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SILVIA MARTINELLI

COLLABORATORE DI RICERCA
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

2023 - Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms [Abstract in Rivista]
Norfo, Ruggiero; Carretta, Chiara; Parenti, Sandra; Badii, Filippo; Bertesi, Matteo; Rontauroli, Sebastiano; Tavernari, Lara; Genovese, Elena; Sperduti, Samantha; Enzo, Elena; Mirabile, Margherita; Pedrazzi, Francesca; Pessina, Chiara; Colugnat, Ilaria; Mora, Barbara; Maccaferri, Monica; Tenedini, Elena; Martinelli, Silvia; Bianchi, Elisa; Casarini, Livio; Potenza, Leonardo; Luppi, Mario; Tagliafico, Enrico; Guglielmelli, Paola; Simoni, Manuela; Passamonti, Francesco; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

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.

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 - 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.

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 - 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.

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.

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 - 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.

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.

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 - 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 - 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.

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.

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.

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 - 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.

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 - 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.

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 - 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.

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.