontana, M; Fiorcari, Stefania; Bonacorsi, G; Ilariucci, F; Torelli, Giuseppe; Marasca, Roberto
abstract

Emerging evidence suggests that angiogenic signalling pathways play important role in the patho-biology of chronic lymphocytic leukemia (CLL). Our goal was to investigate: (i) the spontaneous and hypoxia-induced production of pro-angiogenic factors, VEGF and Ang2, by Real-time PCR and ELISA, (ii) the degree of vascularization in CLL-infiltrated bone marrow (BM) compartment by CD34 immunohistochemical staining of microvessels and (iii) the direct angiogenic effect of CLL-derived VEGF and Ang2 by function-blocking experiments in Matrigel assays. The results demonstrated that CLL cells spontaneously express both VEGF and Ang2 and are able to secrete these factors in surrounding microenvironment. Full-length Ang2 mRNA and truncated form Ang2443 were detectable. Moreover, CLL cells were shown to enhance secretion of both VEGF and Ang2 proteins when subjected to hypoxic condition. Furthermore, increased in vivo and in vitro angiogenesis was induced by CLL cells. Enhanced BM vascularity correlated with Ig-unmutated CLL subset and increased expression of Ang2. Then, we demonstrated that supernatants obtained from CLL cells significantly increase the HUVEC tube formation in Matrigel assays and that this enhanced angiogenic capacity is mediated by both CLL-derived VEGF and Ang2. Taken together, these results suggest that several simultaneous mechanisms may be involved in the CLL capacity to induce the disruption of pre-existing vessel structures to give rise to tumor neoangiogenesis. The preliminary studies in solid tumors, showing that the disruption of Ang2 function can inhibit tumor vessel density and growth, are encouraging and suggest the possibility of new future therapeutic options targeting CLL microenvironment.

2010 - Molecular and clinical features of chronic lymphocytic leukemia with stereotyped B-cell receptors in a Ukrainian cohort [Articolo su rivista]
Bilous, N; Bomben, R; Dal Bo, M; Capello, D; Forconi, F; Laurenti, L; Bertoni, F; Efremov, Dg; Marasca, Roberto; Del Poeta, G; Martina, Z; Kryachouk, I; Dyagil, I; Gaidano, G; Chumak, A; Gattei, V; Abramenko, I.
abstract

A fraction of chronic lymphocytic leukemia (CLL) carries highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity-determining region-3 (HCDR3), often associated with a restricted selection of IG(K/L)V light chains. We analyzed the features of CLL expressing homologous HCDR3 in a cohort of 264 Ukrainian patients by merging them with a recently published reference series of 1426 cases. This approach allowed us to identify 96/264 (36%) cases as expressing homologous HCDR3, subdivided into 47 subsets. Among these, 27 apparently novel subsets were identified, although most of them were composed of two sequences per subset (‘potential subsets’). CLL cases belonging to several stereotyped subsets showed HCDR3 homologies with various autoreactive clones. Our analysis identified molecular and clinical features of a Ukrainian cohort of patients with CLL.

2010 - Pregnancy in PNH: another eculizumab baby. [Articolo su rivista]
Marasca, Roberto; Coluccio, Valeria; Santachiara, R; Leonardi, G; Torelli, Giuseppe; Notaro, R; Luzzatto, L.
abstract

Pregnancy in PNH: another eculizumab baby.

2010 - Stereotyped patterns of B-cell receptor in splenic marginal zone lymphoma. [Articolo su rivista]
Zibellini, S; Capello, D; Forconi, F; Marcatili, P; Rossi, D; Rattotti, S; Franceschetti, S; Sozzi, E; Cencini, E; Marasca, Roberto; Baldini, L; Tucci, A; Bertoni, F; Passamonti, F; Orlandi, E; Varettoni, M; Merli, M; Rizzi, S; Gattei, V; Tramontano, A; Paulli, M; Gaidano, G; Arcaini, L.
abstract

Antigen stimulation may be important for splenic marginal zone lymphoma (SMZL) pathogenesis. To address this hypothesis, the occurrence of stereotyped B-cell receptors (BCR) was investigated in 133 SMZL (26 HCV+) compared with 4,414 HCDR3 sequences from public databases (PDB). Sixteen SMZL (12%) showed stereotyped BCR; 7/86 (8%) SMZL sequences retrieved from PDB also belonged to stereotyped HCDR3 subsets. Three categories of subsets were identified : 1) "SMZL-specific subsets" (n=5), composed only of 12 SMZL (9 HCV- from our series); 2) "Non-Hodgkin lymphoma-like subsets" (n=5), comprising 5 SMZL (4 from our series) clustering with other indolent lymphomas; 3) "CLL-like subsets" (n=6), comprising 6 SMZL (3 from our series) that belonged to known CLL subsets (n=4) or clustered with public CLL sequences. Immunoglobulin 3D modeling of 3 subsets revealed similarities in antigen binding regions not limited to HCDR3. Overall, data suggest that the pathogenesis of SMZL may involve also HCV-unrelated epitopes or an antigenic trigger common to other indolent lymphomas.

2009 - Intrinsic and extrinsic factors influencing the clinical course of B-cell chronic lymphocytic leukemia: prognostic markers with pathogenetic relevance [Articolo su rivista]
Dal Bo, M; Bertoni, F; Forconi, F; Zucchetto, A; Bomben, R; Marasca, Roberto; Deaglio, S; Laurenti, L; Efremov, Dg; Gaidano, G; Del Poeta, G; Gattei, V.
abstract

B-cell chronic lymphocytic leukemia (CLL), the most frequent leukemia in the Western world, is characterized by extremely variable clinical courses with survivals ranging from 1 to more than 15 years. The pathogenetic factors playing a key role in defining the biological features of CLL cells, hence eventually influencing the clinical aggressiveness of the disease, are here divided into "intrinsic factors", mainly genomic alterations of CLL cells, and "extrinsic factors", responsible for direct microenvironmental interactions of CLL cells; the latter group includes interactions of CLL cells occurring via the surface B cell receptor (BCR) and dependent to specific molecular features of the BCR itself and/or to the presence of the BCR-associated molecule ZAP-70, or via other non-BCR-dependent interactions, e.g. specific receptor/ligand interactions, such as CD38/CD31 or CD49d/VCAM-1. A putative final model, discussing the pathogenesis and the clinicobiological features of CLL in relationship of these factors, is also provided.

2009 - Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: Results from an Italian multicentre study [Articolo su rivista]
Bomben, R; DAL BO, M; Capello, D; Forconi, F; Maffei, Rossana; Laurenti, L; Rossi, D; DEL PRINCIPE, Mi; Zucchetto, A; Bertoni, F; Rossi, Fm; Bulian, P; Cattarossi, I; Ilariucci, F; Sozzi, E; Spina, V; Zucca, E; Degan, M; Lauria, F; DEL POETA, G; Efremov, Dg; Marasca, Roberto; Gaidano, G; Gattei, V.
abstract

PURPOSE: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL.EXPERIMENTAL DESIGN: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23-expressing CLL.RESULTS: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non-IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non-IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non-IGHV3-23 CLL.CONCLUSIONS: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features.

2009 - Stereotyped B-cell receptor is an independent risk factor of chronic lymphocytic leukemia transformation to Richter syndrome. [Articolo su rivista]
Rossi, D; Spina, V; Cerri, M; Rasi, S; Deambrogi, C; De Paoli, L; Laurenti, L; Maffei, Rossana; Forconi, F; Bertoni, F; Zucca, E; Agostinelli, C; Cabras, A; Lucioni, M; Martini, M; Magni, M; Deaglio, S; Ladetto, M; Nomdedeu, Jf; Besson, C; Ramponi, A; Canzonieri, V; Paulli, M; Marasca, Roberto; Larocca, Lm; Carbone, A; Pileri, Sa; Gattei, V; Gaidano, G.
abstract

Purpose: Few biological prognosticators are useful for prediction of Richter syndrome (RS), representing the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. Stereotyped B-cell receptors (BCR) may have prognostic effect in CLL progression. We tested the prognostic effect of stereotyped BCR for predicting RS transformation. Experimental Design: The prevalence of stereotyped BCR was compared in RS (n = 69) versus nontransformed CLL (n = 714) by a case-control analysis. Subsequently, the effect of stereotyped BCR at CLL diagnosis on risk of RS transformation was actuarially assessed in a consecutive CLL series (n = 753). Results: RS (n = 69) displayed a higher prevalence of stereotyped BCR (P < 0.001) compared with nontransformed CLL. The actuarial risk of RS transformation was significantly higher in CLL carrying stereotyped BCR (P < 0.001). Among BCR subsets most represented in CLL, subset 8 using IGHV4-39/IGHD6-13/IGHJ5 carried the highest risk of RS transformation [hazard ratio (HR), 24.50; P < 0.001]. Multivariate analysis selected stereotyped BCR (HR, 3.33; P = 0.001) and IGHV4-39 usage (HR, 4.03; P = 0.004) as independent predictors of RS transformation. The combination of IGHV4-39 usage and stereotyped BCR in the same patient identified CLL with a very high risk of RS transformation (5-year risk, 68.7%). The risk carried by stereotyped BCR and IGHV4-39 usage was specific for RS transformation and had no effect on CLL progression without transformation. Conclusions: Analysis of BCR features may help identify CLL patients at risk of RS. A close monitoring and a careful biopsy policy may help early recognition of RS in CLL patients using stereotyped BCR, particularly if combined with IGHV4-39.

2009 - Vorinostat interferes with Wnt and NF-kappaB pathways in the M-07e cell line. [Articolo su rivista]
Galimberti, S; Canestraro, M; Maffei, Rossana; Marasca, Roberto; Guerrini, F; Piaggi, S; Ciabatti, E; Petrini, M.
abstract

Vorinostat interferes with Wnt and NF-κB pathways in the M-07e cell lineLetter to the Editor

2008 - BK virus infection and neurologic dysfunctions in a patient with lymphoma treated with chemotherapy and rituximab [Articolo su rivista]
Ferrari, A; Luppi, Mario; Marasca, Roberto; Potenza, Leonardo; Morselli, M; Volzone, F; Santachiara, R; Forghieri, Fabio; Barozzi, Patrizia; Torelli, Giuseppe
abstract

n.d.

2008 - Bortezomib is able to reduce angiogenesis in half of patients affected by idiopathic myelofibrosis: an ex vivo study [Articolo su rivista]
Galimberti, S; Canestraro, M; Ciancia, E; Fazzi, R; Marasca, Roberto; Petrini, M.
abstract

Bortezomib is able to reduce angiogenesis in half of patients affected by idiopathic myelofibrosis: An ex vivo study

2008 - Chromosome 14q32 translocations involving the immunoglobulin heavy chain locus in chronic lymphocytic leukaemia identify a disease subset with poor prognosis. [Articolo su rivista]
Cavazzini, F; Hernandez, Ja; Gozzetti, A; RUSSO ROSSI, A; DE ANGELI, C; Tiseo, R; Bardi, A; Tammiso, E; Crupi, R; Lenoci, Mp; Forconi, F; Lauria, F; Marasca, Roberto; Maffei, Rossana; Torelli, G; Gonzalez, M; MARTIN JIMENEZ, P; MARIA HERNANDEZ, J; Rigolin, Gm; Cuneo, A.
abstract

Immunophenotypic studies, fluorescence in situ hybridization (FISH) and conventional karyotyping were used to define the clinicobiological significance of 14q32 translocations involving the immunoglobulin gene locus (14q32/IGH) in 252 chronic lymphocytic leukaemia (CLL) patients. The following regions were studied: 13q14, centromere 12, 6q21; 11q22/ATM; 17p13/TP53, 14q32/IGH. Patients were classified as group 1 (favourable, i.e. 13q-single or normal), group 2 (intermediate risk, i.e. +12, 6q-, 1–2 anomalies), group 3 (unfavourable, i.e. 17p-, 11q-, complex karyotype), or group 4 (14q32/IGH translocation). Endpoints were treatment-free survival (TFS) and overall survival (OS). One hundred and ten patients were included in group 1, 99 in group 2, 25 in group 3 and 18 in group 4. 14q32/IGH translocation partners were identified in eight cases (BCL2 in five cases, BCL11A, CCND3 and CDK6 in one case each). group 4 showed shorter TFS versus groups 2 and 1 (25% patients treated at 2 months vs. 12 (P = 0·02) and 20 months (P = 0·002), respectively) and shorter OS (25% patients dead at 18 months versus 50 (P = 0·0003) and >60 months (P < 0·0001) respectively. The 14q32/IGH translocation maintained prognostic significance at multivariate analysis on TFS (P = 0·025) and OS (P < 0·001), along with advanced stage and CD38+. These findings show that the 14q32/IGH translocation predicts for an unfavourable outcome in CLL and that this cytogenetic subset might be included as a separate entity in a hierarchical cytogenetic classification of CLL.

2008 - Development of hypogammaglobulinemia in patients treated with imatinib for chronic myeloid leukemia or gastrointestinal stromal tumor [Articolo su rivista]
Santachiara, Rita; Maffei, Rossana; Martinelli, Silvia; Arcari, Annalisa; Piacentini, Federico; Trabacchi, Elena; Alfieri, Pierluigi; Ferrari, Angela; Leonardi, Giovanna; Luppi, Gabriele; Longo, Giuseppe; Vallisa, Daniele; Marasca, Roberto; Torelli, Giuseppe
abstract

Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukemia and gastrointestinal stromal tumor patients. Although several in vitro and animal studies demonstrated that imatinib affects immune response, few immune alterations are described in humans. We retrospectively studied hematologic and immunological parameters in 72 chronic myeloid leukemia and 15 gastrointestinal stromal tumor patients treated with imatinib at standard dosage and in 20 chronic myeloid leukemia patients treated before the introduction of imatinib in clinical practice. Both chronic myeloid leukemia and gastrointestinal stromal tumor patients developed a significant reduction of gammaglobulin and immunoglobulin serum levels. No significant hypogammaglobulinemia was observed in chronic myeloid leukemia patients in the pre-imatinib era. These data demonstrate that imatinib treatment induces hypogammaglobulinemia that can reach a severe entity in 10% of cases, both in chronic myeloid leukemia and in gastrointestinal stromal tumor patients. Prospective studies are needed to evaluate immune humoral alterations and to define the real incidence of infectious events, including viral reactivations.

2008 - INCIDENCE AND SURVIVAL OF MYELOID MALIGNANCIES IN 1102 PATIENTS IN PROVINCE OF MODENA, NORTHERN ITALY [Abstract in Rivista]
Bari, Alessia; I., Rashid; Marcheselli, Raffaella; G., Bonacorsi; G., Leonardi; Marasca, Roberto; P., Zucchini; F., Giacobbi; Federico, Massimo; Sacchi, Stefano
abstract

Background. Making a research on incidence and survival of myeloid malignancies we realised that few well documented population-based studies exist on epidemiology of myelodisplastic syndromes (MDS) and chronic myeloproliferative disorders (CMPD). Aims. The goal of our population-based study was to add and improve information about epidemiology of myeloid malignancies. In collaboration with Modena Cancer Registry (MCR) we focalized our attention above all on those haematological malignancies which until few years ago were not recorded in cancer registry because not considered neoplastic. Methods. We examined all new cases of AML, MDS, chronic myeloid leukemia (CML) and CMPD diagnosed in the Province of Modena (population 633.993 at 2001 Census) between 1997 and 2006. Death certificate, cytology and histology report, both local and national reports of hospital admission, ICD-9 code reported in medical records were used as sources for identifying new cases and their outcome. All cases were checked and validated by a haematologist (AB) and a pathologist (GB) by a review of the original pathology report. Clinical and follow-up data were retrieved by active search of discharge letters, review of hospital records and interview of general practitioners. Information on vital status was achieved from official population registries. Age-Standardized Rates (ASR) were calculated according to the World Standard population (Doll et al., 1966). The dates of diagnosis and death or the closing date of study (January 2008) were used to estimate survival. Relative survival was calculated according to Hakulinen approach. Results. A total of 1102 myeloid malignancies were identified of which 304 AML, 238 MDS, 29 CMML, 417 CMPD and 114 CML. The ASR (per 100,000 people) was calculated as 2.4 for AML, 1.3 for MDS, 0.1 for CMML, 3.2 for CMPD and 1 for CML. When reported to European Standard Population the incidence was 3.2 for AML, 2 for MDS, 0.2 for CMML, 4.4 for CMPD and 1.3 for CML. Compared with reports from other European countries our series seems to be characterized by a higher incidence of CMPD, by a lower incidence of MDS and similar incidence of AML. After a median follow-up of 55 months (range 7-128) the median survival for AML was 5 months, for MDS 23 months, for CMML 26 months; median survival for CMPD and CML was not reached. Relevant differences were observed among median survival of different subtypes of AML, MDS and CMPD. The 5-year relative survival for AML was 20% (for AML M3 74%), for MDS 27%, for CMML 23%, for CMPD 87% and for CML 53% (for CML Ph1+ 80%). Conclusions. Our population-based study provides the first analysis of incidence, survival and subtypes distribution of myeloid malignancies performed in Northern Italy. Our results may contribute to better understand the true epidemiology of these diseases, avoiding bias related to referral pattern to myeloid malignancy registries and due to recruiting patients into clinical trials. In the time of emerging innovative treatments the availability of precise epidemiological data could help clinicians in choosing the most appropriate and cost-effectiveness treatment.

2008 - Increased expression of angiopoietin-2 characterizes early B-cell chronic lymphocytic leukemia with poor prognosis [Articolo su rivista]
Martinelli, Silvia; Maffei, Rossana; Castelli, Ilaria; Santachiara, R; Zucchini, Patrizia; Fontana, M; Bonacorsi, G; Leonardi, G; Marasca, Roberto; Torelli, Giuseppe
abstract

We measured the angiopoietin-2 (Ang-2) expression in early chronic lymphocytic leukemia (CLL) patients, pointing our attention on the association with immunoglobulin (IgVH) mutational status, CD38 expression and clinical outcome. Our results indicate that Ang-2 expression is heterogeneous among Binet stage A CLL patients. CLL patients can be divided into two subgroups (Ang-2 positive and Ang-2 negative CLL) with 30% of them displaying Ang-2 RNA levels above the cut off. A shorter progression-free survival was observed in Ang-2 positive CLL subset (p = 0.032). Abnormal Ang-2 expression was also associated with unmutated IgVH genes (p < 0.0001) and increased bone marrow angiogenesis (p = 0.028), suggesting a role of Ang-2 in disease-progression of early CLL patients.

2008 - Vorinostat and bortezomib significantly inhibit WT1 gene expression in MO7-e and P39 cell lines [Articolo su rivista]
Galimberti, S; Canestraro, M; Khan, R; Buda, G; Orciuolo, E; Guerrini, F; Fazzi, R; Maffei, Rossana; Marasca, Roberto; Petrini, M.
abstract

Wilms' tumor gene (WT1) can play significant roles both as a tumor suppressor gene and oncogene. The role of tumor suppressor gene can be supported by the finding of leukemic cells showing WT1 mutation, as well as high levels of WT1 RNA were expressed by the 48% of patients, with a negative impact on the clinical outcome.

2007 - Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status [Articolo su rivista]
Maffei, Rossana; Marasca, Roberto; Martinelli, Silvia; Castelli, Ilaria; Santachiara, R; Morandi, E; Zucchini, Patrizia; Fontana, M; Giacobbi, F; Silingardi, P; Bonacorsi, G; Temperani, Paola; Masini, L; Colacci, Am; Serra, Roberto; Torelli, Giuseppe
abstract

Chronic lymphocytic leukemia (CLL) has been considered for several years a disease of accumulation of relentless mature-looking malignant monoclonal B cells due to a presumeddefect in programmed cell death. However, several observations suggest that CLL cells are not immortal and that they require signals from microenvironment to maintain viability. Increased microvessel density in marrow and lymph node and increased levels of certain proangiogenetic factors in blood and urine of CLL patients were observed.2 Angiopoietin-2 (Ang-2) has a pivotal role in the disruption of stability and quiescence of mature vasculature that coincides with the reinitiation of vascular remodelling and angiogenic sprouting in adult, as occurs in tumors.

2007 - Comprehensive characterization of IGHV3-21-expressing B-cell chronic lymphocytic leukemia: an Italian multicenter study [Articolo su rivista]
Bomben, R; DAL BO, M; Capello, D; Benedetti, D; Marconi, D; Zucchetto, A; Forconi, F; Maffei, Rossana; Ghia, Em; Laurenti, L; Bulian, P; DEL PRINCIPE, Mi; Palermo, G; Thorselius, M; Degan, M; Campanini, R; Guarini, A; DEL POETA, G; Rosenquist, R; Efremov, Dg; Marasca, Roberto; Foa, R; Gaidano, G; Gattei, V.
abstract

IGHV3-21–using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3–IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3–IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3–IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3–IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non–IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3–IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3–IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

2007 - SVILUPPO DI IPOGAMMAGLOBULINEMIA IN PAZIENTI TRATTATI CON IMATINIB PER LEUCEMIA MIELOIDE CRONICA O TUMORI STROMALI GASTROINTESTINALI [Abstract in Atti di Convegno]
Santachiara, R.; Leonardi, G.; Vallisa, D.; Maffei, R.; Martinelli, S.; Ferrari, A.; Alfieri, P.; Luppi, G.; Bertolini, F.; Piacentini, F.; Marasca, R.; Torelli, G.
abstract

NA

2006 - Gene expression profiling of acute promyelocytic leukaemia identifies two subtypes mainly associated with Flt3 mutational status [Articolo su rivista]
Marasca, Roberto; Maffei, Rossana; Zucchini, Patrizia; Castelli, Ilaria; Saviola, A; Martinelli, Silvia; Ferrari, Alberto; Fontana, M; Ravanetti, S; Torelli, Giuseppe
abstract

Acute promyelocytic leukaemia (APL) is a well-defined disease characterized by a typical morphology of leukaemic cells, the presence of t(15;17) translocation and the unique sensitivity to the differentiating effect of all-trans retinoic acid. Nevertheless, some aspects are variable among APL patients, with differences substantially related to morphological variants, peripheral leukocytes count, the presence of a disseminated intravascular coagulopathy, different PML/RAR alpha isoforms ( long, variable or short) and Fms-like tyrosine kinase 3 (Flt3) mutations. In order to better define this variability, we investigated the gene expression profiles of 18 APL cases revealing, besides a high uniformity in gene expression pattern, the presence of few robust differences among patients able to identify, by an unsupervised analysis, two major clusters of patients characterized by different phenotypes (hypogranular M3v vs classical M3) and by the presence or absence of Flt3 internal tandem duplications (ITDs). Further supervised analysis confirmed that Flt3 status was the APL parameter best associated with these two subgroups. We identified, between Flt3 wild-type and Flt3-ITDs subsets, 147 differentially expressed genes that were involved in the cytoskeleton organization, in the cell adhesion and migration, in the proliferation and the coagulation/inflammation pathways as well as in differentiation and myeloid granules constitution suggesting a role of Flt3 mutations in the pathogenesis and clinical manifestations of APL.

2006 - Strikingly homologous immunoglobulin gene rearrangements and poor outcome inV(H)3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status [Articolo su rivista]
Thorselius, M; Krober, A; Murray, F; Thunberg, U; Tobin, G; Buhler, A; Kienle, D; Albesiano, E; Maffei, Rossana; Dao Ung, Lp; Wiley, J; Vilpo, J; Laurell, A; Merup, M; Roos, G; Karlsson, K; Chiorazzi, N; Marasca, Roberto; Dohner, H; Stilgenbauer, S; Rosenquist, R.
abstract

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate this further, we analyzed the VH and VL gene rearrangements in 90 VH3-21+ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VH genes and 37% unmutated VH genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased V2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographic origin and mutation status.

2005 - Efficacy of imatinib mesylate as maintenance therapy in adults with acute lymphoblastic leukemia in first complete remission [Articolo su rivista]
Potenza, Leonardo; Luppi, Mario; Riva, Giovanni; Marasca, Roberto; Martinelli, Silvia; Torelli, Giuseppe
abstract

Seven Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) patients in first complete remission received maintenance therapy with imatinib alone. Two-year progression-free survival was 75%. Quantitative polymerase-chain-reaction (qPCR) monitoring of BCR-ABL showed that: (i) persisting molecular complete response (CR) was associated with long-lasting CR; (ii) molecular relapse did not invariably mean hematologic relapse; (iii) only the wide and rapid increment of BCR-ABL values was predictive of leukemia relapse.

2005 - Immunoglobulin mutational status detected through single-round amplification of partial VH region, represents a good prognostic marker for the clinical outcome in chronic lymphocytic leukemia [Articolo su rivista]
Marasca, Roberto; Maffei, Rossana; M., Morselli; Zucchini, Patrizia; Castelli, Ilaria; Martinelli, Silvia; M., Fontana; S., Ravanetti; M., Curotti; G., Leonardi; K., Cagossi; G., Partesotti; Torelli, Giuseppe
abstract

The immunoglobulin (Ig) mutational status in B-cell chronic lymphocytic leukemia (CLL) distinguishes two subsets of patients with different prognosis. Ig status detection is commonly performed with a panel of VH family-specific primers. Although this method detects clonal VDJ rearrangement in virtually all cases, it is technically cumbersome and therefore not widely used clinically. Here, we describe a simple and rapid method to establish the mutational status of IgVH in CLL. The method is based on a consensus VH FR2 primer, used in both polymerase chain reaction (PCR) and sequencing reactions. Overall, monoclonal B-cell populations were detected in 163 of 189 CLL patients (86%). The prognostic value of IgVH mutational status was then evaluated by analyzing survival in 146 CLL cases using different VH homology cutoffs. CLL prognostic groups were best separated by the classical 98% cutoff: median survival was 127 and 206 months in unmutated and mutated CLL cases, respectively (P = 0.0023). VH FR2 consensus and VH family PCR were compared in 41 cases, correctly assigning all cases by both methods. Therefore, we suggest a sequential strategy to detect immunoglobulin mutational status in CLL patients by first using the approach described in this study followed by alternative VH family-specific PCRs for negative cases.

2005 - Role of anti-hepatitis C virus (HCV) treatment in HCV-related, low-grade, B-cell, non-Hodgkin's lymphoma: a multicenter Italian experience [Articolo su rivista]
D., Vallisa; P., Bernuzzi; L., Arcaini; Sacchi, Stefano; V., Callea; Marasca, Roberto; A., Lazzaro; E., Trabacchi; E., Anselmi; A. L., Arcari; C., Moroni; R., Berte; M., Lazzarino; L., Cavanna
abstract

Purpose Hepatitis C virus (HCV) is endemic in some areas of Northwestern Europe and the United States. HCV has been shown to play a role in the development of both hepatocellular carcinoma and B-cell non-Hodgkin´s lymphoma (B-NHL). The biologic mechanisms underlying the lymphomagenic activity of the virus so far are under investigation. In this study, the role of antiviral (anti-HCV) treatment in B-NHL associated with HCV infection is evaluated. Patients and Methods Thirteen patients with histologically proven low-grade B-NHL characterized by an indolent course (ie, doubling time no less than 1 year, no bulky disease) and carrying HCV infection were enrolled on the study. All patients underwent antiviral treatment alone with pegilated interferon and ribavirin. Response assessment took place at 6 and 12 months. Results Of the twelve assessable patients, seven (58%) achieved complete response and two (16%) partial hematologic response at 14.1 +/- 9.7 months (range, 2 to 24 months, median follow-up, 14 months), while two had stable disease with only one patient experiencing progression of disease. Hematologic responses (complete and partial, 75%) were highly significantly associated to clearance or decrease in serum HCV viral load following treatment (P = .005). Virologic response was more likely to be seen in HCV genotype 2 (P = 035), while hematologic response did not correlate with the viral genotype. Treatment-related toxicity did not cause discontinuation of therapy in all but two patients, one of whom, however, achieved complete response. Conclusion This experience strongly provides a role for antiviral treatment in patients affected by HCV-related, low-grade, B-cell NHL.

2004 - Autologous stem cell transplantation for newly diagnosed follicular lymphomas: low toxicity and extended progression free survival [Articolo su rivista]
Narni, Franco; Donelli, A; Cuoghi, A; Bresciani, P; Pozzi, S; Marasca, Roberto; Leonardi, G; Morselli, M; Luppi, Mario; Longo, G; Torelli, Giuseppe
abstract

2004 - AUTOLOGOUS STEM CELL TRANSPLANTATION FOR NEWLY DIAGNOSED FOLLICULAR LYMPHOMAS: LOW TOXICITY AND EXTENDED PROGRESSION FREE SURVIVAL. R1235 Barcelona, Spain. Bone Marrow Transplantation March 2004, Volume 33, Supplement 1 [Abstract in Rivista]
Narni, Franco; A., Donelli; A., Cuoghi; P., Bresciani; Pozzi, Samantha; Marasca, Roberto; G., Leonardi; M., Morselli; Luppi, Mario; G., Longo; Torelli, Giuseppe
abstract

In spite of an indolent course, advanced stage follicularlymphomas (FL) are incurable with conventional chemotherapy.High dose therapy could be investigated in selected patients,provided toxicity were acceptable. Improvements in supporttherapy and the widespread use of peripheral blood stem cells(SC) have made autologous transplantation a relatively safeprocedure. This prompted us to start a pilot study and, since1995, 23 patients have been enrolled at our center. Median agewas 49 (26 - 65). Most patients (80%) had stage IV disease andbone marrow involvement. For patients who achieved a completeresponse or a good partial response after CHOP, SC weremobilized with cyclophosphamide or DHAP plus G-CSF, andhigh dose therapy consisted of BEAM. High risk patients or poorresponders (residual high tumor burden or bone marrowinfiltration >50%) switched to a program of high dose sequentialtherapy. Nine patients received not-manipulated hematopoieticSC, 7 received ''ex vivo'' purged SC (6 by CD34+ positiveselection; 1 by positive+negative selection), and 8 patientsunderwent ''in vivo'' purging with anti-CD20 monoclonal antibody(rituximab). Residual disease was monitored at different steps bynested PCR. Treatment was well tolerated, with a median time toengrafment of 9 days (7-12), and a median stay in hospital of 22days (15-35). RBC and platelet transfusions were necessary,respectively, in 52% and 100% of the patients. The meannumber of RBC and platelet units given to transfused patientswas, respectively, 2.2 (1-5) and 3.7 (1-7). Fever occurred in 52%of the patients (mean 2,8 days in febrile patients) and mucositisin 85%. We did not observe, so far, any case of myelodysplasiaor secondary cancer. With a median FU of 58 (8-95) monthsfrom transplant, all patients are alive, 5 have relapsed, and 5-years estimated PFS is 70%. CD34+ selection had no impact onrelapse. In one patient, molecular response could be restoredand maintained by rituximab alone. Only a few data have beenpublished regarding autologous transplantation in FL patients atdiagnosis. Although the FU is relatively short and the number ofpatients still small, the present data suggest that autologoustransplant is a safe and effective therapy for selected, not heavilypretreated young patients with advanced stage disease.Especially since ''in vivo'' purging strategies can easily be carriedout, autologous SC transplantation should be investigated inrandomized trials.

2004 - Metaboliti mielotossici del benzene nella leucemia umana [Abstract in Rivista]
Vivoli, R; Bergomi, Margherita; Rovesti, Sergio; Marasca, Roberto
abstract

E' stato osservato che alcuni soggetti presentano una più spiccata attitudine metabolica alla biotrasformazione del benzene in un metabolita mielotossico. Si intende verificare se la proporzione di efficienti metabolizzatori sia più elevata nei soggetti affetti da leucemia rispetto ad un gruppo di controllo appaiato per le principali variabili confondenti.

2002 - Distinct genomic events in the myeloid and lymphoid lineages in simultaneous presentation of chronic myeloid leukemia and B-chronic lymphocytic leukemia [Articolo su rivista]
Crescenzi, B.; Sacchi, Stefano; Marasca, Roberto; Temperani, P.; La Starza, R.; Matteucci, C.; Bonacorsi, G.; Romoli, S.; Martelli, M. F.; Mecucci, C.; Emilia, G.
abstract

no abstract

2002 - HOXA homeogene cluster interruption and dysregulation of HOAX9 in the blast crisis of Philadelphia chromosome-positive chronic myeloid leukemia. [Articolo su rivista]
Sinigaglia, B.; Giacobbi, F.; Zucchini, Patrizia; Maffei, Rossana; Marasca, Roberto; Torelli, Giuseppe; Temperani, Paola
abstract

HOXA homeogene cluster interruption and dysregulation of HOAX9 in the blast crisis of Philadelphia chromosome-positive chronic myeloid leukemia

2002 - Long-term salvage therapy with cyclosporin A in refractory idiopathic thrombocytopenic purpura. [Articolo su rivista]
Emilia, G; Morselli, M; Luppi, Mario; Longo, G; Marasca, Roberto; Gandini, G; Ferrara, L; D'Apollo, N; Potenza, Leonardo; Bertesi, M; Torelli, Giuseppe
abstract

Treatment of severe, chronic idiopathic thrombocytopenic purpura (ITP) refractory to most usual therapies Is a difficult challenge. Little information exists on the clinical use of cyclosporin A (CyA) in the treatment of ITP. This report describes long-term treatment with CyA (median, 40 months) and follow-up (median, 36.8 months) in 12 adult patients with resistant ITR CyA used in relatively low doses (2.5-3 mg/kg of body weight per day) led to a clinical Improvement In 10 patients (83.3%). Five had a complete response (41.1%),4 a complete response to maintenance therapy (33.3%), and one a partial response (8.3%). Two patients had no response. Most patients with a response (60%) had a long-term remission (mean, 28.6 months) after discontinuation of CyA. One patient had a relapse of ITP 4 years after CyA therapy was stopped. Side effects were moderate and transient, even In patients dependent on continued CyA treatment. CyA seems to represent reasonable salvage treatment in severe, potentially life-threatening, refractory ITR

2002 - The role of molecular monitoring in autotransplantation for non-Hodgkin's lymphoma [Articolo su rivista]
S., Galimberti; Marasca, Roberto; F., Caracciolo; R., Fazzi; F., Papineschi; E., Benedetti; F., Guerrini; F., Morabito; E., Oliva; N., Di Renzo; Federico, Massimo; M., Petrini; Torelli, Giuseppe
abstract

Seventy-two patients with non-Hodgkin's lymphoma were evaluated for the presence of molecular markers (IgH, bcl-1, bcl-2 rearrangement) on bone marrow, at diagnosis and after PBSCT, and on harvests in order to find a possible predictive role of minimal residual disease on treatment outcome. At diagnosis, 41 (59%) out of 69 available bone marrows showed molecular involvement. Fifty-six percent of leukaphereses were involved, mainly indolent lymphoma (P = 0.001) or advanced disease (P = 0.01). Ex vivo purging cleared only one stem collection out of 31 PCR-positive leukaphereses. Aggressive lymphomas showed both a longer overall survival (OS) (P = 0.03) and relapse-free survival RFS (P = 0.02) when transplanted with unpurged stem cells, whereas indolent NHL survival was not influenced by ex vivo purging. Twenty out of 26 samples taken during follow-up had bone marrow involvement at diagnosis. Of these, 15 cleared their bone marrow; both OS and RFS were significantly longer in the PCR-negative cases (P = 0.05 and P = 0.005). At 1 year after PBSCT, 75% of patients were PCR negative, with 50% molecular remissions; the relapse rate was 55% for patients still PCR positive vs 29% for those who were PCR negative. Thus, after high-dose chemotherapy, close molecular monitoring of MRD using qualitative PCR techniques seems to represent a reliable prognostic indicator.

2001 - BCR-ABL rearrangement is not detectable in essential thrombocythemia. [Articolo su rivista]
Emilia, G.; Marasca, Roberto; Zucchini, Patrizia; Temperani, Paola; Luppi, Mario; Torelli, Giuseppe; Lanza, F.; DE ANGELIS, C.; Gandini, D.; Castoldi, G.; Vallisa, D.; Cavanna, L.; del Senno, L.
abstract

BCR-ABL rearrangement is not detectable in essential thrombocythemia

2001 - Immunoglobulin gene mutations and frequent use of VH1-69 and VH4-34 segments in hepatitis C virus-positive and hepatitis C virus-negative nodal marginal zone B-cell lymphoma [Articolo su rivista]
Marasca, Roberto; Vaccari, Paola; Luppi, Mario; Zucchini, Patrizia; Castelli, Ilaria; Barozzi, Patrizia; A., Cuoghi; Torelli, Giuseppe
abstract

Nodal marginal zone B-cell lymphoma (NMZL) is actually considered as a distinct entity that must be distinguished from extra-nodal and splenic marginal zone Lymphomas. To define the cell origin and the role of antigen stimulation we determined the nucleotide sequence of the tumor-related immunoglobulin heavy chain variable genes in 10 cases of NMZL. The results were also evaluated on the basis of the presence of chronic hepatitis C virus (HCV) infection. All 10 cases harbored VH somatic mutations with a sequence homology compared to the closest germline gene, ranging from 83.33 to 98.28%. Interestingly, different VH segments were preferentially used in HCV-positive and HCV-negative patients: three of five HCV-negative NMZLs used a VH4-34 segment joined with different D and JH segments whereas three of five HCV-positive NMZLs used a VH1-69 gene joined with a D3-22 and a JH4 segment, with very strong similarities in the CDR3s among the three different cases. These data indicate: 1) NMZL is derived from B cells that have experienced the germinal center reaction; 2) the preferential usage of a VH1-69 segment in the majority of the HCV-positive NMZL cases with similar CDR3s suggests the presence of a common antigen, probably a HCV antigen epitope, involved in the B-cell selection; and 3) the use of a VH4-34 segment suggests a role of yet unknown B-cell superantigen(s) in the selection of tumor B-cell precursors in HCV-negative NMZL.

2000 - Bone marrow failure associated with human herpesvirus 8 infection after transplantation [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Schulz, Tf; Setti, G; Staskus, K; Trovato, R; Narni, Franco; Donelli, A; Maiorana, Antonino; Marasca, Roberto; Sandrini, S; Torelli, G.
abstract

BACKGROUND: Human herpesvirus 8 (HHV-8) infection has been linked to the development of Kaposi's sarcoma and to rare lymphoproliferative disorders.METHODS: We used molecular methods, serologic methods, in situ hybridization, and immunohistochemical analyses to study HHV-8 infection in association with nonmalignant illnesses in three patients after transplantation.RESULTS: Primary HHV-8 infections developed in two patients four months after each received a kidney from the same HHV-8-seropositive cadaveric donor. Seroconversion and viremia occurred coincidentally with disseminated Kaposi's sarcoma in one patient and with an acute syndrome of fever, splenomegaly, cytopenia, and marrow failure with plasmacytosis in the other patient. HHV-8 latent nuclear antigen was present in immature progenitor cells from the aplastic marrow of the latter patient. Identification of the highly variable K1 gene sequence of the HHV-8 genome in both the donor's peripheral-blood cells and the recipients' serum confirmed that transmission had occurred. HHV-8 viremia also occurred after autologous peripheral-blood stem-cell transplantation in an HHV-8-seropositive patient with non-Hodgkin's lymphoma. Reactivation of the infection was associated with the development of fever and marrow aplasia with plasmacytosis; there was no evidence of other infections. HHV-8 transcripts and latent nuclear antigen were expressed in the aplastic marrow but not in two normal marrow samples obtained before transplantation.CONCLUSIONS: Primary HHV-8 infection and reactivation of infection may be associated with nonneoplastic complications in immunosuppressed patients.

2000 - Gestational thrombocytopenia - Reply [Articolo su rivista]
Emilia, Giovanni; Luppi, Mario; Marasca, Roberto; Torelli, Giuseppe
abstract

Although patients with essential thrombocythaemia (ET) do not have the Philadelphia chromosome t(9;22), which defines chronic myelocytic leukaemia, the chimeric BCR-ABL transcript mRNA from this translocation has been identified in patients with clinically typical essential thrombocythaemia”.These findings have been detected, by D Blickstein and colleagues2 in 48% of 25 patients negative for the Philadelphia chromosome who had essential thrombocythaemia, investigated by two-step nested RT-PCR. Some workers have not, however, been able to confirm these data by the same technique, in 18 and 20 such patients, respectively. [3] and [4] Moreover, others have shown the absence of the chimeric transcript in 41 patients with essential thrombocythaemia studied by the fluoresence in-situ hybridisation with a BCR-ABL probe.5We investigated 112 patients who had essential thrombocythaemia with long follow-up, by RT-PCR, and detected the BCR-ABL transcript in only one patient who progressed to myeloid blastic crisis 12 years after diagnosis. Actually, whether essential-thrombocythaemia patients negative for the Philadelphia chromosome express the BCR-ABL transcript has been a matter of controversy for several years. In studies, the apparent essential thrombocythaemia carrying the Philadelphia anomaly, cytogenetically or molecularly should probably be considered as a variant of chronic myelocytic leukaemia with thrombocytosis and often long survival, with obvious clinical and therapeutical implications.

2000 - Gestational thrombocytopenia [6] (multiple letters) [Articolo su rivista]
Minakami, H.; Sato, I.; George, J.; Emilia, G.; Luppi, M.; Marasca, R.; Torelli, G.
abstract

2000 - Late-appearing PML/RAR alpha fusion transcript with coincidental t(12;13)(p13.2;q14) in acute promyelocytic leukemia lacking the t(15;17) cytogenetic anomaly [Articolo su rivista]
Temperani, Paola; Luppi, Mario; F., Giacobbi; V., Medici; M., Morselli; Barozzi, Patrizia; Marasca, Roberto; Torelli, Giuseppe; Emilia, Giovanni
abstract

The late appearance of a cytogenetic/molecular hallmark in human leukemias is a rare event. We report on a case of acute myeloid leukemia with morphology, immunophenotype and clinical features typical of promyelocytic subtype (APL), in which the specific PML/RAR alpha gene rearrangement was molecularly detected only at second relapse of disease, without cytogenetic evidence of the t(15;17). The emergence of the PML/RAR alpha gene may be therapy-related or may represent the exceptional result of a clonal evolution during progression of neoplasia. At second relapse, a novel cell clone bearing a t(12;13)(p13.2;q14) was also observed and a molecular deletion and rearrangement of a locus at 13q14, distinct from retinoblastoma (Rb1) locus, was found. In this unusual case, the PML/RAR alpha product seems to be not essential for the expression of the promyelocytic phenotype at diagnosis and, when detectable, it is not the sole genetic defect. (C) 2000 Elsevier Science Inc. All rights reserved.

2000 - Molecular evidence of organ-related transmission of Kaposi sarcoma-associated herpesvirus or human herpesvirus-8 in transplant patients [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; G., Santagostino; R., Trovato; Tf, Schulz; Marasca, Roberto; D., Bottalico; L., Bignardi; Torelli, Giuseppe
abstract

In transplant patients, Kaposi sarcoma (KS)-associated herpesvirus or human herpesvirus-8 (HHV-8) infection is associated with the development of KS, primary effusion lymphoma and Castleman disease. Whether HHV-8 is either reactivated in the recipient or transmitted by the donor has been investigated so far only by serologic studies. Thus, we addressed the issue of HHV-8 transmission in the transplantation setting by molecular methods. We exploited the high level variability of the orf-K1 gene and the polymorphism of the orf-73 gene of the HHV-8 genome to assess the genetic relatedness of the HHV-8 strains identified in the posttransplant KS lesions that developed, simultaneously, 20 months after transplantation, in 2 recipients of twin kidneys from the same cadaver donor. The 100% identity of nucleotide sequence of the most variable viral region and the presence of the same, single orf-73 type in both patients provides strong molecular evidence of organ-related transmission of HHV-8 in the setting of transplantation.

2000 - Nonmalignant disease associated with human herpesvirus 8 reactivation in patients who have undergone autologous peripheral blood stem cell transplantation [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Tf, Schulz; R., Trovato; A., Donelli; Narni, Franco; J., Sheldon; Marasca, Roberto; Torelli, Giuseppe
abstract

Fever, cutaneous rash, and hepatitis-for which an infectious cause was suspected-developed in an Italian patient with non-Hodgkin lymphoma after autologous peripheral blood stem cell (PBSC) transplantation. Polymerase chain reaction (PCR) with degenerate primers for the highly conserved DNA polymerase gene of herpesviruses detected herpesvirus sequences 100% identical to human herpesvirus-8 (HHV-8) in serial cell-free serum samples, collected immediately before or concomitant with the occurrence of clinical symptoms; no other common infections were documented. The presence of the HHV-8 genome (clade C) was confirmed by PCR with HHV-8-specific primers for orf 26 and orf-K1, HHV-8 viremia was undetectable either before transplantation or when the patient was clinically asymptomatic. Semiquantitative PCR analysis showed variations of the viral load correlating with the clinical status. Anti-HHV-8 antibodies were detected before and after transplantation by an immunofluorescence assay for lytic antigens. Active HHV-8 infection may be associated with nonmalignant illness after PBSC/bone marrow transplantation. (Blood. 2000;96:2355-2357)

1999 - Cellular localization of human herpesvirus 8 in nonneoplastic lymphadenopathies and chronic interstitial pneumonitis by in situ polymerase chain reaction studies [Articolo su rivista]
Trovato, R; Luppi, Mario; Barozzi, Patrizia; Da Prato, L; Maiorana, Antonino; Lico, S; Marasca, Roberto; Torricelli, Pietro; Torelli, Giuseppe; Ceccherini Nelli, L.
abstract

Objectives: To study the cellular localization of human herpesvirus 8 (HHV-8) in rare cases of HHV-8 infection from Italy that are associated neither with human immunodeficiency virus (HIV) infection nor Kaposi's sarcoma (KS). Methods: The presence and distribution of HHV-8-infected cells was investigated by direct in situ polymerase chain reaction (PCR) in the lymph node tissues from 2 patients with reactive lymphadenopathies with florid follicular hyperplasia and increased vascularity and in the lung tissue from 1 patient with chronic interstitial pneumonitis. Results: HHV-8 was localized in lymphoid and monocyte-macrophage cells scattered in the interfollicular regions of both lymph nodes but not in endothelial cells. In the lung tissue, HHV-8 was found in the inflammatory cells infiltrating the interalveolar interstitium, in endothelial cells of the pulmonary vasculature, and in rare pneumocytes. Conclusions: HHV-8 can infect nonneoplastic lymph nodes of immunocompetent subjects, and the distribution of infected cells outside of the germinal centers resembles that of Epstein-Barr virus (EBV)-infected cells in the lymph nodes in the course of infectious mononucleosis. Endothelial cells and pneumocytes may be a target of HHV-8 infection out of the KS setting, at least in the presence of a chronic inflammatory process.

1999 - Erratum: Cellular localization of human herpesvirus 8 in nonneoplastic lymphadenopathies and chronic interstitial pneumonitis by in situ polymerase chain reaction studies (Journal of Human Virology (January/February 1999) 2:1 (38-44)) [Articolo su rivista]
Trovato, R.; Luppi, M.; Barozzi, P.; Da Prato, L.; Maiorana, A.; Lico, S.; Marasca, R.; Torricelli, P.; Torelli, G.; Ceccherini-Nelli, L.
abstract

1999 - Expression of cell-homologous genes of human herpesvirus-8 in human immunodeficiency virus-negative lymphoproliferative diseases [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Maiorana, Antonino; R., Trovato; Marasca, Roberto; M., Morselli; K., Cagossi; Torelli, Giuseppe
abstract

Human herpesvirus-8 (HHV-8) genome encodes for genes homologous to human cellular genes such as interleukin-6 (IL-6), Cyclin-D, BCL-5, and IL-8 receptor (G-protein-coupled receptor [GCR]), We used reverse transcriptase-polymerase chain reaction to study the expression of these viral genes in lymphoproliferative disorders associated with HHV-8 infection, None of these genes was expressed in 1 case of benign, localized Castleman´s disease (CD), and only viral IL-6 and viral Cyclin-D were transcribed in 2 cases of benign lymphadenopathies with giant germinal center hyperplasia and increased vascularity. In contrast, all 4 genes were transcribed in 1 case of multicentric CD of plasma cell type with aggressive clinical course and in 1 primary effusion lymphoma cell line. Our study provides the evidence that various HHV-8 genes, homologous to cellular genes involved in control of proliferation and apoptosis, may be differently expressed in different lymphoid disorders in vivo.

1999 - Human herpesvirus 6 latently infects early bone marrow progenitors in vivo [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; C., Morris; Maiorana, Antonino; R., Garber; G., Bonacorsi; A., Donelli; Marasca, Roberto; A., Tabilio; Torelli, Giuseppe
abstract

We have studied the in vivo tropism of human herpesvirus 6 (HHV-6) for hemopoietic cells in patients with latent HHV-6 infection. Having used a variety of cell purification, molecular, cytogenetic, and immunocytochemical procedures, we report the first evidence that HHV-6 latently infects early bone marrow progenitor cells and that HHV-6 may be transmitted longitudinally to cells which differentiate along the committed pathways.

1999 - Lack of confirmation of an association between HTLV-I infection and myelodysplastic syndrome [Articolo su rivista]
M., Morselli; Luppi, Mario; Barozzi, Patrizia; Dominici, Massimo; Temperani, Paola; D., Campione; F., Lanza; R., Trovato; Marasca, Roberto; G., Longo; G., Emilia; Torelli, Giuseppe
abstract

No abstract available

1999 - Missense mutations in the PML/RAR alpha ligand binding domain in ATRA-resistant As2O3 sensitive relapsed acute promyelocytic leukemia [Articolo su rivista]
Marasca, Roberto; Zucchini, Patrizia; S., Galimberti; G., Leonardi; Vaccari, Paola; A., Donelli; Luppi, Mario; M., Petrini; Torelli, Giuseppe
abstract

Background and Objectives. Acute promyelocytic leukemia is characterized by the chromosomal translocation t(15;17) which yields the fusion product PML/RAR alpha. Art-trans retinoic acid probably induces differentiation of atypical promyelocytes and clinical remission in APL patients by binding to the ligand binding domain (LBD) of the RAR alpha portion of the PML-RAR alpha chimeric protein. Structural alterations of the LED of the PML/RAR alpha have been revealed in ATRA-resistant APL cell lines and in a few APL patients with acquired clinical resistance to ATRA therapy. Two APL relapsed patients with clinical resistance to ATRA therapy were evaluated for the presence of nucleotide mutations in the LED of PML/RAR alpha gene and then treated with arsenic trioxide (As2O3). Design and Methods. DNA fragments from the LED of the PML/RAR alpha: chimeric transcript were obtained by reverse-transcribed polymerase chain reaction. Direct sequencing was performed by an unambiguous bidirectional automatic analysis. Samples representative of APL onset and relapse were analyzed from both patients. Results. In both patients, at the ATRA-resistant relapse, a missense point mutation in the LED of the PML/RAR alpha gene was found. The mutations, absent at APL onset, led to an Arg272Gln and to an Arg276Trp amino acid substitution, according to the sequence of the RAR alpha protein. Both patients had complete clinical and hematologic remission after treatment with As2O3. Interpretation and Conclusions. LED missense mutations appear to be a significant mechanism of acquired ATRA-resistance in vivo, closely related to clinical APL relapse. The two cases reported here provide the first in vivo evidence of Apt, relapsed patients, who have become ATRA-resistant for molecular reasons, being sensitive to arsenic trioxide.

1999 - The oncogenic 30 and 69 bp deletion variants of the EBV LMP-1 gene are common in HIV-negative lymphoproliferations, both malignant and benign [Articolo su rivista]
Barozzi, Patrizia; Luppi, Mario; K., Cagossi; Maiorana, Antonino; Marasca, Roberto; T., Artusi; S., Poggi; S. A., Pileri; Torelli, Giuseppe
abstract

BACKGROUND: In vitro studies have shown that the 30 and 69 base pair (bp) deletion variants of the latent membrane protein (LMP)-1 gene of the Epstein-Barr virus (EBV) have a higher transforming capacity than the wild-type variant. In recent years these studies have triggered an in vivo search for such potentially oncogenic variants in lymphoid tissues. PATIENTS AND METHODS: We used polymerase chain reaction (PCR) to investigate the prevalence of LMP-1 gene variants in EBV-positive lymph nodes from 60 HIV-negative Italian patients with benign and malignant lymphoid disorders. RESULTS: The 30 bp variant was detected in 10 of 39 (25.6%) malignant lymphomas but also in 4 of 13 (30%) reactive lymphadenitis with follicular hyperplasia. Of note is the fact that the 69 bp variant was detected in three cases of malignant lymphoproliferation but also in two cases of localized Castleman's disease of hyalin vascular type. CONCLUSIONS: The molecular detection of the oncogenic variants of the LMP-1 gene in a lymph node biopsy as an indicator of the aggressiveness of the EBV-associated lymphoproliferative disease must be considered with caution. The relatively high frequency of the 69 bp variant in our series compared with that reported in the literature probably reflects a different incidence of LMP-1 variants in healthy populations from different geographical areas.

1998 - Chronic myeloid leukemia with thrombocythemic onset may be associated with different BCR/ABL variant transcripts [Articolo su rivista]
Emilia, Giovanni; Luppi, Mario; Ferrari, Mg; Temperani, Paola; Marasca, Roberto; Giacobbi, F; Vaccari, Paola; Bandieri, E; Di Donato, C; Carapezzi, C; Torelli, Giuseppe
abstract

Ph-positive chronic myeloid leukemia (CML) mimicking essential thrombocythemia (ET) at onset seems to be a distinct clinical entity. Whether this rare clinical form of CML is associated with single, specific variants of BCR/ABL transcripts is a matter of debate. Among 82 consecutive patients with Ph-positive CML, we identified 3 patients in which the disease mimicked ET at presentation, because-of marked thrombocytosis and moderate leukocytosis, with few immature myeloid cells in peripheral blood and blood basophilia in 2 of them. Molecular analysis with the reverse transcriptase-polymerase chain reaction technique showed the presence of b2a2, b3a2, and b3a2-b2a2 transcript variants in the three patients, respectively. The results of our study together with a review of literature data suggest that different BCR/ABL transcript variants may occur in CML mimicking ET, without an apparently significant prevalence of one type.

1998 - Clinico-pathological characterization of hepatitis C virus-related B-cell non-Hodgkin's lymphomas without symptomatic cryoglobulinemia [Articolo su rivista]
Luppi, Mario; G., Longo; Mg, Ferrari; Barozzi, Patrizia; Marasca, Roberto; M., Morselli; C., Valenti; Mascia, Maria Teresa; L., Vandelli; D., Vallisa; L., Cavanna; Torelli, Giuseppe
abstract

Background. Epidemiological evidence has suggested an association between hepatitis C virus (HCV) infection and B-cell lymphoproliferation. We studied the prevalence of HCV infection in a series of de novo B-cell non-Hodgkin's lymphoma (B-NHL) cases and correlated virological findings with clinico-histological features. Patients and methods. One hundred fifty-seven patients with de novo B-NHL were included in the study. Their serum was examined by ELISA and RIBA for the presence of anti-HCV antibodies, and either the peripheral blood mononuclear cells or the pathology tissues of all of the patients were examined by reverse transcriptase polymerase chain reaction for the presence of HCV RNA sequences, Results: HCV infection occurred in 22.3% of B-NHL patients and was documented before the diagnosis in about halfof the positive cases. Of interest. HCV infection was more frequently found in follicular center; marginal zone and diffuse large-cell lymphoma types, but was not associated with symptomatic cryoglobulinemia. The median survival time was 48 months in HCV-positive and 52 months in HCV-negative B-NHL patients. Conclusions. Our findings strengthen the pathogenetic link between HCV and B-NHL and show that HCV infection may be associated with the malignant proliferation of defined B-cell subsets other than the immunoglobulin Mk B-cell subset involved in the pathogenesis of mixed cryoglobulinemia type II and associated lymphoplasmacytoid lymphoma type. HCV-related liver disease did not affect the survival of our B-NHL patients.

1998 - Expression of human herpesvirus-6 antigens in benign and malignant lymphoproliferative diseases [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; R., Garber; Maiorana, Antonino; G., Bonacorsi; T., Artusi; R., Trovato; Marasca, Roberto; Torelli, Giuseppe
abstract

Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA, A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin's lymphomas, 14 Hodgkin's disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 Reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called mummified Reed-Sternberg cells, in two Hodgkin's disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the vines in human lymphomagenesis, The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder.

1998 - Might essential thrombocythemia carry Ph anomaly? [Articolo su rivista]
Marasca, Roberto; Luppi, Mario; Zucchini, Patrizia; Longo, G; Torelli, Giuseppe; Emilia, G.
abstract

Might essential thrombocythemia carry Ph anomaly?

1998 - Polymerase chain reaction detection of human herpesvirus 8 sequences in primary central nervous system lymphomas [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Savarino, M; Torelli, Giuseppe
abstract

No abstract available

1997 - Hepatitis C virus-induced leuko-thrombocytopenia and haemolysis [Articolo su rivista]
Emilia, G; Luppi, Mario; Ferrari, Mg; Barozzi, Patrizia; Marasca, Roberto; Torelli, Giuseppe
abstract

Hepatitis C virus (HCV) has been recognized as the cause of thrombocytopenia occurring in patients with chronic hepatitis C, possibly through autoimmune mechanisms. A patient is described with B cell chronic lymphocytic leukaemia, presenting with a marked leuko-thrombocytopenia and an associated mild haemolysis secondary to HCV infection, in the absence of clinical and biochemical signs of hepatitis. Anti-HCV antibodies were detected in the serum both by ELISA and RIBA but not 2 months before the onset of cytopenia. The presence of HCV RNA was documented both in the peripheral blood mononuclear cells and in the bone marrow by reverse transcriptase polymerase chain reaction of the 5´ untranslated region of the viral genome. Of interest, HCV RNA was also found in the serum, showing that viraemia was associated with the presence of circulating anti-HCV antibodies. HCV genotyping, performed by PCR amplification of the core region, revealed the presence of an unclassifiable genotype. The hypothetical mechanisms leading to HCV-induced cytopenia in this patient are briefly discussed. Treatment with corticosteroids was effective in controlling blood cell counts, without increasing viraemia and deterioration of liver disease. HCV infection should be considered in the differential diagnosis of possible causes of cytopenia, mainly in immunosuppressed patients, even in absence of clinical and biochemical signs of hepatitis.

1997 - Human Herpesvirus 8 strain variability in clinical conditions other than Kaposi’s sarcoma [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Ferrari, Mg; Torelli, Giuseppe
abstract

No abstract available

1997 - Prevalence of HCV infection and second neoplasms in marginal zone lymphomas [Articolo su rivista]
Luppi, Mario; Longo, G; Ferrari, Mg; Ferrara, L; Marasca, Roberto; Barozzi, Patrizia; Morselli, M; Emilia, G; Torelli, Giuseppe
abstract

No abstract available

1997 - Relationship between BCR/ABL fusion proteins and leukemia phenotype [Articolo su rivista]
Emilia, G; Luppi, Mario; Marasca, Roberto; Torelli, Giuseppe
abstract

No abstract available

1996 - Additional neoplasms and HCV infection in low-grade lymphoma of MALT type [Articolo su rivista]
Luppi, Mario; Longo, G; Ferrari, Mg; Ferrara, L; Marasca, Roberto; Barozzi, Patrizia; Morselli, M; Emilia, G; Torelli, Giuseppe
abstract

Several chronic inflammatory conditions and genetic alterations are likely to be involved in the pathogenesis of low-grade lymphoma of MALT type. In a well-characterized series of 27 patients with low-grade lymphoma of MALT type, we studied: (1) the incidence of other neoplasms, which might be indicative of genetic instability, apparently a characteristic of this disease; (2) the prevalence of serologic and molecular markers of HCV infection, which has been found in association with other lymphoproliferative disorders. Three patients had one or more additional cancers; a total of eight tumours, five of which occurred in the same patient, suggests the presence of some genetic instability in at least some cases of the disease. Rather unexpectedly, anti-HCV antibodies and HCV RNA sequences were documented in 50% of the patients examined, without elevation of serum transaminases. Of interest, the two patients with parotid and conjunctival MALT lymphomas, respectively, with a previous history of Sjogren's syndrome, were HCV positive. We suggest, for the first time, that HCV may be considered, in addition to Helicobacter pylori, as another potential infectious co-factor in the multistep pathogenesis of low-grade lymphomas of MALT type.

1996 - Frequency and distribution of herpesvirus-like DNA sequences (KSHV) in different stages of classic Kaposi's sarcoma and in normal tissues from Italian population. [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Maiorana, Antonino; G., Collina; M. G., Ferrari; Marasca, Roberto; M., Morselli; E., Rossi; L., Ceccherini Nelli; Torelli, Giuseppe
abstract

The frequency and distribution of herpesvirus-like DNA sequences (KSHV) were investigated by PCR in the pathologic skin lesions of a series of 22 HIV-negative elderly patients with classic Kaposi´s sarcoma (KS) from Italy, one of the few regions of the world where classic KS is prevalent. Viral sequences were clearly identifiable in 15 cases, in particular in 2 of 5 patch, in 3 of 6 plaque and in 10 of 11 nodular lesions. Our findings confirm the association of these herpesvirus-like DNA sequences with KS in unrelated populations, providing evidence of the putative KS-associated agent in all different histologic lesions of the disease, mainly in the nodular stage. The search for other herpesviruses by PCR showed that Epstein-Barr virus (EBV) sequences were present in 7 of 22 pathologic skin lesions. In 4 cases, both EBV and KSHV were present. On the contrary, all 22 classic KS specimens were negative for human herpesvirus-6 sequences. Two of 3 patch and the 1 nodular lesions from AIDS-related KS patients examined were positive for KSHV but negative for both EBV and HHV-6 sequences. Furthermore, we evaluated the prevalence of KSHV sequences in the normal population of the same geographical area. Thirteen peripheral blood mononuclear cell samples, 9 salivary gland tissues and 6 saliva samples from healthy subjects were invariably found negative for KSHV, using the same PCR technique. Of interest, 2 of 11 hyperplastic tonsils harboured these herpesvirus-like sequences, suggesting that, like other herpesviruses, the KS-associated agent may be harboured in a proportion of normal individuals and tonsils may represent at least one of the possible reservoirs of this putative lymphotropic gamma-herpesvirus in vivo.

1996 - Hepatitis C virus infection in subsets of neoplastic lymphoproliferations not associated with cryoglobulinemia [Articolo su rivista]
Luppi, Mario; Ferrari, Mg; Bonaccorsi, G; Longo, G; Narni, Franco; Barozzi, Patrizia; Marasca, Roberto; Mussini, C; Torelli, Giuseppe
abstract

Hepatitis C virus (HCV) is both hepatotropic and lymphotropic and a dear-cut association has been proposed between HCV infection and mixed cryoglobulinemia (MC), a benign lymphoproliferative disorder, which sometimes evolves into a frank malignant B cell non-Hodgkin's lymphoma (B-NHL). Moreover, the presence of antibodies to HCV, as well as of HCV-specific genomes has been reported in the sera of over 37% patients with B-NHL, not associated with MC. Thus, we decided to perform both a serologic and a molecular study to give insights into a possible relationship between HCV infection and neoplastic lymphoproliferations. We used ELISA and RIBA tests to show that anti-HCV antibodies were present in the serum of 29 out of 69 unselected B-NHL patients (42%), while seropositivity in a healthy population was about 1%. The prevalence of anti-HCV antibodies was low in definite subsets of B lymphoid disorders, including multiple myeloma, Waldenstrom's macroglobulinemia and monoclonal gammopathies of undetermined significance. Then, using reverse transcriptase polymerase chain reaction, we detected HCV sequences directly in the pathologic lymph node biopsies in 13 out of 34 B-NHL cases, and in particular in six out of eight low-grade lymphomas of MALT type and in five out of eight centroblastic-centrocytic follicular lymphomas. In contrast, the peripheral blood samples from 10 B cell chronic lymphocytic leukemia patients resulted negative for the presence of HCV genomes. Similarly, viral sequences were absent in 10 T cell NHL, while only one out of the 14 Hodgkin's disease cases tested resulted positive. Finally, we used a PCR-based assay to characterize the genotypes (I-IV) present in the positive lymphomatous tissues. The presence of both serologic and molecular markers of HCV infection in a high percentage of certain types of B-NHL, not associated with cryoglobulinemia, and its absence from other lymphoproliferative diseases extends the spectrum of HCV-associated lymphoproliferations arguing in favor of some role of this viral infection in the pathogenesis of the malignant proliferation of definite B lymphoid populations.

1996 - HHV-8-associated primary cerebral B-cell lymphoma in HIV-negative patient after long-term steroids [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Savarino, M; Torelli, Giuseppe
abstract

No abstract available

1996 - Human herpesvirus-8 DNA sequences in Human Immunodeficiency Virus-negative angioimmunoblastic lymphadenopathy and benign lymphadenopathy with giant germinal center hyperplasia and increased vascularity [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Maiorana, Antonino; T., Artusi; R., Trovato; Marasca, Roberto; M., Savarino; L., Ceccherini Nelli; Torelli, Giuseppe
abstract

Human herpesvirus-8 (HHV-8) DNA sequences have been reported to be strictly associated not only with various forms of Kaposi´s sarcoma but also with an unusual subgroup of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphomas. A possible relation of this putative virus also with multicentric Castleman´s disease (MCD) has been recently suggested. We used polymerase chain reaction to look for the presence of HHV-8 sequences in a well characterized series of benign, atypical, and malignant lymphoid tissues from 45 Hodgkin´s disease and 43 non-Hodgkin´s lymphoma (NHL) cases, as well as from 5 MCD, 15 angioimmunoblastic lymphadenopathy (AILD), and 23 benign lymphadenopathy cases. Among the 38 AIDS-related lymphoid lesions, only 1 NHL and 1 persistent generalized lymphadenopathy (PGL) case were positive. Furthermore, among the 92 non-AIDS-related lymphoproliferative disorders, HHV-8 sequences were detected in 3 classic AILD cases and in 4 reactive lymphadenopathies. Six of 9 HHV-8 positive lymphoid lesions (1 NHL, 1 PGL, 1 AILD, and 3 reactive lymphadenopathy cases) were also positive for Epstein-Barr viral sequences. The four human immunodeficiency virus (HIV) negative lymphadenopathies positive for HHV-8 sequences showed an almost identical histology, characterized by a predominantly follicular lesion, with giant germinal center hyperplasia, and increased vascularity, resembling HIV-related lymphadenopathy and MCD. Our results, while providing the first evidence of the presence of HHV-8 sequences in AILD cases, suggest a possible association of these herpesviral sequences with a distinct hystologic type of non-neoplastic lymphadenopathy, not associated with other common herpes infections.

1996 - Lymphotropic herpes virus (EBV, HHV-6, HHV-8) DNA sequences in HIV negative Castleman's disease [Articolo su rivista]
Barozzi, Patrizia; Luppi, Mario; Masini, L; Marasca, Roberto; Savarino, M; Morselli, M; Ferrari, Mg; Bevini, M; Bonacorsi, G; Torelli, Giuseppe
abstract

Aim-To evaluate the possible involvement of lymphotropic herpes viruses in Castleman's disease. Methods-Archival formalin fixed, paraffin wax embedded biopsy specimens from 16 HIV negative patients (11 with localised and five of multicentric disease) were studied. Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) and human herpes virus-8 (HHV-8) DNA was detected using PCR. PCR was also used to characterise the EBV genomes and the clonal status of the lesions. Results-EBV sequences were identified in nine (56%) cases. The main EBV genotype detected was type 1. Two (12%) cases were positive for both HHV-6 and EBV sequences. HHV-8 sequences were detected in one case of localised Castleman's disease, the sequence of which differed from that of the HHV-8 prototype. No clonal immunoglobulin gene rearrangements were found. Conclusions-EBV DNA was detected in a substantial proportion of cases, suggesting that it may have a role in the pathogenesis of Castleman's disease, unlike HHV-6 which was detected rarely. This is the first report of HHV-8 specific sequences in the localised from of the disease.

1996 - P53 gene mutations in chronic myelogenous leukemia medullary and extramedullary blast crisis [Articolo su rivista]
Marasca, Roberto; Luppi, Mario; Barozzi, Patrizia; Ferrari, Mg; Morselli, M; Torelli, Giuseppe
abstract

Molecular alterations of the P53 gene were investigated in 27 unselected patients with chronic myelogenous leukemia (CML) blast crisis. A rearrangement of the P53 gene was evident by Southern blotting in 3 cases, one of which also showed the same alteration in the chronic phase. Single strand conformation polymorphism and sequencing analysis showed point mutations in 4 blast crisis cases. Of interest, P53 point mutations were evident in all the 3 cases of extramedullary blast crisis examined and the same point mutation was found in the myeloblastoma tissues and in the subsequent peripheral blast cells. These data indicate that: a) P53 gene mutations occur in a significant but not a large number of CML acute phase cases; b) P53 gene point mutations seem to correlate strongly with the infrequent extramedullary presentation of the blast crisis; c) the presence of the same P53 gene point mutation in extramedullary and bone marrow blast cells confirms the common clonal origin of the two cellular populations.

1996 - Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene [Articolo su rivista]
Luppi, Mario; Morselli, M; Bandieri, E; Federico, Massimo; Marasca, Roberto; Barozzi, P; Ferrari, Mg; Savarino, M; Frassoldati, A; Torelli, Giuseppe
abstract

Background: Maspin, a recently identified protein related to the family of serpins, is believed to play a role in human breast cancer. In an effort to improve the present methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for maspin transcript to identify small numbers of mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. Patients and methods: Five non-neoplastic mammary tissue samples, 13 breast cancer specimens as well as 17 peripheral blood and 4 bone marrow samples from normal subjects were screened for the presence of maspin mRNA by RT-PCR. The same assay was applied to peripheral blood or bone marrow samples obtained from 29 patients with stages I to IV breast cancer. Results: By RT-PCR it was possible to amplify maspin mRNA in all of the primary and metastatic breast cancer specimens, but in none of the normal hemopoietic samples from healthy donors. Thus, detection of maspin transcript in the peripheral blood or marrow of a patient known to have breast cancer is indicative of the presence of mammary carcinoma cells. In reconstitution experiments, maspin RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear cells (PBMCs). None of the 9 patients with stages I, II, or III breast cancer had maspin transcript in peripheral blood. Of note, 3 of 9 patients with stage TV breast cancer receiving systemic therapy at the time of sample collection, but only I of 11 patients with stage IV not receiving therapy, had detectable maspin transcript in peripheral blood. Moreover, 3 marrow specimens from stage TV patients tested positive by this assay. Conclusions: This pilot study suggests that maspin RT-PCR assay is a sensitive, specific and sufficiently rapid method for detection of small numbers of circulating cells and marrow micrometastases in breast cancer patients. The possibility of applying this assay in the detection of tumor cell contamination of both marrow and stem-cell apheresis harvests of breast cancer patients merits further investigation.

1995 - DETECTION OF PML-RAR-ALPHA FUSION TRANSCRIPT IN PH POSITIVE LEUKEMIA WITH ACUTE PROMYELOCYTIC PHENOTYPE LACKING THE T(15-17) CYTOGENETIC ABNORMALITY [Articolo su rivista]
Emilia, G; Marasca, Roberto; Longo, G; Ferrari, Mg; Notohamiprodjo, M; Temperani, Paola; Sacchi, Stefano; Torelli, Giuseppe
abstract

A 39-year-old woman was diagnosed with acute promyelocytic leukemia (APL) with disseminated intravascular coagulation syndrome. The hematologic examination showed a morphologic, cytochemical, and immunophenotypic picture typical of an APL, with a marked leukocytosis and a mixed population of hypergranular and microgranular promyelocytes. The cytogenetic analysis showed a 46,XX,t(9;22) karyotype, without any alterations of chromosomes 15 and 17. The t(15;17) translocation was not evident in FISH experiments, while a molecular analysis revealed the presence of a PML-RAR alpha chimera.

1995 - HUMAN HERPESVIRUS-6 - A SURVEY OF PRESENCE AND DISTRIBUTION OF GENOMIC SEQUENCES IN NORMAL BRAIN AND NEUROGLIAL TUMORS [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Maiorana, Antonino; Marasca, Roberto; R., Trovato; Fano, Rita Adriana; L., CECCHERINI NELLI; Torelli, Giuseppe
abstract

In an attempt to study the frequency and distribution of human herpesvirus-6 (HHV-6) infection both in normal and neoplastic brain tissues in vivo, polymerase chain reaction was used to look for HHV-6 genomes: 1) in samples, obtained at necropsy, from different regions of the brain of immunocompetent adult subjects and of patients who died of AIDS; 2) in the surgical biopsies of a well-characterized series of primary brain tumors of neuroglial origin. HHV-6-specific sequences were identified in six of nine brain samples from immunocompetent subjects, and in four of seven brain samples from AIDS patients. Viral sequences were identified in the specimens derived either from the grey (frontal cortex and basal ganglia) or from the periventricular white matter. HHV-6 DNA was found only in 6 of the 37 primary brain tumor biopsies examined. Th is study provides for the first time molecular evidence of a wide distribution of HHV-6 infection in the brain tissues of a high proportion of subjects, both in normal and in impaired immunity. in this large series of tumor biopsies the presence of HHV-6 genomic sequences is a rare phenomenon, arguing against a major role of this herpesvirus in the pathogenesis of primary brain tumors of neuroglial origin in immuno-competent subjects.

1995 - Human herpesvirus-6 genome in acute lymphoblastic leukemia: Evidence against an etiologic relationship [Articolo su rivista]
Barozzi, Patrizia; Luppi, Mario; Marasca, Roberto; Trovato, R; Ceccherininelli, L; Torelli, Giuseppe
abstract

No abstract available

1995 - Human herpesvirus-6 (HHV-6) in blood donors [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Ceccherini Nelli, L; Ceccherelli, G; Torelli, G.
abstract

No abstract available

1995 - P53 GENE POINT MUTATIONS IN RELATION TO P53 NUCLEAR-PROTEIN ACCUMULATION IN COLORECTAL CANCERS [Articolo su rivista]
Costa, A; Marasca, Roberto; Valentinis, B; Savarino, M; Faranda, A; Silvestrini, R; Torelli, Giuseppe
abstract

It is known that structural alterations of the p53 tumour suppressor gene cause malignant transformation and tumour progression in colorectal mucosa. In this study, 38 colorectal cancers were analysed for mutations detected in the p53 gene by single-strand conformational polymorphism and DNA sequence analysis, and the results were compared with p53 protein expression detected by immunohistochemistry. A very strict association (P<0.0001) was found between genetic alterations and protein accumulation, as detected by the PAb 1801 monoclonal antibody. p53 expression and gene mutations were more frequent in rectal than in colonic cancers. No relation was observed with Dukes' stage, even though most of the mutations were at exon 7 in Dukes' A-B cancers and almost all mutations at exon 8 were observed in Dukes' C-D cancers. DNA ploidy was not generally associated with p53 protein expression or gene mutations. However, 83 per cent of cases with exon 5 and 6 mutations were diploid or near-diploid and 71 per cent of cases with mutations at exons 7 and 8 were aneuploid. Tumours with p53 gene mutations at exon 5 had a higher median [H-3]thymidine labelling index (17 per cent) than those with mutations at exons 6, 7, and 8 (11.8 per cent).

1995 - Spontaneous adrenal gland haematoma in a patient with antiphospholipid antibodies [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Torricelli, Pietro; Leonardi, G; Gavioli, Gl; Bocchi, A; Torelli, G.
abstract

No abstract available

1995 - Spontaneous loss of Ph chromosome with maintenance of clonal hemopoiesis in an untreated patient with myeloproliferative disease and a long survival. [Articolo su rivista]
Luppi, Mario; Morselli, M; Emilia, G; Temperani, P; Marasca, Roberto; Barozzi, Patrizia; Selleri, L; Torelli, G.
abstract

The unusual case of myeloproliferative disease described here is characterized by the following features: (1) a clinically completely silent course for 11 years without splenomegaly, marrow fibrosis, or cellular morphologic alterations; (2) the presence, at the onset, of a Philadelphia (Ph) chromosome without DNA breakpoints in the M-bcr region; (3) the spontaneous loss of detectable Ph-positive cells, 5 years after the first finding of leukocytosis, in the absence of any therapy; (4) the maintenance of the clonal nature of hematopoiesis, as revealed by the PGK X-linked inactivation pattern, in the absence of the Ph chromosome; and (5) a biphasic trend in the levels of leukocytes, red cells, and platelets during the years of observation.

1995 - Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Paola, Cocconcelli; Elisa, Merelli; Luca Ceccherini, Nelli; Ferrari, Sergio; Mario, Luppi; Barozzi, Patrizia
abstract

Out of 64 cases of non-Hodgkin's lymphomas (NHL), 55 cases of Hodgkin's disease (HD) and 31 cases of multiple sclerosis (MS), 2 NHL, 7 HD and 1 MS cases were found positive by polymerase chain reaction (PCR) for the presence of HHV-6 sequences in pathologic lymph nodes of the lymphomas and in peripheral blood mononuclear cells (PBMCs) of MS. A further analysis of the PBMCs of the PCR positive cases by standard Southern blot technique revealed only 2 NHL, 3 HD and 1 MS cases as positive, indicating that these six patients have an unusually high viral copy number in the PBMCs. Restriction analysis, carried out using probes representative of different regions of the virus, showed that three cases retain only a deleted portion of the viral genome. In the remaining three cases a complete viral genome was present, containing the right end sequences in which the rep-like gene, possibly crucial to the viral and cellular life cycle, is located. The analysis by pulsed field gel electrophoresis (PFGE) of the total DNA of the PBMCs obtained directly, without culture from PBMCs of these last three cases (1 NHL, 1 HD, and 1 MS), using the same probes, showed the absence of free viral molecules and the association of viral sequences with high molecular weight DNA. These results are consistent with in vivo integration of the entire virus in the cellular genome. A further study of the same patients with chromosome fluorescence in situ hybridization (FISH) showed in all the three cases the presence of a specific hybridization site, located at the telomeric extremity of the short arm of chromosome 17 (17p13), suggesting that this location is at least a preferred site of an infrequent, but possibly biologically important, integration phenomenon.

1994 - Clonal nature of hypereosinophilic syndrome [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Morselli, M; Barozzi, Patrizia; Torelli, Giuseppe
abstract

No abstract available

1994 - DOUBLE P53 POINT MUTATION IN EXTRAMEDULLARY BLAST CRISIS OF CHRONIC MYELOGENOUS LEUKEMIA [Articolo su rivista]
Marasca, Roberto; Longo, G; Luppi, Mario; Barozzi, Patrizia; Torelli, Giuseppe
abstract

A patient with Philadelphia-positive chronic myelogenous leukemia (CML) evolved in extra-medullary blast crisis, was studied for the presence of alterations of the P53 tumor suppressor gene in the different stages of disease progression. No P53 gene aberrations were detected during the chronic and accelerated phases. Two identical missense point mutations, involving codons 249 and 281 and leading to the amino acid substitutions Arg-Ser and Thy-Asp, were identified in cells of an extramedullary mass and then in peripheral blood blast crisis cells. The data indicate that the medullary and extramedullary blast cells belong to the same cellular clone. They also strongly suggest that in this case, the alteration of P53 gene is strictly related to the progression of the disease, although this mechanism is certainly neither the only nor the most frequent molecular event leading to the acute phase.

1994 - Human herpesvirus 6 infection in normal human brain tissue [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Maiorana, Antonino; Marasca, Roberto; Torelli, Giuseppe
abstract

No abstract available

1994 - INTEGRATION OF HUMAN HERPESVIRUS-6 (HHV-6) GENOME IN CHROMOSOME-17 IN 2 LYMPHOMA PATIENTS [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Torelli, Giuseppe
abstract

No abstract available

1993 - CHARACTERIZATION OF HUMAN HERPESVIRUS-6 GENOMES FROM CASES OF LATENT INFECTION IN HUMAN LYMPHOMAS AND IMMUNE DISORDERS [Articolo su rivista]
Luppi, Mario; Barozzi, Patrizia; Marasca, Roberto; Ceccherininelli, L; Torelli, Giuseppe
abstract

No abstract available

1993 - FAILURE TO DETECT GENOMIC MATERIAL OF HTLV-I OR HTLV-II IN MONONUCLEAR-CELLS OF ITALIAN PATIENTS WITH MULTIPLE-SCLEROSIS AND CHRONIC PROGRESSIVE MYELOPATHY [Articolo su rivista]
Merelli, E; Sola, P; Marasca, Roberto; Salati, R; Torelli, Giuseppe
abstract

To contribute to the undecided question if a retrovirus of the human T-cell lymphotropic virus (HTLV) family may be involved in the development of multiple sclerosis (MS), we investigated by the polymerase chain reaction (PCR) the presence of HTLV-I and HTLV-II sequences in the peripheral blood mononuclear cell DNAs from 30 patients affected by MS and 15 by chronic progressive myelopathy. Moreover a control group of 14 blood donors was examined. All these patients were devoid of anti-HTLV-I antibody in the serum and cerebrospinal fluid at ELISA. For the PCR, primers and probes, specific for the tax region common to HTLV-I and HTLV-II, for the pol region of HTLV-I, and for the pol region of HTLV-II were used. In spite of the high sensitivity of the technique used, the three groups of subjects were negative for HTLV-I and HTLV-II genomic sequences.

1993 - FREQUENT DETECTION OF HUMAN HERPESVIRUS-6 SEQUENCES BY POLYMERASE CHAIN-REACTION IN PARAFFIN-EMBEDDED LYMPH-NODES FROM PATIENTS WITH ANGIOIMMUNOBLASTIC LYMPHADENOPATHY AND ANGIOIMMUNOBLASTIC LYMPHADENOPATHY-LIKE LYMPHOMA [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Barozzi, Patrizia; Artusi, T; Torelli, Giuseppe
abstract

In search of a possible involvement of viral agents in angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) and AILD-like lymphoma (AILD-L), we studied the presence of human herpesvirus-6 (HHV-6) in the lymph node biopsies of 12 cases by polymerase chain reaction (PCR). Given the rarity of this lymphoproliferative disorder, we investigated archival specimens, consisting of formalin-fixed and paraffin-embedded tissues, obtained from patients with a clinical and histologic diagnosis of AILD and AILD-L. HHV-6 sequences were detected in the lymph node biopsies of 7 out of the 12 AILD and AILD-L cases examined. HHV-6 sequences were identified also in the involved liver and spleen tissues of one patient and in the PBMCs of two patients, all carrying viral sequences in the affected lymph nodes. We also used PCR to characterize the HHV-6 genomes, showing that two different viral strains are represented in the pathologic tissues. This study provides evidence of the presence of HHV-6 specific sequences in an unexpectedly high proportion of our series of AILD and AILD-L cases, suggesting a possible involvement of this lymphotropic virus in the pathogenesis of at least some cases of the disease.

1993 - HUMAN HERPESVIRUS-6 AND MULTIPLE-SCLEROSIS - SURVEY OF ANTI-HHV-6 ANTIBODIES BY IMMUNOFLUORESCENCE ANALYSIS AND OF VIRAL SEQUENCES BY POLYMERASE CHAIN-REACTION [Articolo su rivista]
Sola, P; Merelli, E; Marasca, Roberto; Poggi, M; Luppi, Mario; Montorsi, M; Torelli, Giuseppe
abstract

A possible involvement of human herpesvirus 6 (HHV-6) infection in the pathogenesis of multiple sclerosis (MS) was investigated. The immunofluorescence analysis of sera from 126 MS patients showed significantly higher anti-HHV-6 antibody titres in MS sera than in 500 normal controls. A polymerase chain reaction (PCR) assay of the peripheral blood mononuclear cell (PBMC) DNAs of 31 MS patients and 24 normal subjects was positive in one normal control and in one MS patient. The Southern blot analysis indicated an unexpectedly high level of viral sequences in the MS patient, but not in the control. Since viral sequences are rarely present in MS subjects, the high anti-HHV-6 antibody titres found in MS are likely to be related to immune impairment rather than reactivation of a latent infection.

1993 - 3 CASES OF HUMAN HERPESVIRUS-6 LATENT INFECTION - INTEGRATION OF VIRAL GENOME IN PERIPHERAL-BLOOD MONONUCLEAR CELL-DNA [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Barozzi, Patrizia; Ferrari, Sergio; Ceccherininelli, L; Batoni, G; Merelli, E; Torelli, Giuseppe
abstract

Saliva and peripheral blood mononuclear cells from three patients, two with lymphoproliferative disorders and one suffering from multiple sclerosis, were examined for the presence of human herpesvirus-6 (HHV-6) genome by using the polymerase chain reaction and Southern blot analysis. The search for anti-HHV-6 antibodies, carried out in the sera of the same cases by an immunofluorescence assay, was negative in two cases at the lowest dilution used (1:40). These three patients had a high number of HHV-6 specific sequences in uncultured peripheral blood mononuclear cells, which are thought to be a normal site of viral latency although, in healthy individuals, the infected cells are extremely rare. In order to gain some insight into the state of the viral genome in this latent HHV-6 infection, we used pulsed field gel electrophoresis to separate HHV-6 DNA directly from HHV-6 (strain GS) infected HSB-2 cells and from the peripheral blood mononuclear cells of these three patients. Our study showed the presence of intact viral genome, of the expected length of 170 kb, persisting as free extrachromosomal element in the HSB-2 cells but not in patients' peripheral blood mononuclear cells. On the other hand, in strong contrast with the results obtained in infected HSB-2 DNA, the restriction analysis of the three patients' peripheral blood mononuclear cell DNA showed fragments of molecular weight constantly higher than the 170 kb segment, indicating that the viral sequences are linked to high molecular weight cellular DNA. Our findings are consistent only with a latent infection in which HHV-6 is integrated in vivo and suggest that pulsed field gel electrophoresis analysis is well worth using to evaluate the presence of integrated, intact, or fragmented viral genomes in HHV-6 associated lymphoproliferative diseases and immune disorders.

1992 - HUMAN HERPESVIRUS-6 IN NON-AIDS RELATED HODGKINS AND NON-HODGKINS-LYMPHOMAS [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Montorsi, M; Luppi, Mario; Barozzi, Patrizia; Ceccherini, L; Batoni, G; Bendinelli, M; Muyombano, A.
abstract

Human herpesvirus 6 in non-AIDS related Hodgkin's and non-Hodgkin's lymphomas.

1991 - HHV-6 INFECTION AND HUMAN HODGKIN AND NON-HODGKIN LYMPHOMAS [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Selleri, L; Luppi, Mario; Montorsi, M; Federico, Massimo; Narni, Franco; Ceccherininelli, L; Ferrari, Sergio
abstract

1991 - Human Herpes virus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Luppi, Mario; L., Selleri; Ferrari, Sergio; Narni, Franco; Mt, Mariano; Federico, Massimo; L., CECCHERINI NELLI; M., Bendinelli; G., Montagnani; M., Montorsi; Artusi, Tullio
abstract

In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV-6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.

1990 - IDENTIFICATION OF SEQUENCES OF HUMAN HERPES VIRUS-6 (HHV-6) IN A CASE OF HODGKINS-DISEASE BY POLYMERASE CHAIN-REACTION [Articolo su rivista]
Marasca, Roberto; Luppi, Mario; Montorsi, M; Fancinelli, M; Sabbatini, R; Mariano, Mt; Selleri, L.
abstract

In the last decades new approaches to the diagnosis and treatment of Hodgkin's Disease (HD) have contributed to improved rates of survival and cure but the pathogenesis of the disease still remains unknown. Data have been collected suggesting a relationship between viral infections and HD. HD patients with evidence of Epstein-Barr virus genomes in their affected tissues have been recently reported. Human Herpes Virus six (HHV-6) is a newly isolated virus, derived from patients with lymphoproliferative disorders. In order to investigate the possible role of this virus in the pathogenesis of HD we looked for a specific segment of HHV-6 genome by means of the polymerase chain reaction (PCR) in tissue samples obtained from peripheral blood and lymphnodes in HD patients: one clearly positive case has been identified. This result is the first indication of HHV-6 sequences associated with a case of HD and raises the possibility that this virus might be involved in the pathogenesis of this lymphoproliferative disorder. The relationship between HHV-6 and HD therefore warrants further investigation.

1990 - MITOCHONDRIAL-DNA DELETION IN A PATIENT WITH PROGRESSIVE OPHTHALMOPLEGIA [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Sola, P; Corradi, M; Fancinelli, M; Montorsi, M; Manfredini, Rossella; Selleri, L.
abstract

Mitochondria are unique among intracellular organelles because they contain their own DNA, which can be transcribed and translated to form proteins. Mitochondrial diseases include myopathies and multisystem disorders. The case of a patient showing bilateral ophthalmoplegia with proximal limb weakness, severe dysphagia and short stature, without family history, is described. The analysis of mitochondrial DNA of the patient muscle revealed a deleted form accounting for 65% of the total mitochondrial DNA. The Southern Blot Analysis of mtDNA allows a rather precise localization of deletions giving new insights in the pathogenesis of mitochondrial myopathies and representing a new precious diagnostic tool in these diseases.

1990 - SURVEY OF THE PRESENCE OF ANTI-HHV6 ANTIBODIES AND HHV6 SEQUENCES IN NON-HODGKIN LYMPHOMA CASES [Articolo su rivista]
Selleri, L; Marasca, Roberto; Luppi, Mario; Montorsi, M; Ceccherininelli, L; Bendinelli, M; Torelli, Giuseppe
abstract