Annalisa Chiarenza

Analysis of the coding genome of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. Although a number of structural alterations have been associated with the pathogenesis of this malignancy, the full spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains, on average, more than 30 clonally represented gene alterations per case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2; 24% of samples) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

B-cell non-Hodgkin’s lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types—follicular lymphoma and diffuse large B-cell lymphoma—harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin’s lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Rituximab Maintenance Compared With Observation After Brief First-Line R-FND Chemoimmunotherapy With Rituximab Consolidation in Patients Age Older Than 60 Years With Advanced Follicular Lymphoma: A Phase III Randomized Study by the Fondazione Italiana Linfomi

PURPOSEnTo evaluate the efficacy of rituximab maintenance in 60- to 75-year-old patients with advanced follicular lymphoma responding to brief first-line chemoimmunotherapy followed by rituximab consolidation.nnnPATIENTS AND METHODSnA total of 234 treatment-naive 60- to 75-year-old patients began chemoimmunotherapy with four monthly courses of rituximab, fludarabine, mitoxantrone, and dexamethasone (R-FND) followed by four weekly cycles of rituximab consolidation. Of these, 210 patients completed the planned treatment, and 202 responders were randomly assigned to rituximab maintenance (arm A) for 8 months, once every 2 months for a total of four doses, or to observation (arm B).nnnRESULTSnMedian ages in arms A and B were 66 and 65 years, respectively. After induction and consolidation therapy, the overall response rate was 86%, with 69% complete remissions (CR). After a 42-month median follow-up from diagnosis, 3-year progression-free survival (PFS; the primary end point) and overall survival (OS) were 66% (95% CI, 59% to 72%) and 89% (95% CI, 85% to 93%), respectively. After randomization, 2-year PFS was 81% for rituximab maintenance versus 69% for observation, with a hazard ratio of 0.74 (95% CI, 0.45 to 1.21; P = .226), although this was not statistically significant. No differences between the two arms were detected for OS. Overall, the regimen was well-tolerated. The most frequent grade 3 to 4 toxicity was neutropenia (25% of treatment courses), with 13 infections. Two toxic deaths (0.8%) occurred during induction treatment.nnnCONCLUSIONnA brief R-FND induction plus rituximab consolidation achieved excellent results with high CR and PFS rates, supporting the feasibility of this regimen in patients older than 60 years. A short rituximab maintenance did not achieve a statistically significant PFS improvement over observation.

Circulating myeloid-derived suppressor cells correlate with clinical outcome in Hodgkin Lymphoma patients treated up-front with a risk-adapted strategy

In the attempt to find a peripheral blood biological marker that could mirror the dysregulated microenvironment of Hodgkin Lymphoma (HL), we analysed the amount of myeloid‐derived suppressor cells (MDSC), including the three main sub‐types (monocytic, granulocytic and CD34 + fraction). The absolute MDSC count was investigated in 60 consecutive newly diagnosed HL patients and correlated with clinical variables at diagnosis and outcome. Patients received standard‐of‐care chemotherapy with the exception of interim fluorodeoxyglucose positron emission tomography (PET‐2)‐positive patients, who were switched early to a salvage regimen. All MDSC subsets were increased in HL patients compared to normal subjects (P < 0·0001) and were higher in non‐responders. However, a strong prognostic significance was limited to immature (CD34+) MDSC. A cut‐off level of 0·0045 × 109/l for CD34+MDSC resulted in 89% (95% confidence interval [CI] 52–99%) sensitivity and 92% (95% CI 81–98%) specificity. The positive predictive value to predict progression‐free survival was 0·90 for PET‐2 and 0·98 for CD34+MDSC count; the negative predictive value was 0·57 for PET‐2 and 0·73 for CD34+MDSC. PFS was significantly shorter in patients with more than 0·0045 × 109 CD34+MDSC cells/l at diagnosis and/or PET‐2 positivity (P < 0·0001). In conclusion, all circulating MDSC subsets are increased in HL; CD34+MDSC predict short PFS, similarly to PET‐2 but with the advantage of being available at diagnosis.

Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC)

Granulocytic-Myeloid-derived suppressor cells (G-MDSC) are increased in Multiple Myeloma (MM) patients but the mechanisms of G-MDSC generation are still unknown. There are many evidences of the role of mesenchymal stem cells (MSC) in promoting MM cell growth, survival and drug-resistance. We here used a specific experimental model in vitro to evaluate the ability of MSC to induce G-MDSC. We found that although MSC derived from healthy donors (HD), MGUS and MM were able to generate the same amount of MDSC, only MM-MSC-educated G-MDSC exhibited suppressive ability. In addition, in comparison with MSC derived from HD, MM-MSC produce higher amount of immune-modulatory factors that could be involved in MDSC induction. Compared to G-MDSC obtained from co-culture models with MSC from healthy subjects, both MGUS and MM-MSC-educated G-MDSC showed increase of immune-modulatory factors. However, only MM-MSC educated G-MDSC 1) up-regulated immune-suppressive factors as ARG1 and TNFα, 2) expressed higher levels of PROK2, important in angiogenesis and inflammatory process, and 3) showed ability to digest bone matrix. Our data demonstrate that MM-MSC are functionally different from healthy subjects and MGUS-MSC, supporting an evolving concept regarding the contribution of MM-MSC to tumor development and progression.

Salvage Therapy of Multiple Myeloma: The New Generation Drugs

During the past decade, overall results of treatment of multiple myeloma (MM) have been improved and survival curves are now significantly better with respect to those obtained with historical treatment. These improvements are linked to a deeper knowledge of the biology of disease and to the introduction in clinical practice of drugs with different mechanism of action such as proteasome inhibitors and immunomodulatory drugs (IMiDs). However, MM remains in most cases an incurable disease. For patients who relapse after treatment with novel agents, the prognosis is dismal and new drugs and therapeutic strategies are required for continued disease control. In this review, we summarize new insights in salvage therapy for relapsed/refractory MM as emerging from recent clinical trials exploring the activity of bendamustine, new generation proteasome inhibitors, novel IMiDs, monoclonal antibodies, and drugs interfering with growth pathways.

Endoscopic features of gastro-intestinal lymphomas: From diagnosis to follow-up

Many progresses have been done in the management of gastrointestinal (GI) lymphomas during last decades, especially after the discovery of Helicobacter pylori-dependent lymphoma development. The stepwise implementation of new endoscopic techniques, by means of echoendoscopy or double-balloon enteroscopy, enabled us to more precisely describe the endoscopic features of GI lymphomas with substantial contribution in patient management and in tailoring the treatment strategy with organ preserving approaches. In this review, we describe the recent progresses in GI lymphoma management from disease diagnosis to follow-up with a specific focus on the endoscopic presentation according to the involved site and the lymphoma subtype. Additionally, new or emerging endoscopic technologies that have an impact on the management of gastrointestinal lymphomas are reported. We here discuss the two most common subtypes of GI lymphomas: the mucosa-associated lymphoid tissue and the diffuse large B cell lymphoma. A general outline on the state-of-the-art of the disease and on the role of endoscopy in both diagnosis and follow-up will be performed.

Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application.

BackgroundTP53 defects, i.e. 17p13 deletion and/or nucleotide mutations, associate with short survival and chemorefractoriness in chronic lymphocytic leukemia (CLL). In this context, since direct sequencing of the TP53 gene does not evaluate TP53 functionality, a functional assessment of TP53 pathway may be of interest to identify high risk CLL. By taking advantage of a training cohort of 100 CLL and a validation cohort of 40 CLL with different patterns of TP53 mutation/deletion by FISH and sequencing, we propose an in-vitro assay in which the modulation of TP53 protein and CDKN1A mRNA were investigated upon 24-hour exposure of CLL cells to Nutlin-3.MethodsThe functional assay was set-up on cell lines recapitulating all TP53 genotypes (EHEB, TP53wt/wt; RAJI, TP53mut/wt; MEC-1 and MAVER1, TP53mut/del; HL-60, TP53del/del) and evaluated in two multi-institutional cohorts, purposely enriched in CLL bearing TP53 disruption: a training cohort of 100 cases and a validation cohort of 40 cases, both characterized by FISH and TP53 direct sequencing. Cells were exposed to 10xa0μM Nutlin-3 for 24xa0hours; TP53 accumulation was evaluated by Western blotting; TP53 transcriptional activity was determined by quantitative realtime PCR (qRT-PCR) of the TP53 target gene CDKN1A.ResultsAccording to TP53 protein modulation, in the training cohort we identified: i) 63 cases (51 TP53wt/wt, 12 TP53del/wt) with absence of basal TP53 and induction after treatment (normal pattern); ii) 18 cases (3 TP53mut/wt, 15 TP53mut/del) with high basal TP53 without increase after treatment (mutant pattern); iii) 19 cases (5 TP53mut/wt; 3 TP53mut/del; 11 TP53wt/wt) with basal TP53 that increases upon treatment (intermediate pattern). Evaluation of CDKN1A mRNA levels upon Nutlin-3 exposure showed that the 26 TP53 mutated (TP53mut/del or TP53mut/wt) cases had lower induction levels than the majority (57/63) of cases with normal pattern, and 10/12 cases with intermediate pattern without evidence of TP53 derangement by FISH and sequencing. These results were confirmed in the independent validation cohort of 40 cases (13 TP53wt/wt, 3 TP53del/wt, 12 TP53mut/del, 12 TP53mut/wt).ConclusionsThe proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.

Salvage therapy with pegylated liposomal doxorubicin, bortezomib, cyclophosphamide, and dexamethasone in relapsed/refractory myeloma patients

In vitro studies have shown synergistic anti‐myeloma effects of bortezomib combined with alkylating agents or anthracycline. We tested safety and efficacy of the combination of bortezomib, doxorubicin cyclophosphamide, and dexamethasone (ABCD) in the treatment of relapsed/refractory myeloma.

Mutations in the 3′ untranslated region of NOTCH1 are associated with low CD20 expression levels in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 coding region (coding NOTCH1 mutations) have been associated with impaired degradation of NOTCH1 protein[1][1],[2][2] and, clinically, with shorter time to first treatment, shorter overall survival,[1][1]–[3][3] and resistance to anti-