Classical Mantle Cell Lymphoma Research Articles

Genomic and Transcriptomic Profiles of Blastoid and Pleomorphic Mantle Cell Lymphoma Are Distinct from Classic Histology Mantle Cell Lymphoma

Introduction - Patients (pts) with mantle cell lymphoma (MCL) with blastoid (B-MCL) and pleomorphic (P-MCL) histology are traditionally considered as high risk; however, there is a paucity of information regarding the differences between P-MCL and B-MCL compared to classic MCL (C-MCL) histology. In this study, we present a comprehensive genomic and transcriptomic portrait of B-MCL and P-MCL. Methods - We conducted comprehensive molecular profiling using whole exome (WES) and bulk RNA sequencing (RNA seq) among the evaluable pt samples (n=260) from our center (MDA cohort) and previously reported cohorts from Bea et al ( PNAS 2013) (n=29) and Agarwal et al ( Nature Medicine 2019) (n=15) respectively. Pts with WES were (n=195) including n=151, n=29 and n=15 for MDA, Bea and Agarwal respectively while 175 pts underwent RNA sequencing (all MDA). This cohort study was conducted under an IRB approved protocol for MCL patients at our center. Patient characteristics, somatic mutation profiles, copy number abnormalities and gene expression profiles (GEP) were analysed. Lymph node, bone marrow, blood samples and any other tissue biopsies were utilized. DNA and RNA were extracted from FFPE sections from lymph nodes and non-nodal tissues. Joint WES and RNA-seq mutation calling, GEP, and transcriptomic cell deconvolution were performed using the BostonGene automated pipeline. WES and bulk RNA sequencing were performed with Illumina HiSeq4000 using a 76bp paired end configuration. Results - Among the 195 pts with WES, the distribution of tissue types was 133 nodal, 33 bone marrow, 25 blood, 4 spleen and other tissues. Mutation distribution and copy number analysis was performed for 99 C-MCL , 72 B-MCL, and 20 P- MCL pts. In the mutation profile, we found that the percentage of ATM mutations was comparable among the 3 pt groups (51%/50%/39%). The frequency of TP53 mutations was significantly lower in C-MCL (23%) compared to B-MCL (51%) and P-MCL (39%) (p < 0.001; Figure 1). NOTCH2 gain of function mutations were found in 11% of B-MCL and 4.6% of C-MCL but not detected in P-MCL. The NFkBIE Y254Sfs*13 mutation was exclusively found in B-MCL. Blastoid samples were enriched with CCND1 E36K mutations (7 of 16 CCND1 mut), while C-MCL had 2 of 16. P-MCL had a higher prevalence of SMARCA4 and DNMT3A mutations compared to B-MCL and C- MCL. We further observe that P-MCL pts exhibited higher polyploidy and aneuploidy than B-MCL and C-MCL ( p < 0.001; Figure 2), indicating that compared to C-MCL, genomic instability decreased from P-MCL to B-MCL. The most frequent arm level amplifications in P-MCL compared to B-MCL were 8q (=MYC) (38%) and 18q (=BCL2) (38%). In C-MCL and B-MCL the 3q (=BCL6) was frequently amplified (28% and 30%). MALT1, Bcl2 and CARD11 gene amplifications were highest in P-MCL. These differences were further supported when the data were compared at the gene level. Comparison of the deletion frequencies revealed that 9p (CDKN2A/B) and 17p (TP53) were frequently lost in B and P-MCL (~40%). Del8p, 6q, 10q, and 16q were frequently deleted in P-MCL compared to B-MCL. At the segmental level, mostly altered segments included del9p21.1-24.3, del13q14.2-14.3, and del17p12-13.3 that span genes like CDKN2A/B, miR15A, and TP53. Next, we performed GSEA analysis using RNA-seq data. The metabolic reprogramming pathway was upregulated in both B and P-MCL compared to C-MCL (p = 0.0096). The proportion of pts with an immune desert (D) “cold” tumor microenvironment associated with refractory disease was higher in P-MCL (50%) than in C or B-MCL. Conclusions -B and P-MCL exhibit a distinct molecular profile compared to C-MCL. P and B-MCL have the highest degree of aneuploidy and exhibit an immune cold tumor microenvironment. Further studies are ongoing to refine the molecular differences among B and P-MCL compared to C-MCL.

Prognostic Role of CDKN2A Deletion and p53 Expression and Association With MIPIb in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare type of lymphoma, which despite improvements in therapies in recent decades, remains an incurable disease. There is currently no reliable marker of chemoresistance available. In this study, we investigated the prognostic role of MIPIb and the association with biological markers including SOX11, p53 expression, Ki-67, and CDKN2A. This retrospective study was focused on 23 patients with newly diagnosed classical MCL, treated at the University Hospital of Bari (Italy) between January 2006 and June 2019. We identified a MIPIb value ≥ 5.4440 as a prognostic parameter that correlates with p53 expression and CDKN2A deletion. We also observed that patients with p53 overexpression had a significantly higher MIPIb (5.52 ± 0.53) which in 80% of patients had a value higher than 5.4440. On the other hand, CDKN2A deletion was found more frequently (75%) associated with MIPIb ≥5.4440. Only the CDKN2A deletion was associated with a higher proliferation index, with 66.7% of samples having Ki67 ≥30%. From the survival analysis we found that patients with p53 overexpression and CDKN2A deletion have a significantly worse prognosis with a median overall survival of 50 (P=.012) and 52 months (P=.018), respectively. p53 expression and CDKN2A deletion represent a reliable pretreatment prognostic factor that identifies patients who do not benefit from currently used immunochemotherapy-based therapies and who are candidates for diversified treatments with the aim of improving prognosis. The MIPIb represents a prognostic index that correlates well with these biological alterations and can be used in clinical practice as their surrogate.

Clinical and biological features and prognosis of patients with leukemic non-nodal mantle cell lymphoma

Objective: To investigate the clinical, biological and prognostic characteristics of leukemic non-nodal mantle cell lymphoma (nnMCL). Methods: The clinical data of 14 patients with nnMCL and 238 patients with classical mantle cell lymphoma (cMCL) in Blood Diseases Hospital, Chinese Academy of Medical Sciences from November 2000 to October 2020 were retrospectively analyzed. Among the 14 patients with nnMCL, there were 9 males and 5 females, with the age [M (Q1, Q3)] of 57.5 (52.3, 67.0) years. Among the 238 patients with cMCL, there were 187 males and 51 females, with the age of 58.0 (51.0, 65.3) years. The clinical and biological characteristics of the two groups were recorded and compared. Follow-up and efficacy evaluation were conducted by re-examination during hospital stay and telephone follow-up and so on. Results: The proportion of CD200 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [14.6% (19/130)] (P=0.001). The proportion of CD23 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [13.5% (23/171)] (P<0.001). The proportion of CD5 expression in nnMCL patients was 10/14, which was lower than that in cMCL patients [97.4% (184/189)] (P=0.001). The proportion of CD38 expression in nnMCL patients was 4/14, which was lower than that in cMCL patients [69.6% (112/161)] (P=0.005). The expression proportion of sex-determining region of Y chromosome-related high-mobility-group box 11 (SOX11) in nnMCL patients was 1/5, which was lower than that in cMCL patients [77.9% (60/77)] (P=0.014). The proportion of immunoglobulin heavy chain variable region (IGHV) mutations in nnMCL patients was 11/11, which was higher than that in cMCL patients [26.0% (13/50)] (P<0.001). As of April 11, 2021, the follow-up time for nnMCL and cMCL patients was 31 (8-89) months and 48 (0-195) months, respectively. Among the 14 nnMCL patients, 6 patients were still under observation, and 8 patients were treated. The overall response rate (ORR) was 8/8, including 4 patients with complete remission and 4 patients with partial response. The median overall survival and median progression-free survival were not reached in nnMCL patients. In the cMCL group, 50.0% (112/224) patients achieved a complete response, 24.6% (55/224) patients achieved a partial response, and ORR was 74.6% (167/224). There was no statistically significant difference in ORR between the two groups (P=0.205). Conclusions: nnMCL patients have an indolent progression, with higher expression rates of CD23 and CD200 and lower expression rates of SOX11, CD5 and CD38. Most patients have IGHV mutations, with a relatively good prognosis, and"watch and wait"approach is an optional treatment.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

The Therapeutic Synergy of Selinexor and Venetoclax in Mantle Cell Lymphoma Through Induction of DNA Damage and Perturbation of the DNA Damage Response.

Introduction: Mantle cell lymphoma (MCL) can be stratified into blastoid and classical subtypes based on morphological features, with the former subtype having a poorer prognosis. Despite recent advances in targeted approaches, including multiple bruton tyrosine kinase inhibitors which yield impressive clinical responses and improve prognoses, MCL remains an incurable disease with frequent relapses. Additional therapeutic interventions are therefore unmet medical needs for the management of patients with MCL. Methods: Cell viability and apoptosis assays were employed to analyze the therapeutic interaction of venetoclax combined with selinexor in MCL cells. Western blot was used to investigate the potential mechanism of action for the synergy of venetoclax in combination with selinexor in MCL cells. Results: In this study, we revealed that both blastoid and classical MCL cells were vulnerable to the cytotoxic effects of selinexor, a well-established XPO1 inhibitor, manifested by loss of cell viability and induction of cell apoptosis. Moreover, our data indicated that the addition of venetoclax to selinexor showed synergistically decreased cell viabilities and increased cell deaths in blastoid and classical MCL cells compared to each single drug treatment. Either selinexor or venetoclax treatment alone decreased MCL1 expressions and increased BAX levels in MCL cells, and these effects were further enhanced by their combined regimen. Mechanistically, our findings demonstrated that induction of DNA damage and inactivation of DNA damage response were involved in the synergistic interaction of the drug combination regimen. Conclusion: Collectively, this study might provide a potential attractive therapy option for the treatment of MCL. However, the conclusion needs additional experimental validation in in vivo models and clinical evaluations are mandatory.

Updates in the treatment of mantle cell lymphoma: A Canadian expert framework

Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin Lymphoma that develops in the mantle zone of the lymph node. It is more common in men and is usually diagnosed at an advanced stage with involvement of lymph nodes, bone marrow, and potentially the gastrointestinal tract. MCL accounts for 5–10% of all new NHL cases per year in Canada, which is estimated at 11,400 for 2022. While most patients respond to initial treatment, relapses occur early and MCL generally shows a variable response to subsequent treatments, often with limited duration of benefit. Two main subtypes of MCL can be distinguished that arise from in situ MCL lesions. The most common subtype, classic MCL, arises from these cells with limited or no immunoglobulin heavy chain variable region (IGHV) mutations. Cells from this subtype express SOX11, are genetically unstable, and have naïve B cell-like characteristics. Classic MCL is more often nodal and extranodal and may eventually progress to aggressive blastoid or pleomorphic MCL. The other subtype, leukemic non-nodal MCL, arises from cells that have undergone IGHV somatic hypermutations, do not express SOX11, and exhibit characteristics of memory B cells. This subtype can have an indolent clinical behavior for a long time, often several years, but frequently acquires TP53 and other mutations and progresses to a more aggressive subtype. Treatment options have expanded significantly over the past decades, with improvements in both overall survival (OS) and progression-free survival (PFS) compared to earlier treatment eras. This Canadian expert framework aims to discuss the management considerations for patients with MCL, and will present both front-line treatment options as well as those for relapsed and refractory disease.

Differential Phosphoproteomics Deciphers Physiopathology of High-Risk Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare adult-onset non-Hodgkin lymphoma of which genetic hallmark is CCND1 translocation with IG genes, although CCND2, CCND3 or CCNE translocations are alternatives. The median survival of patients with MCL has lately increased due to improved therapies. However, the identification of high-risk MCLs characterized by pleomorphic or blastoid appearance, high proliferation index and TP53 deletions/mutations, remains mandatory due to their highly aggressive features and dismal clinical course even with intensified regimens. Therefore, specific therapeutic approaches are still warranted for such patients, but clinical and biological data are scarce since high-risk MCLs represent only 10-20% of all MCLs. A valuable source of rare samples for clinical/translational research is archived collections of formalin-fixed paraffin-embedded (FFPE) tissue blocks which enable retrospective studies to be performed. However, FFPE samples remain challenging for omics studies, especially phosphoproteomics. Here we implemented an original process to overcome this technical roadblock and took full advantage of FFPE samples to investigate biological processes differentially involved in high-risk and classical MCL (cMCL) to highlight rationale for innovative targeted therapies for these aggressive forms. Material and methods: We employed an unbiased differential label-free phosphoproteomic approach using archived FFPE MCL samples. Briefly, archived FFPE lymph nodes conclusive for cMCL or blastoid MCL (bMCL) diagnoses were retrieved from the Biological Resource Center of Strasbourg University Hospital from 1996 to 2016. H&E slides were reevaluated, and areas enriched in tumoral cells were selected to be punched from FFPE blocks. Our original sample preparation procedure entailed 1/ xylene-based deparaffinization, 2/ protein extraction with high content SDS-based lysis buffer, 3/ sample reduction/alkylation followed by cleanup and protein digestion on carboxylate paramagnetic beads and Lys-C/trypsin proteolytic enzyme mix, 4/ phosphopeptide enrichment on the AssayMap Bravo platform (Agilent) using IMAC Fe(III)-NTA cartridges. All samples were then analyzed on a NanoElute coupled to a TimsTOF Pro mass spectrometer (Bruker). Label-free quantification was performed using MaxQuant with the human database downloaded from SwissProt in April 2022. Further bioinformatics analyses were conducted in Perseus and Prostar. Data were functionally annotated with Reactome Pathway Database and STRING for association networks. Results: For this monocentric study we retrieved 30 FFPE blocks of lymph node biopsies conclusive for MCL diagnosis, including 15 bMCL and 15 cMCL. Although FFPE samples represented a challenge to work with for phosphoproteomics, we extracted enough protein from three 2mm-punches to proceed to the phosphopeptide enrichment according to our original sample preparation procedure. Overall, we successfully identified and quantified 7,220 class I phosphosites, i.e., sites of phosphorylation for which the probability of the phosphate group localization is greater than 0.75. No significant difference was found considering neither the archived period length of FFPE blocks nor the MCL subtypes (bMCL or cMCL). Differential analysis of bMCL and cMCL identified 180 differentially phosphorylated phosphosites (p-value < 0.05) corresponding to 103 proteins. Among these phosphosites, 139 were more phosphorylated in bMCL. Functional annotation of their corresponding proteins highlighted an over-representation of proteins involved in cell cycle, DNA repair and RNA metabolism within bMCL differential phosphoproteome (Fig.1). In addition, the kinase enrichment analysis of these differentially phosphorylated proteins predicted an increased activity of cyclin-dependent kinases in bMCL compared to cMCL. Conclusion: Here we performed for the first time phosphoproteomics from archived FFPE MCL samples. Differential analysis revealed that high-risk MCLs are characterized by an increased phosphorylation of proteins involved in cell cycle and an increased activity of cyclin-dependent kinases. Further studies are mandatory to fully understand to which extent cyclin-dependent kinases could be suitable targeted therapeutic candidates for patients with high-risk MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deep Learning–Based Nuclear Morphometry Reveals an Independent Prognostic Factor in Mantle Cell Lymphoma

Blastoid/pleomorphic morphology is associated with short survival in mantle cell lymphoma (MCL), but its prognostic value is overridden by Ki-67 in multivariate analysis. Herein, a nuclear segmentation model was developed using deep learning, and nuclei of tumor cells in 103 MCL cases were automatically delineated. Eight nuclear morphometric attributes were extracted from each nucleus. The mean, variance, skewness, and kurtosis of each attribute were calculated for each case, resulting in 32 morphometric parameters. Compared with those in classic MCL, 17 morphometric parameters were significantly different in blastoid/pleomorphic MCL. Using univariate analysis, 16 morphometric parameters (including 14 significantly different between classic and blastoid/pleomorphic MCL) emerged as significant prognostic factors. Using multivariate analysis, Biologic MCL International Prognostic Index (bMIPI) risk group (P=0.025), low skewness of nuclear irregularity (P=0.020), and high mean of nuclear irregularity (P=0.047) emerged as independent adverse prognostic factors. Additionally, a morphometric score calculated from the skewness and mean of nuclear irregularity (P=0.0038) was an independent prognostic factor in addition to bMIPI risk group (P=0.025), and a summed morphometric bMIPI score was useful for risk stratification of patients with MCL (P=0.000001). These results demonstrate, for the first time, that a nuclear morphometric score is an independent prognostic factor in MCL. It is more robust than blastoid/pleomorphic morphology and can be objectively measured.

Combined epigenetic and immunotherapy for blastic and classical mantle cell lymphoma.

Classical MCL (cMCL) constitutes 6–8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870 CCND1 polymorphism is predictive of blastic disease, nuclear localization of cyclinD1 and response to SCR therapy. The major resistance mechanisms to SCR therapy are loss of CD20 expression and evasion of treatment by sanctuary in the CNS. These data indicate that administration of epigenetic agents improves efficacy of anti-CD20 immunotherapies. This approach is promising in the treatment of MCL and potentially other previously treatment refractory cancers.

Clinicopathological and molecular genetic features of cyclin D1-negative mantle cell lymphoma

Objective: To investigate the clinicopathological features and molecular genetics of cyclin D1-negative mantle cell lymphoma (MCL). Methods: The clinicopathological features and molecular genetics of CyclinD1-negative MCL diagnosed between January 2016 and July 2021 at the First Affiliated Hospital of Zhengzhou University were analyzed using immunohistochemistry and fluorescence in situ hybridization. Clinical information was collected and analyzed. Results: A total of five Cyclin D1-negative MCL cases from all 212 MCL patients (5/212, 2.4%)were included. There were three male and two female patients,age ranged from 59 to 70 years (median 64 years). All patients presented with nodal lesions. None of the patients had B symptoms but four had bone marrow involvement. Histopathologically, four cases were classic MCL and one case was pleomorphic variant type. All five cases were negative for Cyclin D1 but SOX-11 were positive in all cases. CD5 was positive in four cases and one case was weakly positive for CD23. CD10 and bcl-6 were negative in all cases. CCND1 translocation was identified in three cases and CCND2 translocation in one case by FISH analysis. However,CCND3 translocations were not found in the five cases. Conclusions: Cyclin D1-negative MCL are uncommon, its accurate diagnosis needs combined analysis with morphologic and immunophenotypic characteristics and genetic changes. It may be particularly difficult to distinguish from other small cell type B cell lymphomas. FISH analyses for CCND1/CCND2/CCND3 translocations and immunohistochemistry for SOX-11 are helpful to resolve such a difficult distinction.

Mantle cell lymphoma with chronic lymphocytic leukemia-like features: a diagnostic mimic and pitfall

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by t(11;14) (q13;q32) and cyclin D1 overexpression in >95% of cases. Classic MCL cases are composed of a monotonous population of small- to medium-sized lymphocytes with irregular nuclear contours that are positive for cyclin D1 and SOX11and negative for CD23 and CD200. By contrast, occasional MCL cases express CD23 and CD200 but lack SOX11and morphologically and immunophenotypically resemble chronic lymphocytic leukemia (CLL), termed as CLL-like MCL in this study. These neoplasms pose a diagnostic challenge and are easy to be diagnosed as CLL in daily practice. We studied 14 cases of CLL-like MCL to define their clinicopathologic features and compared them with 33 traditional CLL cases. There were 8 men and 6 women with a median age of 62 years (range, 44-80). Compared with CLL, patients with CLL-like MCL have lower levels of peripheral blood and bone marrow involvementand more frequently had mutated IGHV. Immunophenotypically, CLL-like MCL often showed moderate to bright expression of B-cell antigens and surface immunoglobulin light chain, dim and partial expression of CD23 and CD200, infrequent CD43 positivity, and lack of LEF1. The overall survival of patients with CLL-like MCL was similar to that of CLL patients. In conclusion, CD23+, CD200+, and SOX11-negative MCL closely resemble CLL, both clinically and pathologically, including a similar indolent clinical course. They may pose a diagnostic challenge. However, patients with CLL-like MCL also have distinctive immunophenotypic features that are useful to distinguish these neoplasms from CLL.

Impact of p53 Disruption on Mantle Cell Lymphoma (MCL) Treatment out-Come, Multi-Centre Retrospective Study

Background: MCL is a rare type of non-Hodgkin lymphoma (NHL) with an aggressive clinical course. MCL is associated with poor prognosis and incurable disease in the majority of patients. However, a minority of patients will not require any treatment for many years. TP53 mutations continue to confer a dismal prognosis in MCL with a median survival of 1.3 years. However, targeted therapy and, more recently, promising CAR-T treatment have revolutionised the outcome.Methods: Patients were identified through our pathology database, and informed consent was obtained. The patient's demographic characteristics, clinical features, laboratory findings, MIPI score, initial / subsequent treatment and survival were analysed. p53 expression percentage and its concordance with the mutational status were evaluated in a subset of patients.Results: One hundred and one patients were included in this study, with a male predominance (80%), median age 67 years (Range 37-89 years) and majority with advanced disease. Fifty-one patients (50%) presented with extra-nodal disease. Most of the patients presented with Classical MCL ( 75%) histological sub-type, 20 patients (20%) with blastoid variant and 6 patients (6%) with indolent disease. Seventy-four patients (73%) had a high MIPI risk score, while intermediate and low scores were noted in 17 patients (17%) and 10 patients (10%), respectively. Chemo-immunotherapy (CIT) was the main initial treatment modality in our cohort (79 patients / 78%). Watch and wait approach was applied in 6 patients with the indolent disease, 4 patients (4%) were not fit for any form of therapy. Forty-four patients (44%) had Rituximab maintenance and autologous stem cell transplant (Auto-SCT ) consolidation was used in 12 patients (12%). Five patients (5%) had allogeneic SCT (Allo-SCT) post-remission.Initially, 29 patient samples were used to validate immunohistochemistry (IHC) p53 expression percentage and its correlation with TP53 mutational data from genomic sequencing (SS and NGS). p53 expression of > 30% had 100% concordance with TP53 mutational status.A total of 128 patient samples, including 27 samples with relapsed disease, were screened for TP53 alterations using IHC p53 expression as a surrogate marker. Twenty samples (16%) showed p53 overexpression (15/12% diagnostic & 5/19% relapsed samples).At the final data compilation (31/01/2021), 44 patients were still alive, and 57 patients had died. The OS and PFS for the whole cohort were 69 and 47 months, respectively. (Figure 1)The prognostic impact of age (<65 years), MIPI score & disease sub-type were confirmed in this cohort with a P-value of P.00046, P.0.036 and P.0.0192, respectively. In the treated cohort, p53 disruption revealed a dismal prognosis and poor treatment outcome, with a median OS and PFS in the p53 wild-type cohort of 95 months and 50 months. In contrast, in patients harbouring p53 disruption, the median OS & PFS were 38months (P.0323) and 25 months (P.0383 ), respectively. (Figure 2)ConclusionThis study reflects real-life MCL experience and the potential use of p53 expression using IHC in routine practice in assessing MCL disease prognosis. It also confirms the dismal outcome of MCL patients with TP53 mutations. Participation in clinical trials based on genetic risk stratification is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Validation of MCL35 in Mantle Cell Lymphoma Patients ≥65 Years Receiving Bendamustine-Rituximab

Clinical Validation of MCL35 in Mantle Cell Lymphoma Patients ≥65 Years Receiving Bendamustine-Rituximab

Complex Structural Variation Associated with Enhancer Hijacking and Loss of Tumor Suppressors in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a rare, incurable mature B-cell lymphoma that can have either an aggressive or indolent clinical course. The hallmark aberration of MCL is the t(11;14)(q13;q32) translocation that places CCND1 under control of the immunoglobulin heavy chain (IGH) locus resulting in its constitutive expression. CCND1/IGH translocation occurs in nearly all MCL cases and arises during VDJ recombination. Whole genome sequencing (WGS) of MCLs has shown the prevalence of additional structural variations (SV), particularly in tumors with poor outcome. Complex rearrangements, such as chromothripsis and chromoplexy have been observed in MCL but their role in lymphomagenesis has not been determined. We hypothesized that some of these complex rearrangements afford a selective advantage to the tumor by disrupting tumor suppressor genes or by placing oncogenes in proximity to regulatory elements in cis, thereby resulting in ectopic expression.We performed WGS on tumor DNA extracted from 106 RCHOP-treated MCL patients. Matching ribosomal-depleted RNA-seq data was available for all tumor samples. Gene expression count values were obtained using Salmon and counts were normalized using the DESeq2 method. Structural variants were identified using a consensus between GRIDSS and Manta and these were analyzed to identify the topology of complex rearrangements using JaBbA. This allows the annotation of rearrangements such as chromothripsis and chromoplexy and enables the detection of genes near new regulatory elements through complex (multi-breakpoint) rearrangements. We supplemented this analysis by including 57 published classical and non-nodal leukemic MCL genomes.Chromothripsis and chromoplexy were observed in 8 and 12 tumors, respectively. The majority of the genomes were associated with isolated structural variations such as large deletions, inversions and reciprocal translocations were more common. Six genes (BCL10, TRAF6, TRAF3, MAP3K7, BTK, RELB) coding for canonical and non-canonical NFκB signaling proteins were found rearranged to within 1Mbp of a naïve B-cell super-enhancer region. The TRAF6 rearrangement was part of a chromothripsis and chromoplexy event involving both chr9 and chr11. A separate chromothripsis example involving chr1 placed each of BCL10 and NOTCH2 within 100-200kbp of super-enhancers. A 1Mbp region containing RELB was amplified and inserted into chr19p approximately 150kbp downstream a super-enhancer. An 800kbp deletion brought MAP3K7 to within 400kbp of a super-enhancer. DAZAP1, a gene known to be recurrently mutated in MCL, was translocated upstream to within 300kbp of a super-enhancer by t(5;19)(p13.3;p15.33). TRAF3 was translocated 400kbp upstream a super-enhancer by t(14;20)(q32.32;q13.13). None of the aforementioned SVs were associated with a detectable increase in expression of the affected gene. In contrast, we identified one genome in which the MYC oncogene was relocated 500kbp upstream of a super-enhancer via an unbalanced t(4;8)(q21.23;q24.21) translocation. In this case, MYC expression was in the 95 th percentile of MYC expression across the cohort. Focal deletions and amplifications were also found affecting lymphoma driver genes. Focal amplifications affecting 3q (34 tumors) and 5p (9 tumors), were among the most common recurrent events. These respectively affect the TERC and TERT genes, both involved in telomerase function. All tumors with TERT amplifications also showed TERC amplifications. TERC and TERT were expressed higher on average in amplified tumors than in unamplified tumors. Two tumors showed focal deletions affecting the 3‘ end of BIRC3. The focal deletion in one tumor was found to span to the 3‘ end of BIRC2 resulting in a BIRC2/BIRC3 fusion. Aberrant splicing across the two genes was evident in matching tumor RNA-seq data.Complex rearrangements in MCL have been found to link distant super-enhancer elements with a variety of lymphoma oncogenes. We noted a recurrence of such events affecting known regulators of NFκB signaling. We are using nanopore-based long-read PromethION sequencing to validate the structure of the derivative chromosome in these cases. Although these genes were not detectably overexpressed, deregulation of genes may be occurring by other means. The full extent of deregulation of NFκB and other oncogenic pathways will be revealed as complex rearrangements are studied in additional MCL tumors. DisclosuresCoyle: Allakos, Inc.: Consultancy. Melnick: Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Scott: BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling–licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; AstraZeneca: Consultancy; Abbvie: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; Rich/Genentech: Research Funding; Celgene: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding. Morin: Epizyme: Patents & Royalties; Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees.

Ibrutinib Plus Rituximab and Venetoclax (IRV) Followed By Risk-Stratified Observation or Short Course R-Hypercvad/MTX in Young Patients with Previously Untreated Mantle Cell Lymphoma - Phase-II Window-2 Clinical Trial

Ibrutinib Plus Rituximab and Venetoclax (IRV) Followed By Risk-Stratified Observation or Short Course R-Hypercvad/MTX in Young Patients with Previously Untreated Mantle Cell Lymphoma - Phase-II Window-2 Clinical Trial

Mantle cell lymphoma involving tonsils: a clinicopathologic study of 83 cases

We report 83 cases of mantle cell lymphoma (MCL) involving the tonsil as initial manifestation (IM). The median age at the time of tonsillar involvement was 58 years (range, 35-79 years). Most (85%) patients presented similar to acute tonsillitis. Lymphadenopathy (84%) and advanced stage of disease (81%) were frequent. With a median follow-up of 6.1 years (range, 0.5-18.4 years), the median overall survival (OS) was 11.3 years for all patients. Cases with classic MCL morphology demonstrated a superior OS (median OS: 11.7 years versus 7.8 years for aggressive morphology, P=0.0361). Approximately 20% of patients had limited stage of disease, and they had excellent outcomes (median OS: not reached versus 11.3 years for advanced-stage MCL, P=0.0479). All the patients were alive after a median follow-up of 6.6 years (range, 1-16.2 years). There were no differences in relapse-free survival in morphology and stage (P>0.05). When tonsils were involved by relapsed MCL, patients less commonly had acute tonsillitis-like symptoms, lymphadenopathy, and advanced stage of disease compared to MCL as IM. Patients in the relapse group had poorer OS than patients in the IM group from the time of tonsillar involvement by MCL to the date of death or last follow-up (7.8 versus 11.7 years, P=0.003). Compared with a group of 93 patients whose initial biopsy specimen was a lymph node, patients whose initial biopsy specimen was tonsil had similar OS (11.7 versus 8.8 years, P=0.1764). However, patients with tonsillar MCL more commonly had limited stage disease (19% versus 8%, P=0.0385) and a low-risk Mantle Cell Lymphoma International Prognostic Index score (71% versus 47%, P=0.0025).

Leukemic Variant of Mantle Cell Lymphoma: Clinical Presentation and Management.

This review summarizes the unique presentation and management of the leukemic variant of mantle cell lymphoma (LV-MCL, also referred to as non-nodal MCL) and highlights the biologic and clinical differentiation from classical mantle cell lymphoma (cMCL) in biomarker expression, clinical features, prognosis, disease course, and treatment. Several studies have evaluated the gene expression profile of mantle cell lymphoma, differentiating LV-MCL from cMCL. The typical immunophenotypic profile is CD5-positive, SOX 11-negative, CD23-low, CD200-low, and cyclin D1 overexpressed. LV-MCL commonly has mutated immunoglobulin heavy chain variable region genes. Data on treatment of LV-MCL is limited to retrospective analyses; the ideal treatment for these patients is unknown although many have a clinically indolent, asymptomatic presentation and often may be observed for an extended period without active treatment. LV-MCL is a clinically and biologically distinct entity. Clinically, it must be distinguished from chronic lymphocytic leukemia and cMCL. Future prospective, randomized clinical trials are required to optimize management, define the initial treatment, and appropriately sequence treatment modalities.

CD5-Negative Mantle Cell Lymphoma Defines a Distinct Disease Entity Characterized By an Indolent Clinical Course Irrespective of Known Prognostic Markers

Introduction: Mantle Cell Lymphoma (MCL) is an aggressive CD5-positive B-cell malignancy with a median survival of 3 to 5 years. A small subset of patients present with an indolent disease and can be managed on a “watch and wait” approach. Indolent MCL remains ill-defined, but is classically associated with hypermutated IGHV, non-nodal presentation, and absence of SOX11 expression. Up to 10% of MCL lack CD5 expression, but there has been limited attention given to its clinical and biological implications. We performed an extensive review of all reported cases of CD5-negative MCL to further characterize this disease subset.Patients and Methods: A systematic literature search was performed and included studies published from June 1994 to July 2017 in Pubmed, Embase, and Web of Science. We used the key words CD5 negative, CD5-, mantle cell lymphoma, and MCL. Data was tabulated regarding 20 variables: t(11;14)(q13;32), cyclin-D1, CD5, CD10, CD19, CD20, CD23, BCL2, BCL6, and Ki-67 (higher or lower than 30%), IGHV mutation status (mutated defined as >3% discordance with germline), SOX11, morphology, initial site of diagnosis, gender, age, light chain expression, presence of leukemic phase, stage, and overall survival (OS). Data was reported as percent and total number of cases with available data of each variable, and compared to historic data on CD5-positive MCL (referred to as conventional MCL). We used the Kaplan-Meier method to study OS, and log-rank test to compare OS between disease subsets.Results and discussion: 140 of 818 screened articles included CD5-negative MCL cases. After exclusion of duplicates and non-English articles, 70 sources were included. Additionally, we included 2 cases identified at our institution. Data of 323 cases of CD5-negative MCL was collected (Figure 1A). Conventional and CD5-negative MCL shared certain similarities: both had a median age of diagnosis between 60 and 70 years, showed male predominance, and presented as stage IV disease. They shared overexpression of cyclin-D1 (100%, 241/241), possessed the t(11;14)(q13;32) (96%, 107/111: 107 of 111 cases with available data on translocation), were CD19-positive (86%, 32/37), CD20-positive (97%, 107/110), BCL2-positive (98%, 44/45), CD10-negative (91%, 77/85), CD23-negative (84%, 91/109), and BCL6-negative (71%, 29/41). Presence or absence of leukemic phase was reported in 31 cases, 14 of those were positive for circulating MCL cells (45%). Lymph node was frequently the initial site of MCL diagnosis (59%, 57/97). Majority of cases showed the classical MCL morphology with small to medium sized lymphoid cells (59%, 61/104). The proliferation marker Ki-67 was reported in 82 cases, 48 (59%) of which were <30%.There were considerable clinical and biological differences between CD5-negative and conventional MCL. Most notably, the median OS (mOS) of 79 CD5-negative MCL cases with available follow-up was >14 years (Figure 1B), which compares very favorably to the historic survival of conventional MCL. SOX11, an MCL-specific biomarker that is positive in 90% of conventional cases was expressed in only 44% (11/25) of CD5-negative MCL cases. Lack of SOX11 expression in MCL predicts an indolent MCL subset. Surprisingly, OS was not significantly different between SOX11-positive and SOX11-negative CD5-negative MCL (Figure 1C, P=0.88). SOX11 stains were performed by expert hematopathologists at Tulane (2 cases) or other reliable institutions. Akin to SOX11 expression, λ light chain restriction and unmutated IGHV, two variables known topredict worse outcomes in conventional MCL, did not significantly impact OS in CD5-negative MCL. Similarly, higher Ki-67 expression (>30%) did not affect OS of CD5-negative MCL. Variables more prevalent in CD5-negative MCL compared to conventional MCL included κ light chain restriction (70%, 26/37) and IGHV hypermutation (79%, 15/19). Though sample size is limited, our survival analysis highly suggests that lack of CD5-expression might in fact represent an independently good prognostic marker in MCL.Conclusion: Lack of CD5 appears to improve survival in MCL independently of other favorable prognostic markers such as IGHV hypermutation, absence of SOX11 expression, low Ki-67, and κ restriction. Reasons for this indolent behavior remain unknown. Our analysis indicates that CD5-negative MCL deserves deeper investigation to further understand its biological implications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RAC2 Links B-Cell Receptor Signaling and Cell Adhesion in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.Methods: We used RNA-sequencing to identify pathways that have differential responses to ibrutinib in MCL cell lines that are either intrinsically sensitive or resistant to ibrutinib. We then validate the RNA-Seq findings in sensitive and resistant cell lines via RNA interference and transfection, xenograft mouse models, and human primary MCL samples.Results: We found that sensitivities of six MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. These include sensitive cell line Jeko-1, intermediate cell lines Mino, REC-1 and JVM2, and resistant lines Maver-1 and Granta 519. RNA-sequencing revealed that BCR signaling and cell adhesion transcriptional programs were coordinately down-regulated by ibrutinib in ibrutinib-sensitive cells, but not in ibrutinib-resistant cells. Among genes that are differentially regulated in the sensitive and resistant cell lines, we identified RAC2, a regulator of cell adhesion this is also involved in BCR signaling. To determine whether RAC2 is relevant, we examined RAC2 expression in primary MCL tumors by immunohistochemical staining (IHC). We found that RAC2 was weakly expressed in 7/10 classic MCL tumors and strongly expressed in 3/3 aggressive MCL. In contrast, in tonsilar lymphoid reactive tissues, RAC2 positivity was restricted only to mantle zone and T-zone of the follicles. For further validation, we found that RAC2 expression, at the protein level, was down-regulated by ibrutinib treatment in sensitive, but not resistant, cell lines. More interestingly, using co-immunoprecipitation, we found that RAC2 is physically associated with BLNK, an adaptor of early BCR signaling pathway, in the sensitive cells but not in the resistant cells. To establish that RAC2 is a regulator of MCL cell adhesion in the context of MCL, we demonstrated that siRNA knock-down of RAC2 impaired cell adhesion while exogenous introduction of RAC2 reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib showed slower tumor growth with reduced RAC2 expression by IHC. Lastly, we show that in primary human MCL cells, RAC2 was down-regulated by ibrutinib, and this down-regulation correlated well with cell adhesion impairment following ibrutinib treatment.Conclusions: Our findings suggest the RAC2 mediates the link between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC expression and via disruption of RAC2-BLNK interaction. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development. DisclosuresLu:Portola Pharmacueticals, Inc.: Research Funding. Wang:Portola: Research Funding; Karyopharm: Research Funding.

Brexucabtagene autoleucel therapy induces complete remission in a primary refractory blastoid mantle cell lymphoma with neurolymphomatosis

Brexucabtagene autoleucel therapy induces complete remission in a primary refractory blastoid mantle cell lymphoma with neurolymphomatosis