Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1767-1767
Author(s):  
Tomas Stopka ◽  
Jarmila Vargova ◽  
Karina Vargova ◽  
Nina Dusilkova ◽  
Vojtech Kulvait ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Differentially Expressed Genes ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Mrna Levels ◽  
Kinase Substrate ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases. In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells. In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker. Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. Disclosures No relevant conflicts of interest to declare.

Analysis of Mantle Cell Lymphoma Patients Reveals Novel Regulatory Circuits Involving MicroRNAs and Their Targets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4624-4624
Author(s):  
Vojtech Kulvait ◽  
Vit Pospisil ◽  
Karin Vargova ◽  
Marek Trneny ◽  
Pavel Klener ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Target Gene ◽  
Zinc Finger Protein ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Differentially Expressed ◽  
Regulatory Pathways ◽  
Mantle Cell

Abstract Abstract 4624 Mantle cell lymphoma (MCL) represents B-cell lymphoma derived from the mantle zone that surrounds normal germinal center follicles. Pathophysiology of this hardly curable disease involves t(11,14)(q13,q32) translocation which leads to upregulation of Cyclin D1(CCND1). Recently, microRNAs were demonstrated to significantly modify MCL pathogenesis (Jian-Jun Zhao et al. 2010) and therapy responsiveness (Jiang et al. 2010). In order to broaden our knowledge of regulatory pathways in MCL we searched for differentially expressed microRNAs and their differentially expressed mRNA targets between MCL samples and control samples. Samples consist of magnetically separated B cells derived from peripheral blood. We used 1) Microarray mRNA hybridization [Affymetrix Human Genome U133 Plus 2.0 Array, N(MCL)=5, N(control)=5] and 2) microRNA profiling [TaqMan® Array Human MicroRNA Card A v2.0 technology, N(MCL)=5, N(control)=5] followed by statistical analysis [limma (Smyth 2005)]. Differentially regulated targets of the deregulated microRNAs were selected from the databases involving both predicted (Betel et al. 2007) or confirmed (Hsu et al. 2010) target mRNAs. Among the most significant upregulated microRNAs (exceeding 10 fold) are miR-9, miR-124 and miR-183. We have found that upregulated miR-9 has confirmed downregulated target gene PRDM1 (PR domain zinc finger protein 1) which plays a role in B cell maturation (Turner et al. 1994) and may act as a tumor suppressor (Pasqualucci et al. 2006). Upregulation of miR-9 and downregulation of its targets was recently demonstrated in Burkitt lymphoma (Onnis A et al. 2010) and Hodgkin lymphoma (Nie K et al. 2008). Two additional upregulated microRNAs, miR-124 and miR-183, yet not associated with lymphomas, have downregulated target gene Integrin beta-1 (CD29) which regulates survival (Fukumori et al. 2003). Among most significant downregulated microRNAs is miR-101 (FC=-7). Downregulated microRNA miR-101 has known oncogene N-Myc (MYCN) as upregulated confirmed target and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) as upregulated target. Overexpression of a well known hematopoietic oncogene MYCN and epigenetic repressor DNMT3A may represent additional pathogenesis-related factors in MCL. Our data support importance of the candidate mechanisms involving microRNAs and their target programs in pathogenesis of MCL. They are currently extended on a larger patient cohort by analyzing expression and functional significance of the candidate microRNAs. (Grants: NT10310-3/2009, MPO FR-TI2/509, NPVII 2B06077, MSM 0021620806, LC 06044, SVV-2011-262507). Disclosures: No relevant conflicts of interest to declare.

CD5+ B-cell lymphoproliferative disorders: Beyond chronic lymphocytic leukemia and mantle cell lymphoma

2010 ◽  
Vol 34 (9) ◽  
pp. 1235-1238 ◽  
Author(s):  
Dragan Jevremovic ◽  
Roxana S. Dronca ◽  
William G. Morice ◽  
Ellen D. McPhail ◽  
Paul J. Kurtin ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Mantle Cell

Flavopiridol, Fludarabine and Rituximab Is a Highly Active Regimen in Indolent B-Cell Lymphoproliferative Disorders Including Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 944-944 ◽  
Author(s):  
Thomas S. Lin ◽  
Beth Fischer ◽  
Mollie E. Moran ◽  
Maureen M. Buckner ◽  
Roshini Shank ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Dose Escalation ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Stage Iii ◽  
Clinical Activity ◽  
Mantle Cell ◽  
Grade 3

Abstract The cyclin-dependent kinase inhibitor flavopiridol was inactive when given by 24–72-hr infusion, but 1-hr IV bolus dosing demonstrated clinical activity in mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Flavopiridol induces apoptosis independent of p53 and may be able to eliminate tumor cells resistant to fludarabine and rituximab. We performed a phase I dose escalation study of flavopiridol in combination with fludarabine and rituximab (FFR) in patients (pts) with MCL, CLL and indolent B-cell non-Hodgkin’s lymphoma (NHL). Pts had ANC ≥ 1500, hemoglobin ≥ 9.0, platelets ≥ 100,000, adequate organ function, and ECOG performance status 0–2, and provided informed consent. Pts received fludarabine 25 mg/m2 IV on day 1–5 and rituximab 375 mg/m2 on day 1 every 28 days for up to 6 cycles. The planned dose escalation of flavopiridol was 50 mg/m2 by 1-hr IV bolus on day 1 (cohort 1), day 1–2 (cohort 2), or day 1–3 (cohort 3) of each cycle. Pts were placed on prophylactic Bactrim and Valtrex. Growth factor support was prohibited. Twenty-one pts were enrolled and are evaluable for toxicity and response. Median age was 62 years (range, 43–81), and 10 pts were male. Pts had the following diagnoses: CLL (8), MCL (5), follicular lymphoma (FL; 4), small lymphocytic lymphoma (SLL; 3), and lymphoplasmacytic lymphoma (1). Nine pts had received 1-2 prior therapies; 12 pts were previously untreated. CLL pts were Rai stage III/IV (5) or required treatment for Rai stage I/II disease (3) by NCI 96 criteria. NHL pts were stage III/IV (10) or had progressive stage II disease (3). Three pts were treated in cohort 1, and dose limiting toxicity (DLT) was not observed. Six pts were treated in cohort 2. Two pts developed DLT; 1 pt developed grade 3 confusion and grade 3 seizures, and 1 pt developed nausea and diarrhea resulting in grade 3 acute renal failure. Three pts in cohort 2 did not receive flavopiridol after cycles 2, 2 and 3, due to life threatening tumor lysis in our single agent flavopiridol study. Twelve additional pts were enrolled at the cohort 1 dose level, to better define toxicity and efficacy. Pts received a median of 4 cycles (range 1–6), and 9 of 21 pts completed all 6 planned cycles. Therapy was stopped early due to cytopenias (7), infection (2), DLT (2) or progressive disease (1). One patient who received only 2 cycles of FFR due to cytopenias subsequently received 4 cycles of fludarabine and rituximab from his local oncologist. Response was graded by NCI 96 criteria (CLL) or IWG criteria (NHL). Overall response rate (ORR) was 90%; 15 pts achieved CR (71%), and 4 pts achieved PR (19%). Six pts relapsed a median of 7.5 months (range 4–18) after completing therapy; 13 pts remain in remission a median of 11 months (range 4–23) after completing therapy. Of note, all 9 MCL/FL pts responded (8 CR, 1 PR), and 8 pts remain in remission a median of 15 months (6–23) after finishing therapy. In conclusion, FFR exhibited significant clinical activity in a small group of pts, but cytopenias limited the administration of therapy. We are currently studying a modified FFR regimen administering a more active flavopiridol schedule (30-min IV bolus followed by 4-hr IV infusion) and allowing the use of prophylactic filgrastim, prior to phase II clinical study.

Flavopiridol, Fludarabine and Rituximab (FFR): An Active Regimen in Indolent B-Cell Lymphoproliferative Disorders and Mantle Cell Lymphoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1571-1571 ◽  
Author(s):  
Thomas S. Lin ◽  
Beth Fischer ◽  
Kristie A. Blum ◽  
Pierluigi Porcu ◽  
Eric H. Kraut ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Response Rate ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Mantle Cell ◽  
Grade 3

Abstract Background: The cyclin-dependent kinase inhibitor flavopiridol (alvocidib) induces p53-independent apoptosis and may be able to eliminate tumor cells resistant to fludarabine and rituximab. Study Design and Treatment: We report final results of a phase I dose escalation study of flavopiridol in combination with fludarabine and rituximab (FFR) in patients (pts) with mantle cell lymphoma (MCL), indolent B-cell non-Hodgkin’s lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL). Pts had ANC 3 1500, hemoglobin 3 9.0, platelets 3 100,000, adequate organ function, and ECOG performance status 0–2, and provided informed consent. Pts received fludarabine 25 mg/m2 IV on day 1–5 and rituximab 375 mg/m2 on day 1 every 28 days for up to 6 cycles. Flavopiridol was administered 50 mg/m2 by 1-hr IV bolus on day 1 (cohort 1, n=15) or day 1 and 2 (cohort 2, n=6) of each cycle. Based on promising results with a novel single agent dosing schedule in CLL, the study was amended to give flavopiridol by 30-min IV bolus followed by 4-hr IV infusion at a dose of 20 mg/m2 + 20 mg/m2 (cohort 3, n=3) or 30 mg/m2 + 30 mg/m2 (cohort 4, n=14) beginning with cycle 2. Pts were placed on prophylactic Bactrim and Valtrex. Growth factor support was allowed in cohorts 3 and 4. Results: Thirty-eight pts were enrolled. Median age was 62 years (range, 38–81), and 22 pts were male (58%). Pts had CLL (11), MCL (10), follicular (FL, 9), small lymphocytic (3), marginal zone (4) or lymphoplasmacytic lymphoma (1). Sixteen pts had received 1 or 2 prior therapies; 22 pts were previously untreated. Two of 6 pts in cohort 2 developed dose limiting toxicity; 1 pt developed grade 3 confusion and grade 3 seizures, and 1 pt developed nausea and diarrhea resulting in grade 3 acute renal failure. Fifteen pts were enrolled in cohort 1 and 14 pts were enrolled in cohort 4, to better define toxicity and efficacy. Pts received a median of 4 cycles (range 1–6), and 16 of 38 pts completed all 6 planned cycles. Cytopenias (10), fatigue (3), fever (2) and progression (2) were the most common reasons for early discontinuation of therapy. Response was graded by NCI 96 criteria (CLL) or IWG criteria (NHL). Overall response rate (ORR) was 82% (CR 50%, CRu 5%, PR 26%). Median progression-free survival (PFS) of responders was 25.5 months. ORR (82% vs. 81%), CR (50% vs. 50%) and median PFS (25.7 vs. 25.1 months) were similar for previously untreated and relapsed pts. Thirteen pts remain in remission with a median PFS of 33.5 months (range, 17.5–59.5), and 3 other pts died of unrelated causes. Eight of 10 MCL pts (median age 68, range 62–81) responded (7 CR, 1 PR). Two responders with blastoid variant MCL relapsed within 1 year, but median PFS of the other 6 responding MCL pts was 33.5 months. All 9 FL pts responded (5 CR, 2 CRu, 2 PR) with a median PFS of 25.1 months (range, 4.0–46.3). Conclusions: FFR exhibited significant clinical activity in indolent B-NHL, MCL and CLL. FFR was effective in both relapsed and previously untreated pts and showed promising clinical activity in older MCL pts. Changing from 1-hr IV bolus dosing to 30- min IV bolus followed by 4-hr IV infusion did not improve the response rate, suggesting that 1-hr IV bolus dosing may be effective when flavopiridol is given as part of combination chemotherapy. This regimen warrants further study.

Safety and Efficacy of Bendamustine with or without Rituximab in the Treatment of Heavily Pretreated Patients with Haematological Malignancies: A Multicenter Retrospective Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4945-4945 ◽  
Author(s):  
Luigi Rigacci ◽  
Benedetta Puccini ◽  
Alberto Bosi ◽  
Sergio Cortelazzo ◽  
Sergio Storti ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Large B Cell Lymphoma ◽  
Mantle Cell ◽  
Heavily Pretreated ◽  
Pretreated Patients ◽  
Large B Cell

Abstract Bendamustine is an purine analog alkylating agent with marked efficacy in haematological malignancies either when given as monotherapy or in combination with rituximab. The efficacy and safety of this drug was investigated in heavily pretreated patients (pts) with hematological malignancies. A total of 44 patients (median age 63 years ranging from 22–87) from 6 Italian centers treated with bendamustine alone or in combination with rituximab were analyzed in this retrospective study. The diagnoses were multiple myeloma (n=2), chronic lymphocytic leukemia or small lymphocytic lymphoma (n=19), diffuse large B cell lymphoma (n=7), follicular lymphoma (n=8), mantle cell lymphoma (n=4), marginal zone lymphoma (n=2), Hodgkin’s disease (n=1) and peripheral T cell lymphoma (n=1). All pts received bendamustine 60–90 mg/m2 at day 1+2, alone or in combination with rituximab 375 mg/m2 (n=35) at day 1 of each cycle given every 21 or 28 days. The pts were heavily pretreated with a median of 3 previous treatments (range 1–8); 37 pts had previously received rituximab and 9 pts had undergone autologous transplantation. Prior to receiving bendamustine, 14 pts had relapsed disease, 7 had refractory disease and 23 were progressing during therapy. The median number of bendamustine cycles was 3 (range 1–8); 11 pts were still on treatment at the time of this analysis. Patients who completed therapy with at least 1 cycle of chemotherapy were evaluated for response and toxicity; pts in continuous therapy were evaluated for toxicity only. Of 33 pts evaluable for response 7 pts achieved a CR (21%) and 14 a PR (42%) resulting in an ORR of 64%. The remaining 12 pts were non-responders. No differences in the results were observed between groups with different bendamustine doses or scheduling. The best results were obtained in 10 evaluable pts with indolent lymphoma (4 CR, 6 PR) and in 9 pts with chronic lymphocytic leukemia (1 CR, 6 PR). Two evaluable pts with mantle cell lymphoma obtained a response (1 CR, 1 PR). By contrast, only 1 pt with diffuse large B cell lymphoma of 6 patients evaluable for response obtained a CR: the other 5 were non-responders. No pt with myeloma, Hodgkin’s disease or T cell lymphoma achieved a response. After a median follow-up of 4 months, 80% of pts were alive. During 150 treatment cycles, 2 pts experienced grade 4 thrombocytopenia and 1 experienced grade 4 neutropenia; non-hematological toxicity was mild. In conclusion, this retrospective analysis shows that treatment with bendamustine, alone or in combination with rituximab, is a safe and effective regimen in heavily pretreated pts. The best results were obtained in indolent lymphoma: the data in mantle cell lymphoma were also encouraging. No lack of efficacy can be inferred in pts with diffuse large B cell lymphoma, due to the refractory nature of their disease and the advanced age of this particular group (median age 76 years ranging from 67–87).

Genome-Wide Array-Based Methylation Profiling Reveals Preferential Methylation of Homeobox Transcription Factor Genes In Mantle Cell Lymphoma and Pro-Apoptotic Genes In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 536-536
Author(s):  
Anna M Halldorsdottir ◽  
Meena Kanduri ◽  
Millaray Marincevic ◽  
Hanna Göransson ◽  
Anders Isaksson ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Aberrant Methylation ◽  
B Cell Malignancies ◽  
Genome Wide ◽  
Mantle Cell ◽  
Apoptotic Genes

Abstract Abstract 536 Introduction: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies of different postulated origin, genetics, clinical presentation and prognosis. Several studies have reported that both MCL and CLL individually exhibit aberrant methylation in comparison to normal B-cells. However, a comprehensive comparison of the methylation profiles of these two B-cell disorders has not been performed yet. This strategy has the potential to identify cellular pathways and genes that are specifically targeted in each disease. Methods: We applied the genome-wide Illumina Infinium HumanMethylation27 BeadChip array (Illumina, San Diego, USA) which measures methylation levels at 27,578 CpG dinucleotides covering 14,495 genes, to compare the methylation profiles in: (i) 20 MCL cases; and, (ii) 30 CLL cases, 15 each with unmutated stereotyped subset #1 (IGHV1-5-7/IGKV1(D)-39) B cell receptors (BCRs) or mutated stereotyped subset #4 (IGHV4-34/IGKV2-30) BCRs, where these two subsets represent prototypes of unmutated and mutated CLL. The methylation status for each detected CpG site ranged between 0.1 (completely unmethylated) to 1 (completely methylated). Results: As expected, major differences in methylation patterns between MCL and CLL were observed. When the methylation profiles of the two entities were compared, 51 genes were identified as differentially methylated in all comparisons (MCL versus both CLL subsets combined and each subset separately). Among the 19 genes highly methylated in MCL were six (32%) homeobox or homeodomain-containing transcription factors (e.g. POU4F1, PITX3), whereas genes enhancing cell proliferation and tumor progression such as MERTK and CAMP were hypomethylated in MCL. Of the 32 genes hypermethylated in CLL were six pro-apoptotic genes, including DYRK2 and CYFIP2, the tumor suppressor PRDM2 and the cell cycle regulator CCND1. Conclusions: We report for the first time disease-biased methylation profiles for different functional classes of genes in MCL or CLL. Homeobox genes were highly methylated in MCL, whereas CLL was characterized by methylation of apoptosis-related genes. The identified differences in global methylation profiles between MCL and CLL may assist in unfolding distinct epigenetic silencing mechanisms involved in the pathogenesis of these B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

Somatic Mutation and Isotype Switch Events Outside Igv Gene Loci Provide New Evidence for a Role of Antigenic Challenge in Mantle Cell Lymphoma Etiology

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2647-2647
Author(s):  
Sandrine Roulland ◽  
Nathalie Jouve ◽  
Agnes Bru ◽  
Marie-Helene Delfau-Larue
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
High Rate ◽  
Chain Gene ◽  
Functional Allele ◽  
Antigenic Challenge ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) represents 5-10% of B-cell lymphomas and generally exhibits an aggressive clinical behavior. t(11;14) translocation, the genetic hallmark and early initiating event of MCL pathogenesis, lead to the constitutive CCND1 expression and consequently to cell-cycle deregulation. However, t(11;14) is also present in the blood from rare healthy individuals indicating that alone the t(11;14) is not sufficientfor malignant transformation. It has long been assumed in MCL that t(11;14)+ cells were actually carried by circulating naive and antigen-inexperienced B-cells. Yet, several reports, including long-term persistence of t(11;14)+ cells in lymphoma free-patients, biased immunoglobulin heavy variable chain gene (IgVH) repertoire and evidence of subset of cases with mutated IgVH genes led us to question this model and further investigate the role of antigenic challenge in MCL etiologic origin. As a first approach, we sought to determine the class-switch recombination (CSR) status of t(11;14)+ cells in MCL patients. CSR, as somatic hypermutation (SHM), are diversification mechanisms of the B cell receptor induced upon antigen encounter in the germinal center (GC) reaction, and absent in naive cells. Evidence for ongoing switch events in MCL has been previously reported with detection of mature switch transcripts, however in MCL cells, sIgM(D) expression remains clearly evident suggesting that effective deletional switch events occur infrequently on the functional allele. To characterize CSR in t(11;14)+ cells, we thus took advantage of their unique BCL1/IGH translocation signature assuming that BCL1/IGH fusions do not prevent CSR to occur on the downstream constant region of the IGH locus. Using 2 long-range PCR assays designed to amplify unswitched BCL1/Sµ and switched BCL1/Sγ regions, DNA from 30 MCL patients differing by their IgVH status were tested. Of the MCL cases, we confirmed that 7 of 30 (23%) were unmutated (UM, 100% germline homology) and 23 of 30 (77%) were mutated (M), considering as mutated all cases exhibiting any level of SHM (ranging from minimal: >0.3% to high rate: 7.0%). Although mutated BCRs were common in most MCL cases, we found that only 1 out of 30 MCL cases underwent CSR to IgG on the non-productive, translocated allele. Accordingly, this case carried a mutated IgVH gene with 97.5% germline identity in line with a GC/antigen-experienced signature of the tumor cells. Contrary to SHM events of the IgVH gene region, CSR deletional events appeared very infrequent in the bulk of MCL cells both on the functional and translocated allele, contrasting from findings in other GC-derived non-Hodgkin lymphomas where the same selective pressure to maintain a sIgM is accompanied by deletional switch events on the non-functional allele. To get more insights into a role of antigenic drive in MCL and AID targeting outside the VH gene loci, we next evaluated the clonal evolution of t(11;14)+ cells from 11 MCL cases (2 UM and 9 M) by analyzing SHM in the Sμ region on the translocated allele using sequencing of LR-PCR amplicons (with 2 subclones/patient). We found that 63% (7 out of 11) of BCL1/IGH clones display significant levels of SHM in the Sµ together with intra-Sµ indels (2/11) compatible with off-target AID-mediated events linked to chronic(?) antigen encounter. In addition, we found a good correlation between mutated BCR and the SHM levels in Sμ regions for a given patient. Importantly, in one instance subclones from a MCL case displayed intraclonal diversification (despite the fact that the expected rate of SHM is much lower in the Sµ region than in the IgVH region), indicative of an ongoing AID activity at least in a subset of MCL cases. In conclusion, we found that CSR events, although detectable in some MCL cases, do not appear to participate predominantly or to be selected during MCL lymphomagenesis. By contrast, analysis of SHM profiles both on the functional and the non-functional translocated allele provide evidence for an active AID activity in MCL cells inside and outside V-gene loci, highlighting the propensity of some t(11;14)+ clones to acquire a complementary oncogenic hit over time as a result of recurrent antigen challenge. Disclosures No relevant conflicts of interest to declare.

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