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Monica CIVALLERO

Assegnista di ricerca
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2023 - The SALENTO prognostic model for limited-stage peripheral T-cell lymphoma from the International T-Cell Project Network [Articolo su rivista]
Hapgood, Greg; Civallero, Monica; Stepanishyna, Yana; Vose, Julie M; Cabrera, Maria Elena; Advani, Ranjana H; Pileri, Stefano A; Manni, Martina; Horwitz, Steven M; Foss, Francine M; Hitz, Felicitas; Radford, John; Dlouhy, Ivan; Chiattone, Carlos Sérgio; Kim, Won-Seog; Skrypets, Tetiana; Nagler, Arnon; Trotman, Judith; Luminari, Stefano; Federico, Massimo
abstract

: The natural history of limited-stage peripheral T-cell lymphomas (PTCLs) remains poorly defined. We investigated outcomes and prognostic variables in patients registered in the T-Cell Project (TCP)(NCT01142674) to develop a model to predict overall survival (OS) for the common nodal PTCL subtypes (PTCL-NOS, AITL, ALCL). The model was validated in an independent data set from Australian and Brazilian registries. 211 patients registered in the TCP between 2006-2018 were studied. The median age was 59 years (range 18-88) and median follow-up was 49 months. 127 patients (78%) received anthracycline-based regimens, 5 patients (3%) radiotherapy alone (RT), 24 patients (15%) chemotherapy+RT. 5-year OS and PFS were 47% and 37%, respectively. Age >60y, elevated LDH and low serum albumin were independent prognostic factors. The model identified three groups with low- (26%, score 0), intermediate- (41%, score 1), and high-risk (33%, score 2-3) with 5-yr OS of 78% [95% CI 29-127], 46% [95% CI 24-68], and 25% [95% CI 20-30], respectively (P<0·001) and 5-yr PFS of 66% [95% CI 33-99], 37% [95% CI 9-65], and 17% [95% CI 9-25], respectively (P<0·001). The model demonstrated greater discriminatory power than established prognostic indices and an analogous distribution and outcomes in the three groups in the validation cohort of 103 patients. The SALENTO Model (Limited Stage Peripheral T Cell Lymphoma Prognostic Model) is an objective, simple and robust prognostic tool. The high-risk group has poor outcomes, comparable to advanced stage disease, and should be considered for innovative first-line approaches.

2022 - COVID-related disruption in mammographic screening: a year later. [Articolo su rivista]
Toss, A; Callegari, V; Cortesi, G; Civallero, M; Armocida, C; Piacentini, F.
abstract

2022 - Characteristics and clinical outcomes of patients with ALK-positive anaplastic large cell lymphoma: Report from the prospective international T-cell lymphoma project [Articolo su rivista]
Chiattone, C.; Civallero, M.; Fischer, T.; Miranda, E.; Manni, M.; Zing, N. P. C.; Pileri, S. A.; Montoto, S.; Horwitz, S. M.; Cabrera, M. E.; De Souza, C. A.; Nagler, A.; Luminari, S.; Ferreri, A. J. M.; Carson, K. R.; Re, A.; Rigacci, L.; Nassi, L.; Stepanishyna, Y.; Federico, M.; Inghirami, G.
abstract

The T-cell Lymphoma Project is an international registry prospective study that enrolled patients with newly diagnosed peripheral T-cell and NK-cell lymphomas (PTCL). The main objective was to define the clinical features and outcomes, establishing a robust benchmark for future clinical trials. Seventy-four institutions from 14 countries in North America, South America, Europe, and Asia collected data on patients diagnosed and treated at their respective centers between September 2006 and February 2018. Among 1553 PTCL patients, 131 (8.4% of the total cohort) were confirmed to have anaplastic large cell lymphoma - kinase positive (ALCL, ALK+). The median age of the patients was 39 years (18–84). Sixty-five patients (66%) had advanced-stage disease, although majority (45 patients, 54%) had a low-risk International Prognostic Index (IPI) score (0–1). Of 97 patients treated with chemotherapy, 97% received anthracycline-containing regimens. The overall response rate was 81%, with 69 patients (70%) achieving complete remission. Estimated OS and PFS at 3 years were 77% (95% CI: 54%–99%) and 68% (95% CI: 46%–90%), respectively, and at 5 years were very similar, 77% of OS (95% CI: 62%–92%) and 64% of PFS (95% CI: 34%–94%). Multivariate analysis for PFS showed advanced stage (hazard ratios [HR]: 4.72, 95% CI: 1.43–23.9, p = 0.015), elevated lactate dehidrogenade (LDH) (HR 4.85; 95% CI: 1.73–13.60, p = 0.001), and Eastern Cooperative Oncology Group Performance Status scale (ECOG-PS) ≥2 (HR: 5.25; 95% CI: 1.68–16.4, p = 0.024). For OS, elevated LDH (HR: 3.77; 95% CI: 1.98–14.17, p = 0.014) and ECOG-PS ≥2 (HR: 4.59; 95% CI: 1.46–14.39, p = 0.004) were identified. In summary, although the outcome of ALK+ ALCL is superior to that of other PTCLs, it remains sufficiently favorable, given the young median age of the patients. Our results confirm the usefulness of both IPI and Prognostic Index for T-cell Lymphoma (PIT) in identifying groups of patients with different outcomes. Clinical Trials ID: NCT01142674.

2022 - Predictive factors for relapse in triple-negative breast cancer patients without pathological complete response after neoadjuvant chemotherapy [Articolo su rivista]
Toss, A.; Venturelli, M.; Civallero, M.; Piombino, C.; Domati, F.; Ficarra, G.; Combi, F.; Cabitza, E.; Caggia, F.; Barbieri, E.; Barbolini, M.; Moscetti, L.; Omarini, C.; Piacentini, F.; Tazzioli, G.; Dominici, M.; Cortesi, L.
abstract

IntroductionTriple-negative breast cancer (TNBC) patients who do not obtain pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) present higher rate of relapse and worse overall survival. Risk factors for relapse in this subset of patients are poorly characterized. This study aimed to identify the predictive factors for relapse in TNBC patients without pCR after NACT. MethodsWomen with TNBC treated with NACT from January 2008 to May 2020 at the Modena Cancer Center were included in the analysis. In patients without pCR, univariate and multivariable Cox analyses were used to determine factors predictive of relapse. ResultsWe identified 142 patients with a median follow-up of 55 months. After NACT, 62 patients obtained pCR (43.9%). Young age at diagnosis (<50 years) and high Ki-67 (20%) were signi!cantly associated with pCR. Lack of pCR after NACT resulted in worse 5-year event-free survival (EFS) and overall survival (OS). Factors independently predicting EFS in patients without pCR were the presence of multifocal disease [hazard ratio (HR), 3.77; 95% CI, 1.45-9.61; p=0.005] and residual cancer burden (RCB) III (HR, 3.04; 95% CI, 1.09-9.9; p=0.04). Neither germline BRCA status nor HER2-low expression were associated with relapse. DiscussionThese data can be used to stratify patients and potentially guide treatment decision-making, identifying appropriate candidates for treatment intensi!cation especially in neo-/adjuvant setting.

2021 - ALK-negative anaplastic large cell lymphoma: Features and outcomes of 235 patients from the International T-Cell Project [Articolo su rivista]
Shustov, A.; Cabrera, M. E.; Civallero, M.; Bellei, M.; Ko, Y. H.; Manni, M.; Skrypets, T.; Horwitz, S. M.; de Souza, C. A.; Radford, J. A.; Bobillo, S.; Prates, M. V.; Ferreri, A. J. M.; Chiattone, C.; Spina, M.; Vose, J. M.; Chiappella, A.; Laszlo, D.; Marino, D.; Stelitano, C.; Federico, M.; Savage, K.; Connors, J.; Gascoyne, R.; Chhanabhai, M.; Wilson, W.; Jaffe, E. S.; Armitage, J. O.; Weisenburger, D. D.; Anderson, J.; Ullrich, F.; Bast, M.; Hochberg, E.; Harris, N.; Smogorzews ka, A.; Levine, A.; Nathwani, B. N.; Miller, T.; Rimsza, L.; Montserrat, E.; Lopez-Guillermo, A.; Campo, E.; Cuadros, M.; Ferreira, J. A.; Delgado, B. M.; Holte, H.; Delabie, J.; Rudiger, T.; Muller-Hermelink, K.; Reimer, P.; Adam, P.; Wilhelm, M.; Schmitz, N.; Nerl, C.; Lister, A.; Norton, A.; Maclennan, K. A.; Zinzani, P. L.; Pileri, S. A.; Bellai, M.; Luminari, S.; Coiffier, B.; Berger, F.; Tanin, I.; Wannakrairot, P.; Au, W. Y.; Liang, R.; Loong, F.; Rajan, S.; Sng, I.; Tobinai, K.; Matsuno, Y.; Morishima, Y.; Nakamura, S.; Seto, M.; Tanimoto, M.; Yoshino, T.; Suzumiya, J.; Ohshima, K.; Kim, W. -S.
abstract

Anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (ALK- ALCL) is an aggressive neoplasm of T-cell/null-cell lineage. The T-Cell Project is a global prospective cohort study that consecutively enrolled patients newly diagnosed with peripheral T-cell lymphoma, registered through a centralized computer database between September 2006 and February 2018. Of 1553 validated cases from 74 sites in 13 countries worldwide, 235 were reported as ALK- ALCL. The median age at diagnosis was 54 years (range, 18-89 years), with a male predominance (62%). Stage III to IV disease was identified in 71% of patients, bulky disease and bone marrow involvement were uncommon, and 66% of patients presented with a low (0-1) International Prognostic Index score. Of all treated patients, 85% received multiagent initial chemotherapy, and 8% were consolidated with autologous hematopoietic cell transplantation. The initial overall and complete response rates were 77% and 63%, respectively. After a median follow-up of 52 months (95% confidence interval [CI], 41-63), the median progression-free survival (PFS) and overall survival (OS) were 41 months (95% CI, 17-62) and 55 months (95% CI, 36-75), respectively. The 3- and 5-year PFS rates were 52% and 43%, and the 3- and 5-year OS rates were 60% and 49%. Treatments containing both anthracycline and etoposide were associated with superior OS (P 5.05) but not PFS (P 5.18). In this large prospective cohort study, outcomes comparable to those previously reported in the retrospective International Peripheral T-Cell Lymphoma Project were observed. The study underscores the need for introducing novel platforms for ALK- ALCL and establishes a benchmark for future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT01142674.

2021 - Outcomes and prognostic factors in angioimmunoblastic T-cell lymphoma: final report from the international T-cell Project [Articolo su rivista]
Advani, R. H.; Skrypets, T.; Civallero, M.; Spinner, M. A.; Manni, M.; Kim, W. S.; Shustov, A. R.; Horwitz, S. M.; Hitz, F.; Cabrera, M. E.; Dlouhy, I.; Vassallo, J.; Pileri, S. A.; Inghirami, G.; Montoto, S.; Vitolo, U.; Radford, J.; Vose, J. M.; Federico, M.
abstract

Angioimmunoblastic T-cell lymphoma (AITL) is a unique subtype of peripheral T-cell lymphoma (PTCL) with distinct clinicopathologic features and poor prognosis. We performed a subset analysis of 282 patients with AITL enrolled between 2006 and 2018 in the international prospective T-cell Project (NCT01142674). The primary and secondary end points were 5-year overall survival (OS) and progression-free survival (PFS), respectively. We analyzed the prognostic impact of clinical covariates and progression of disease within 24 months (POD24) and developed a novel prognostic score. The median age was 64 years, and 90% of patients had advanced-stage disease. Eighty-one percent received anthracycline-based regimens, and 13% underwent consolidative autologous stem cell transplant (ASCT) in first complete remission (CR1). Five-year OS and PFS estimates were 44% and 32%, respectively, with improved outcomes for patients who underwent ASCT in CR1. In multivariate analysis, age ≥60 years, Eastern Cooperative Oncology Group performance status >2, elevated C-reactive protein, and elevated β2 microglobulin were associated with inferior outcomes. A novel prognostic score (AITL score) combining these factors defined low-, intermediate-, and high-risk subgroups with 5-year OS estimates of 63%, 54%, and 21%, respectively, with greater discriminant power than established prognostic indices. Finally, POD24 was a powerful prognostic factor with 5-year OS of 63% for patients without POD24 compared with only 6% for patients with POD24 (P < .0001). These data will require validation in a prospective cohort of homogeneously treated patients. Optimal treatment of AITL continues to be an unmet need, and novel therapeutic approaches are required.

2021 - The Value and relevance of T-cell lymphoma registries [Capitolo/Saggio]
Skrypets, Tetiana; Manni, Martina; Civallero, Monica; Kriachok, Iryna; Federico, Massimo
abstract

2020 - Citarinostat and Momelotinib co-target HDAC6 and JAK2/STAT3 in lymphoid malignant cell lines: a potential new therapeutic combination [Articolo su rivista]
Cosenza, M.; Civallero, M.; Marcheselli, L.; Sacchi, S.; Pozzi, S.
abstract

Histone deacetylase (HDAC) inhibitors represent an encouraging class of antitumor drugs. HDAC inhibitors induce a series of molecular and biological responses and minimal toxicity to normal cells. Citarinostat (Acy-241) is a second generation, orally administered, HDAC6-selective inhibitor. Momelotinib (CYT387) is an orally administered inhibitor of Janus kinase/signal transducer of transcription-3 (JAK/STAT3) signaling. Momelotinib showed efficacy in patients with myelofibrosis. We hypothesized that both HDAC and JAK/STAT pathways were important in lymphoproliferative disorders, and that inhibiting JAK/STAT3 and HDAC simultaneously might enhance the efficacy of momelotinib and citarinostat without increasing toxicity. Accordingly, we tested the citarinostat + momelotinib combination in lymphoid cell lines. Citarinostat + momelotinib showed strong cytotoxicity; it significantly reduced mitochondrial membrane potential, down-regulated Bcl-2 and Bcl-xL, and activated caspases 9 and 3. Caspase-8 was upregulated in only two lymphoid cell lines, which indicated activation of the extrinsic apoptotic pathway. We identified a lymphoid cell line that was only slightly sensitive to the combination treatment. We knocked down thioredoxin expression by transfecting with small interfering RNA that targeted thioredoxin. This knockdown increased cell sensitivity to the combination-induced cell death. The combination treatment reduced Bcl-2 expression, activated caspase 3, and significantly inhibited cell viability and clonogenic survival.

2020 - Incidence and outcomes of rare T cell lymphomas from the T Cell Project: hepatosplenic, enteropathy associated and peripheral gamma delta T cell lymphomas [Articolo su rivista]
Foss, Francine M; Horwitz, Steven M; Civallero, Monica; Bellei, Monica; Marcheselli, Luigi; Kim, Won Seog; Cabrera, Maria E; Dlouhy, Ivan; Nagler, Arnon; Advani, Ranjana H; Pesce, Emanuela A; Ko, Young-Hyeh; Montoto, Silvia; Chiattone, Carlos; Moskowitz, Alison; Spina, Michele; Cesaretti, Marina; Biasoli, Irene; Federico, Massimo
abstract

The T Cell Project was the largest prospective trial to explore the incidence, treatment patterns, and outcomes for T cell lymphomas. The rare subtypes of T cell lymphomas, including hepatosplenic T cell lymphoma (HSTCL), enteropathy associated T cell lymphoma (EATL), and peripheral gamma delta T cell lymphomas (PGDTCLs) are poorly represented in most studies and there is little data regarding treatment patterns. We report results from 115 patients with hepatosplenic (n = 31), enteropathy associated (n = 65), and PGDTCLs (n = 19). While anthracycline regimens were most commonly used as first line therapy, response rates ranged from 20%-40% and were suboptimal for all groups. Autologous stem cell transplantation was performed as a consolidation in first remission in a small number of patients (33% of HSTCL, 7% of EATL, and 12% of PGDTCL), and four patients with HSTCL underwent allogeneic stem cell transplantation in first remission. The progression free survival at 3 years ranged from 28%-40% for these rare subtypes, and the overall survival at 3 years was most favorable for PGDTCL (70%). These data highlight the need for novel treatment approaches for rare subtypes of T cell lymphomas and for their inclusion in clinical trials.

2020 - Survival outcomes for extranodal natural-killer T-cell lymphoma – Authors' reply [Abstract in Rivista]
Fox, C. P.; Civallero, M.; Federico, M.; Kim, W. S.
abstract

2020 - Survival outcomes of patients with extranodal natural-killer T-cell lymphoma: a prospective cohort study from the international T-cell Project [Articolo su rivista]
Fox, C. P.; Civallero, M.; Ko, Y. -H.; Manni, M.; Skrypets, T.; Pileri, S.; Kim, S. J.; Cabrera, M. E.; Shustov, A. R.; Chiattone, C. S.; Horwitz, S. M.; Dlouhy, I.; Spina, M.; Hitz, F.; Montoto, S.; Nagler, A.; Martinez, V.; De Souza, C. A.; Fernandez-Alvarez, R.; Ballova, V.; Gabus, R.; Inghirami, G.; Federico, M.; Kim, W. S.
abstract

Background: Extranodal natural killer (NK) T-cell lymphoma (ENKTL) is a unique clinicopathological entity, typically associated with poor survival outcomes. Most published data have come from east Asian study groups, with little information available from international cohorts. The effects of treatment advances on routine clinical practice across continental territories has not been clear. We aimed to improve understanding of the clinical characteristics and outcomes of patients with ENKTL. Methods: We did a substudy of patients with ENKTL from the T-cell Project, a global prospective cohort study. The T-cell Project registered consecutively diagnosed adults (>18 years) with newly diagnosed, untreated mature T-cell or NK lymphomas (WHO 2001 or 2008 classifications) from 74 centres in 13 countries (in Asia, Europe, North America, and South America). In total, 1695 patients with mature T-cell or NK lymphomas were enrolled between Oct 12, 2006 and Feb 28, 2018 in the T-cell Project. The first patient with ENKTL was enrolled on Feb 15, 2007, and the last on May 26, 2017. Data on baseline characteristics, first-line treatment, treatment response, and survival outcomes were recorded in a central database (locked March 30, 2019). The primary outcome was 5-year overall survival. The T-cell Project is registered on ClinicalTrials.gov, NCT01142674. Findings: 166 patients were diagnosed with ENKTL, comprising 11% of 1553 eligible registered cases and distributed across 40 participating centres in four continents. At a median follow-up of 44 months (IQR 20–61), overall survival at 5 years was 54% (95% CI 44–63) in patients with nasal disease (n=98) and 34% (27–46) in patients with extranasal disease (n=68). Interpretation: To our knowledge, this study presents the largest international cohort of patients with ENKTL. We describe a clinically significant improvement in the survival of patients with ENKTL treated in routine clinical practice over the past decade, likely to be attributable to the increasing use of treatment protocols specific for ENKTL. Funding: The Fondazione Cassa di Risparmio di Modena, the Associazione Angela Serra per la Ricerca sul Cancro, the Fondazione Italiana Linfomi, Allos Therapeutics, Spectrum Pharmaceuticals, Associazione Italiana per la Ricerca sul Cancro, and the National Cancer Institute at the National Institutes of Health.

2018 - Cell-penetrating CaCO3nanocrystals for improved transport of NVP-BEZ235 across membrane barrier in T-cell lymphoma [Articolo su rivista]
Vergaro, Viviana; Civallero, Monica; Citti, Cinzia; Cosenza, Maria; Baldassarre, Francesca; Cannazza, Giuseppe; Pozzi, Samantha; Sacchi, Stefano; Fanizzi, Francesco Paolo; Ciccarella, Giuseppe
abstract

Owing to their nano-sized porous structure, CaCO3nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

2017 - Ricolinostat, a selective HDAC6 inhibitor, shows anti-lymphoma cell activity alone and in combination with bendamustine [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Marcheselli, Luigi; Sacchi, Stefano; Pozzi, Samantha
abstract

Histone deacetylase inhibitors (HDACis) have emerged as a new class of anticancer agents, targeting the biological process including cell cycle and apoptosis. We investigated and explained the anticancer effects of an HDAC6 inhibitor, ricolinostat alone and in combination with bendamustine in lymphoma cell lines. Cell viability was measured by MTT assay. Apoptosis, reactive oxygen species (ROS) generation, Bcl-2 protein expression, cell cycle progression and tubuline expression were determined by flow cytometry. The effects of ricolinostat alone and in combination on the caspases, PI3K/Akt, Bcl-2 pathways, ER stress and UPR were assessed by immunoblotting. Ricolinostat shows anti lymphoma activity when used as single agent and its capability to induce apoptosis is synergistically potentiated by the bendamustine in lymphoma cell lines. Drug combination reduced the proportion of cells in the G0/G1and S phases and caused an increase of “sub-G0/G1” peak. The synergistic effect accompanied with the increased ROS, activation of caspase-8, -9, and -3, the cleavage of PARP and modulated by Bcl-2 proteins family. In addition, the exposure of ricolinostat induced the acetylation level of α-tubulin, the extend of which was not further modified by bendamustine. Finally, the apoptosis effect of ricolinostat/bendamustine may be mediated by a corresponding effect on microtubule stabilization. Our data suggest that ricolinostat in combination with bendamustine may be a novel combination with potential for use as an antitumor agent in lymphoma.

2017 - Ruxolitinib combined with vorinostat suppresses tumor growth and alters metabolic phenotype in hematological diseases [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Pozzi, Samantha; Sacchi, Stefano
abstract

JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms.

2016 - The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Fiorcari, Stefania; Pozzi, Samantha; Marcheselli, Luigi; Bari, Alessia; Ferri, Paola; Sacchi, Stefano
abstract

We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, −9, −3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.

2015 - Activity of BKM120 and BEZ235 against lymphoma cells [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Pozzi, Samantha; Bari, Alessia; Ferri, Paola; Sacchi, Stefano
abstract

Non-Hodgkin lymphomas encompass a heterogeneous group of cancers, with 85-90% arising from B lymphocytes and the remainder deriving from T lymphocytes or NK lymphocytes. These tumors are molecularly and clinically heterogeneous, showing dramatically different responses and outcomes with standard therapies. Deregulated PI3K signaling is linked to oncogenesis and disease progression in hematologic malignancies and in a variety of solid tumors and apparently enhances resistance to antineoplastic therapy, resulting in a poor prognosis. Here, we have evaluated and compared the effects of the pan-PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitor BEZ235 onmantle, follicular, and T-cell lymphomas. Our results suggest that BKM120 and BEZ235 can effectively inhibit lymphoma cell proliferation by causing cell cycle arrest and can lead to cell death by inducing apoptosis and autophagy mediated by ROS accumulation. Despite great advances in lymphoma therapy after the introduction of monoclonal antibodies, many patients still die from disease progression. Therefore, novel treatment approaches are needed. BKM120 and BEZ235 alone and in combination are very effective against lymphoma cells in vitro. If further studies confirm their effectiveness in animal models, they may be promising candidates for development as new drugs.

2015 - Calcium-Carbonate Nanocapsules Improve the Efficacy of BEZ235 in Lymphoma a Cell Line: A Promising New Technology of Drug Delivery [Abstract in Rivista]
Civallero, Monica; Vergaro, Viviana; Citti, Cinzia; Cosenza, Maria; Cannazza, Giuseppe; Parenti, Carlo; Bari, Alessia; Ciccarella, Giuseppe; Sacchi, Stefano; Pozzi, Samantha
abstract

Nanotechnology is a promising branch of the medical field, directed to improve diagnostic and therapeutics strategies, applying nanovectors as drug delivery systems. Efficient encapsulation of anticancer drugs in nanocolloids and microcapsules was recently developed by G. Ciccarella research group (1). Based on our collaboration with the Nantional Nanotechnology Laboratory of the University of Salento and our previous experience with target therapies, we encapsulated BEZ235, a phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR). BEZ235 efficiently blocks the dysfunctional activation of the PI3K/mTOR pathway in cellular and in vivo settings, thus inhibiting the growth and proliferation of various cancer cells, and phase I/II clinical trials were open in solid cancer. However the scarse solubility limited further development of this promising compound. In order to overcome the solubility issue BEZ235-loaded nanocapsules were generated by the stepwise adsorption of oppositely charged polyelectrolytes into biocompatible CaCO3 cores. First nanocapsules were tested for biocompatibility. The exposition of lymphoma cell lines to empty nanocapsules up to 48 hours, did not induce any cititoxicity, confirming their biocompatibility. Second, encapsulated BEZ235 was compared with free-drug to test the cytotoxicity in a T lymphoma cell line (HUT78) by MTT assay. The results suggested that nanoencapsulated-BEZ235 was extremely efficient compared with free-BEZ235, reaching IC50 just after 5 hours of exposure compared with an IC65% at 48 hours with the free drug. A validated LC-MS/MS method was developed in order to quantify intracellular concentration of BEZ235 over time. Intracellular concentration of BEZ235 in the lymphoma cell line was consistent with biological results since the internalization kinetic and efficiency was increased by the coating. In order to confirm that the encapsuled-BEZ235 was still effective on cell apoptosis, we tested free BEZ and encapsulated BEZ235 at a concentration of 1µM in T cell lymphoma cell lines. Encapsulated-BEZ235 induced apoptosis evidenced by the cleavage of caspase 8, 9 and 3 at an earlier time point compared with free BEZ235 and at significantly lower concentration. We also confirmed that the encapsulated-BEZ235 maintained its effect on the target mTOR/AKT pathway: p-AKT was dephosphorylated at 5h while the free BEZ235 operates at least after 24 hours at concentrations 100 times higher, as previously demonstrated. Keeping in mind a future clinical application of these polymeric particles/capsules, our data can be regarded as a promising new nanotechnology-based strategy to improve the efficacy and bioavailability of old and new drugs. Functional biological studies of BEZ235-encapsulated carrier and its mechanism of internalization are already under way, and animal in vivo studies to evaluated toxicity and distribution of the nanocapsuled compound are ongoing.

2015 - The combination of bortezomib with enzastaurin or lenalidomide enhances cytotoxicity in follicular and mantle cell lymphoma cell lines [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Pozzi, Samantha; Marcheselli, Luigi; Bari, Alessia; Sacchi, Stefano
abstract

We analyzed the combination of a proteasome inhibitor (bortezomib) with enzastaurin (PKC/AKT-inhibitor) or lenalidomide (immunomodulatory agent) for the inhibition of proliferation and induction of apoptosis in B-cell lymphoma cell lines and primary malignant cells. The effects of bortezomib, enzastaurin or lenalidomide, alone or in combinations, on cell viability and apoptosis were evaluated using the Cell Proliferation Kit and flow cytometry analysis. The interaction between drugs was examined by the Chou-Talalay method. Cell cycle analysis was performed by flow cytometry. The PI3K/AKT, PKC and MAPK/ERK signaling pathways were analyzed using western blot. Bortezomib with either enzastaurin or lenalidomide synergistically induced anti-proliferative and pro-apoptotic effects in B-cell lymphoma cells, even in the presence of the bone marrow microenvironment. The direct cytotoxicity is mediated by signaling events involving the PI3K/AKT, PKC and MAPK/ERK pathways leading to cell death. The significant increase of apoptosis was mediated by an increased ratio of pro-apoptotic proteins (Bax, Bad and Bim) to anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), triggering the cleavage of caspases -3, -9, -8 and PARP. Further evaluation of the combination of bortezomib with enzastaurin or lenalidomide for the treatment of B-cell lymphoma is warranted, with the goal to improve the quality of life and survival of patients. Copyright © 2014 John Wiley & Sons, Ltd.

2012 - Combination of low doses of Enzastaurin and Lenalidomide has synergistic activity in B-non-Hodgkin lymphoma cell lines [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Grisendi, Giulia; Marcheselli, Luigi; Roat, Erika; Bari, Alessia; Sacchi, Stefano
abstract

Less toxic and more active treatments are needed for indolent lymphomas as there is no curative treatment, and patients eventually die due to complications related to their disease. The purpose of the present study was to assess the antitumour activity of the combination of low doses of Enzastaurin and Lenalidomide (Revlimid) on B-lymphoma cell lines. The combination of Enzastaurin and Lenalidomide, at doses as low as 1 μM, showed strong synergism against indolent lymphomas by reducing cell growth, producing an increase in G0-G1 phase followed by significant decrease in S phase, increasing apoptosis, and inhibiting PI3K/AKT, PKC and MAPK/ERK pathways. These preclinical findings, together with promising results obtained with Lenalidomide for the treatment of non-Hodgkin lymphoma, suggest that further evaluation of the combination of Enzastaurin and Lenalidomide for the treatment of indolent lymphomas is warranted. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents, causing severe side effects, and could be considered an example of a new innovative attempt of an anti-cancer 'soft treatment'.

2012 - NVP-BEZ235 alone and in combination in mantle cell lymphoma: an effective therapeutic strategy [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Marcheselli, Luigi; Pozzi, Samantha; Sacchi, Stefano
abstract

Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma; the complete response rate for standard therapies in use today is 85 - 90%. NVP-BEZ235 inhibits the PI3K/Akt/mTOR signaling axis at the level of both PI3K and mTOR. In this study, we analyzed the inhibitory effects of NVP-BEZ235 on mantle cell lines and its effects in combination with enzastaurin, everolimus and perifosine.

2011 - Genomic profiling of enzastaurin-treated B cell lymphoma RL cells [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Neri, Antonino; Bari, Alessia
abstract

No abstract is available for this article.

2011 - Rational combinations of enzastaurin with novel targeted agents for patients with B-cell non-Hodgkin's lymphoma [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Bari, Alessia; Sacchi, Stefano
abstract

Non-Hodgkin’s lymphoma (NHL) is the most common hematologic malignant neoplasm in adults. Combination chemotherapy regimens have been the main-stay of treatment for NHL for several decades. In the 1990s, the introduction of rituximab marked the beginning of the era of immunotherapy with monoclonal antibodies and revolutionized the treatment of B-cell NHL (B-NHL). Chemotherapy combined with anti-CD20 monoclonal antibodies has improved survival in both indolent and aggressive B-NHL; this combination has become the standard of care for these patients. However, a substantial subset of patients does not achieve a cure or long-term disease remission. In recent years, advances in the knowledge of NHL biology have improved our understanding of cell growth, proliferation and cell death in malignant cells. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future B-NHL therapies. Protein kinase C beta (PKC-b), a serine/threonine kinase, is involved in several signal transduction pathways, from differentiation and cell growth to survival and cell migration. It has been shown that PKC-b isoverexpressed in 20-25% of diffuse large B-cell lymphomas (DLBCLs) at diagnosis and 90% of DLBCLs at relapse. Therefore, PKC represents a potential targeted therapy for lymphomas. Enzastaurin (LY317615.HCl), an acyclic bisindolylmaleimide,is one of several new molecules directed against PKC-b. Enzastaurin is an ATP-competitive selective inhibitor of PKC. Enzastaurin suppresses the phosphoinositide 3-kinase (PI3K)/acutely transforming retrovirus (AKT) pathway,which blocks the phosphorylation of glycogen synthase kinase 3 beta (GSK3-b), mammalian target of rapamycin (mTOR) and ribosomal protein S6. It also suppresses cyclin D1 synthesis, induces dephosphorylation of p90 and ribosomal S6 kinase (RSK), regulates the MAPK pathway and seems to be involved inthe interferon-regulated JAK/STAT pathways. Furthermore, it affects BAD, a pro-apoptotic member of the Bcl-2 family proteins, which is particularly important in lymphoma.Based on promising preclinical results and the good tolerability profile, enzastaurin has been introduced in clinical trials as a treatment for patients. Enzastaurin has been used alone or in combination with other agents in more than 40 clinical trials (source: www.clinicaltrials.gov), and 9 of these focused on indolent or aggressive NHL.....

2010 - Effects of enzastaurin, alone or in combination, on signaling pathway controlling growth and survival of B-cell lymphoma cell lines. [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Grisendi, Giulia; Marcheselli, L; Todoerti, K; Sacchi, Stefano
abstract

AbstractImmunochemotherapies have improved outcomes in indolent lymphoma. However, response durations progressively shorten following each treatment, and the majority of patients eventually die from the disease. Thus, new, less toxic, and more active treatments are needed. Protein kinase C (PKC), which has been repeatedly implicated in B-cell lymphoma progression, may be a new target for lymphoma cell growth inhibition. Enzastaurin, a PKC-beta inhibitor, has toxic effects on a variety of cancer cells. The purpose of the present study was to assess the antitumor activity of enzastaurin on B-cell lymphoma cell lines and to investigate the underlying antitumor mechanisms. Enzastaurin induced apoptosis and inhibited phosphorylation of PKC, RSK, AKT, and downstream proteins. Moreover, our results reveal a new mechanism for enzastaurin-induced apoptosis via BAD activation. Finally, enzastaurin synergizes in its effects with chlorambucil and fludarabine, respectively. Taken together, our results strongly support clinical evaluation of enzastaurin in patients with B-cell lymphoma.

2009 - Molecular targeting of the PKC-β inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression [Articolo su rivista]
Verdelli, D; Nobili, L; Todoerti, K; Intini, D; Cosenza, Maria; Civallero, Monica; Bertacchini, Jessika; Deliliers, Gl; Sacchi, Stefano; Lombardi, L; Neri, A.
abstract

The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin-treated KMS-26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B-cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin-treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM.

2008 - Effects of Enzastaurin, Alone or in Combination, on Signalling Pathway Controlling Growth and Survival of B-Cell Lymphoma Cell LinesBlood (ASH Annual Meeting Abstracts), Nov 2008; 112: 4978 [Abstract in Rivista]
Sacchi, Stefano; Civallero, Monica; Cosenza, Maria; Grisendi, Giulia; Luigi, Marcheselli; Antonino, Neri
abstract

Background. Although, the recent introduction of Rituximab in combination with chemotherapy has improved outcome of patients with indolent lymphomas, in particular FL, these diseases are still considered incurable and the majority of patients still have a fatal course. Enzastaurin, an acyclic bisindolylmaleimide, is a potent and selective competitive inhibitor of protein kinase C beta, which has been shown to inhibit cell proliferation and angiogenesis in human cancer cell lines. The purpose of the present study was to test enzastaurin for its effects on proliferation and survival of B-cell lines established from NHL patients. Methods. The WSU-NHL and Karpas 422 were kindly provided Dr M Introna ( Division of Haematology, Ospedali Riuniti, Bergamo, Italy); the RL was purchased by DSMZ. All three lines are carrying the t(14; 18) and Karpas 422 is EBV+. We decided to conduct all the experiments under the optimal culture conditions with 10% FCS to avoid subjecting the cells to further stress stimuli. IC50 values were calculated from curves based on enzastaurin concentration ranging from 1 to 10µM using MTT assay and cell viability assessment by Trypan Blue exclusion. Cell apoptosis was assessed by flow cytometer after staining with Annexin V-FITC and propidium iodide. The effects of enzastaurin on caspases activation as well as on AKT phosphorilation were evaluated by Western blotting. We also investigated the interaction of enzastaurin with chlorambucil and fludarabine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the StaCorp8.2 software program based upon the Chou-Talalay method to determine if the combinations were additive or synergistic. Results. We found that enzastaurin alone inhibits the proliferation of B-cell lymphomas cell lines at IC50 values ranging from 5 to 7.5 µM after 48 h and from 2.5 to 3.2 µM after 72 h. Induction of apoptosis by enzastaurin evaluated on WSU-NHL and RL, by flow cytometry analysis of membrane permeability, showed that enzastaurin induces an increase in the percentage of apoptotic cells compared with untreated controls in a time-dependent fashion. Furthermore, enzastaurin induces the appearance of the cleaved caspase-3 fragment in the same cell lines in a time dependent fashion. Activation of the apoptotic pathway was confirmed by cleavage of the PARP enzyme. The apoptosis is partially prevented by the ZVAD–fmk broad caspase inhibitor. Phosphorilation status analysis of AKT up to 72h of treatment showed a decrease of AKT phosphorilation starting from 48h after treatment. We tested the effect of enzastaurin combined with chlorambucil and fludarabine,drugs that are active against B-cell lymphoma and we demonstrated that these agents enhanced the cytotoxicity triggered by enzastaurin in a dose-dependent fashion. A clear synergistic interaction (CI=0.87) appeared using low concentrations of the drugs and increased (CI=0.1) at high concentrations. Conclusions. Our data suggest that in B lymphoma cell lines carrying t(14; 18), enzastaurin elicits its antitumor effect suppressing AKT phosphorilation, inducing apoptosis and inhibiting proliferation. Furthermore, enzastaurin synergizes with chlorambucil and fludarabine. These results support the potential use of enzastaurin in patients with NHL, and in particular provide a rationale for combination with chlorambucil and fludarabine

2006 - The Oral PKC-ß Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway. [Abstract in Rivista]
Antonino, Neri; Marmiroli, Sandra; Pierfrancesco, Tassone; Luigia, Lombardi; Lucia, Nobili; Donata, Verdelli; Civallero, Monica; Cosenza, Maria; Jessika, Bertacchini; Federico, Massimo; DE POL, Anto; Giorgio Lambertenghi, Deliliers; Sacchi, Stefano
abstract

The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-ß inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-ß I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 µM to 12,5 µM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT. Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3ß, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin. In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 ß. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.

2005 - Biological effects of Atra and Arsenic Trioxide on short term cultures of non-M3 leukemic blasts [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Sacchi, Stefano; Marcheselli, Raffaella; Pozzi, Samantha
abstract

The efficacy of All-Trans Retinoic Acid (Atra) and Arsenic Trioxide (As2O3) in the treatment of Acute Promyelocytic Leukemia (APL) is well known. Further, these drugs inhibit cell growth and induce apoptosis in several cell lines, but few data are reported on leukemic blasts. The aim of this study was to evaluate the biological effects on non-M3 Acute Myeloid Leukemia (AML) cells. Blasts of six patients, after exposition to Atra and As2O3 were tested for growth inhibition, induction of apoptosis and change in cell cycle distribution, evaluating cell viability, percentage of apoptotic cells and of blasts positive for Ki-67 and BrdU. In the present study we demonstrated that either Atra or As2O3 inhibit leukemic cells proliferation by induction of apoptosis. The effects are time and dose dependent. We did not observe additive or synergistic effects with the combination of the drugs. Further, our results showed that Atra and As2O3 have effects on cell cycle distribution reducing S-phase and proliferating cells. These results should be taken in to account preparing future laboratory and clinical experimental protocols that associate these drugs with antineoplastic agents with different cell cycle specificity.