MCL Cases Research Articles

Abstract 777: Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties

Abstract Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cancer stem cells (CSC) able to self-renew and responsible for drug resistance. SOX11 embryonic transcription factor is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-CSC have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL clinical features and aggressive behavior remains unknown. We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. Here, we integrated the differential gene expression profile (GEP) between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, and specific SOX11 ChIP-chip and epigenetic data in MCL cell lines and primary cases, to identify potential mediators of CSC directly regulated by SOX11 in MCL. We observed an enrichment of hematopoietic (HSC) and leukemic stem cells (LSC) gene signatures in SOX11+ compared to SOX11- MCL primary cases. MSI2, an RNA binding protein that maintains self-renewal and prevents differentiation in HSC, emerged as one of the most significant upregulated CSC-related gene in SOX11+ compared to SOX11- MCLs, positively correlating with SOX11 expression in MCL primary cases. Our in vitro experiments showed that SOX11 binds to MSI2 regulatory regions increasing its expression in MCL cells. MSI2 enhancers located in intronic regions were activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/TP53, 11q/ATM and 9p/CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models. Overall, our results suggest that SOX11+ MCL cases acquire stemness features through the upregulation of MSI2 expression, which promotes chemoresistance, self-renewal and tumor survival. MSI2 represents a novel biomarker for MCL-CSC and therapeutic target for relapsed MCL. Citation Format: Marta Sureda-Gómez, Patricia Balsas, Marta Leonor Rodríguez, Ferran Nadeu, Anna De Bolòs, Álvaro Eguileor, Marta Kulis, Giancarlo Castellano, José Ignacio Martin-Subero, Santiago Demajo, Pedro Jares, Eva Giné, Elias Campo, Virginia Amador. Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 777.

TP53 Mutations, Deletions, and CN-LOH: Comparison of TP53 single Hit and Double Hit Events and Their Impact on Prognosis in Hematological Malignancies

Background: TP53 is altered in ~50% of human cancers. Alterations mainly include mutations and/or deletions, but also copy-neutral loss of heterozygosity (CN-LOH) was reported. Frequently, both TP53 alleles are altered (by mutation + deletion, mutation + CN-LOH or ≥2 mutations), leading to a “double hit” event.Aim: Analysis of TP53 aberrations using WGS in 4646 cases with 29 different hematological malignancies, comparing (1) the frequencies of TP53 alterations, (2) occurrence of single hit vs. double hit, (3) correlation with complex karyotype and (4) impact on survival.Methods: Whole-genome sequencing (WGS) was performed for all 4,646 patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p<0.05.Results: In the total cohort of 4,646 cases, in 582 (13%) at least one alteration (alt) involving TP53 was detected (comprising mutations (mut), deletions (del) and CN-LOH (LOH); Fig 1A,B). Cases were categorized as follows: cases with (1) 1 TP53 mut only (n=166), (2) del only (n=100), (3) LOH only (n=15), constituting the single hit events. Further, (4) cases with mut+del (might include 1 or more mut, n=211), cases with mut+LOH (≥1 mut, n=41), cases with ≥2 mut only (without del or LOH, n=49), resulting in double hit events (Fig 1B). Regarding the respective entities, high frequencies of TP53 alt were mainly detected in lymphoid malignancies (e.g. HGBL, MPAL, vHZL, MZL, MCL), whereas they were infrequent or absent in many myeloid malignancies (e.g. aCML, MPN, CMML, CML, MLN_eo; Fig 1A). For further analysis, only entities in which >10 cases showed TP53 alt events were used. Comparison of single hit vs. double hit revealed that T-NHL, MM, MPN and MDS predominantly showed a single hit, whereas the double hit was frequent in MPAL, MZL, MDS/MPN-U, CLL and MCL cases (Fig 1A). However, the type of double hit differed between myeloid and lymphoid malignancies, as myeloid neoplasms showed a high frequency of cases with ≥2 mut only, whereas in many lymphoid malignancies the double hit was predominantly generated by mut+del (Fig 1A,C). All TP53-associated events (mut, del, LOH and the respective combinations) were found to be associated with a complex karyotype in the total cohort (LOH: 14% complex karyotype in cases without TP53 alt vs. 59% in cases with TP53 alt, p<0.001). This association was also detected for most of the selected entities (exceptions: MZL, T-NHL). Regarding overall survival (OS), in the total cohort, all events involving TP53 impact on OS (TP53 alt: 22 months vs. 84 months, p<0.001; TP53 mut: 20 vs. 82 months, p<0.001; TP53 del: 20 vs. 79 months, p<0.001; TP53 LOH: 20 vs. 75 months, p<0.001). Moreover, although the single hit already impacts on OS, the double hit leads to an even inferior outcome (no hit vs. single hit vs. double hit: 84 vs. 39 vs. 14 months, p<0.001). In the selected entities, an influence of TP53 alt on OS was detected for all malignancies except HGBL, MZL and T-NHL, for which also the presence of a double hit did not show an effect on OS. For the majority of the other entities, the double hit leads to a shorter OS than the single hit (as observed for the total cohort), with the exceptions of MCL and MPAL: in these entities, the single hit did not impact on OS, but only a double hit is associated with inferior outcome.Conclusions: (1) Frequency of TP53 alterations and of double hit vs. single hit differs markedly between entities. (2) The kind of TP53 complexity differs between both lineages (double hit in myeloid neoplasms: often ≥2 mut only; in lymphoid malignancies: predominantly mut+del). (3) In 7% (41/582) of cases with TP53 alt, CN-LOH transforms a single hit into a double hit. (4) In the total cohort and in the majority of selected entities (except MZL and T-NHL), TP53 alt are associated with complex karyotype. (5) In the total cohort, all events involving TP53 impact on OS; cases with double hit show an inferior outcome compared to single hit. (6) Regarding OS, the selected entities can be divided into three categories: (i) no influence of TP53 alt (HGBL, MZL, T-NHL); (ii) double hit required for impact on OS (MCL, MPAL); (iii) influence of both single hit and double hit with inferior outcome of double hit (all other). [Display omitted] DisclosuresKern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

SOX11, CD70, and Treg cells configure the tumor immune microenvironment of aggressive mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11− primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11− tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory T (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.

Management of BTK Inhibitor Associated Adverse Events: Current Practice Trends Among Healthcare Providers and Concordance with Expert Recommendations

Introduction: The advent of BTK inhibitors (BTKi; ibrutinib, acalabrutinib, zanubrutinib) has dramatically improved outcomes for many patients with B-cell malignancies including CLL/SLL, MCL, WM, and MZL. To ensure optimal patient outcomes with BTKi therapy, it is essential to maintain both ongoing therapy and patient quality of life. These dual goals require prompt recognition and management of the unique adverse events (AEs) associated with BTKi therapy. An online Interactive Decision Support Tool was developed to provide healthcare providers (HCPs) with case specific guidance on managing BTKi AEs (available at: www.clinicaloptions.com/BTKiTool). Here, we report HCP practice trend data for the management of various BTKi AEs collected with this online tool compared with recommendations from a panel of experts. Methods: The online tool was developed with input from 5 experts who provided BTKi AE management recommendations for specific patient scenarios. The online tool captured 164 unique clinical scenarios based on criteria including disease type, BTKi used, and the type and severity of the BTKi AE. To use the online tool, HCPs enter information about their patient case and their planned management strategy. The HCPs were then shown the management recommendation from the 5 experts for that specific BTKi AE scenario. After viewing the expert management recommendation, HCPs were asked if it had an impact on their intended management approach. Results: Between September 2019 and July 2020, 754 unique case scenarios were entered into the BTKi AE tool, with 319 cases related to a specific BTKi AE, by 432 HCPs. Of HCPs who answered the optional demographics questions (n = 96), 65% of cases were entered by clinicians who treat ≤ 10 patients/year with BTKi and 51% of cases entered were for specific patients in their clinical practice. Most cases entered in the tool were for patients with CLL (69%) followed by MCL cases (15%). Cases involving ibrutinib treatment were most common in this analysis (82% of cases) with acalabrutinib treatment entered for the remaining 18% of cases. Zanubrutinib was not yet approved when this tool was created and therefore was not included as a BTKi option. The most common AEs selected by HCPs using the tool regardless of BTKi therapy were atrial fibrillation, bruising or bleeding, and diarrhea [Figure]. For patient scenarios treated with acalabrutinib, HCPs also frequently selected headache (24%) as an AE of interest [Figure]. Overall, the planned BTKi AE management strategy of HCPs for mild to moderate AEs matched the expert recommendations in 56% of cases with ibrutinib treatment and 79% with acalabrutinib treatment. There was lower concordance between HCPs and experts for the management of grade 3/4 events with either ibrutinib (42%; P &amp;lt; .0001) or acalabrutinib (36%; P &amp;lt; .0001). For example, for patient cases with grade 3/4 atrial fibrillation (n = 49), only 39% of HCPs would hold therapy whereas 41% indicated that they would continue therapy and 14% were unsure how to manage the AE. For HCPs who differed from expert recommendations, only 42% of respondents who entered mild to moderate AEs indicated that the expert recommendations provided by the tool changed their management plan, but 81% of respondents who entered grade 3/4 events would change their management plan. Conclusions: These data suggest that management of BTKi AEs by HCPs often diverges from evidence-based expert recommendations, especially grade 3/4 AEs. Use of an online tool providing easy access to BTKi AE management recommendations may improve patient care and safety. A detailed analysis of the tool, including case entries and planned management vs expert recommendations will be presented. Figure Disclosures Awan: Blueprint medicines: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Astrazeneca: Consultancy; Sunesis: Consultancy; Karyopharm: Consultancy; MEI Pharma: Consultancy; Genentech: Consultancy; Gilead Sciences: Consultancy; Kite Pharma: Consultancy; Dava Oncology: Consultancy. Leonard:MEI Pharma: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; BMS/Celgene: Consultancy; Roche/Genentech: Consultancy; Epizyme: Consultancy; Sutro: Consultancy; Miltenyi: Consultancy; AstraZeneca: Consultancy; Regeneron: Consultancy; ADC Therapeutics: Consultancy. Vose:AbbVie: Consultancy, Honoraria; Kite, a Gilead Company: Honoraria, Research Funding; Incyte: Research Funding; Novartis: Research Funding; Seattle Genetics: Research Funding; Epizyme: Honoraria, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Allogene: Honoraria; Bristol-Myers Squibb: Research Funding; Miltenyi Biotec: Honoraria; Wugen: Honoraria; Roche/Genetech: Consultancy, Honoraria, Other; Celgene: Honoraria; Loxo: Consultancy, Honoraria, Research Funding; Verastem: Consultancy, Honoraria; Janssen: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding. Flowers:Denovo Biopharma: Consultancy; Kite: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; OptumRx: Consultancy; Karyopharm: Consultancy; Pharmacyclics/Janssen: Consultancy; Spectrum: Consultancy; Acerta: Research Funding; Millennium/Takeda: Consultancy, Research Funding; TG Therapeutics: Research Funding; Burroughs Wellcome Fund: Research Funding; Eastern Cooperative Oncology Group: Research Funding; National Cancer Institute: Research Funding; V Foundation: Research Funding; Cancer Prevention and Research Institute of Texas: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; BeiGene: Consultancy; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy.

Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferation through reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma.

ObjectiveTo explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and the underlying mechanisms.MethodsIn this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated them to clinical outcome in 41 MCL patients. Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1 and EZH2 in well-characterized MCL cell lines which were compared to those of naïve B cells. Then we manipulated the expression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL. We also evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycle distribution and apoptosis in vitro. Finally, we performed RNA-sequencing (RNA-Seq) and Chromatin immunoprecipitation (ChIP) assay to further gain insight into the underlying molecular mechanisms. ResultsWe found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases. More importantly, the overexpression of EZH2 is associated with poor OS in the patients. Nevertheless, simple EZH2 depletion in vitro has little impact on the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1. Consistently, UNC1999, a dual EZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells. Furthermore, we discover CDKN1C and TP53INP1 as the two important cell cycle regulators, the expression of which are repressed by EZH1/2 mediated epigenetic regulation and are restored by EZH1/2 dual inhibition. ConclusionsOur study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker for prognosis prediction. The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL.

TP53 mutations are common in mantle cell lymphoma, including the indolent leukemic non-nodal variant

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell neoplasm, but clinically indolent subtypes are also recognized. Data on the utility of mutation profiling in the context of routine workup and its role in risk-stratification of MCL patients are limited. In this study, we describe the mutational landscape and clinicopathologic correlates of a series of MCL cases at a single-institution setting. MethodsSamples from 26 patients with MCL were evaluated by NGS using DNA extracted from peripheral blood (PB) or bone marrow (BM). Evaluation of extent of PB or BM involvement was performed using flow cytometry immunophenotyping. ResultsThe study group included 17 (65%) men and 9 (35%) women with a median age of 65 years (range, 50–94). Twenty-one (81%) patients had nodal MCL (N-MCL) and 5 (19%) had the “leukemic variant” (L-MCL). Mutated genesincluded TP53 (35%), ATM (27%), CARD11 (10%); and FBXW7, NOTCH1, SPEN, BIRC3 (~5% each). Most mutations were clonal in nature. Ten unique TP53 mutations were identified in 9 samples, including 3 L-MCL cases. There was no difference in the frequency of TP53 mutations between L-MCL and N-MCL groups (p = 0.3), but TP53 mutations were subclonal in 2/3 L-MCL cases. Identification of clonal TP53 alterations in L-MCL patients prompted initiation of therapy despite low tumor burden. ConclusionsTP53 is commonly mutated in MCL. TP53 mutations may be clonal or subclonal. Seemingly indolent L-MCL may harbor subclonal TP53 mutations which may serve as a useful biomarker for prognostication, therapeutic planning, follow-up monitoring, and early detection of clonal expansion.

CCND2 and CCND3 hijack immunoglobulin light-chain enhancers in cyclin D1− mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1− MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1−/D2− MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1−/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1− MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1− MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1− MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1−/D2−/D3− MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1− MCL.

Which Markers Should the used for Diagnostic Chronic Lymphocytic Leukemia Immunophenotyping Scoring System by Flow Cytometry?

The scoring system used for chronic lymphocytic leukemia (CLL) cannot make an accurate diagnosis in some cases. Novel markers are available for the differential diagnosis of CLL, especially from MCL. However, these markers are still not incorporated into diagnostic algorithms. We investigated the role of CD43, CD81, CD200, and ROR1 in the differential diagnosis of CLL and their expression in non-CLL cases. We investigated the role of CD43, CD81, CD20, and ROR1 in the differential diagnosis of CLL by incorporating them into the diagnostic panel after studying peripheral blood or bone marrow samples of 165 patients with 8-color flow cytometry. CD43 positivity was a sensitive marker but had a lower specificity for CLL. CD43 had high diagnostic value for CLL (sensitivity 100%, specificity 88.5%, AUC 98.0%). CD200 was a specific marker for CLL (sensitivity 98%, specificity 90%, AUC: 96%). CD81 expression was highest in the MCL cases, with a median expression rate of 68.5% (range: 54 - 82.5%). It was negative in all the CLL cases. For CLL, CD81 negativity had a sensitivity of 95%, a specificity of 82% and an AUC of 92%. ROR1 was positive in all CLL and MCL cases. CD79b, on the other hand, was a fairly sensitive and specific marker for MCL. CD43, CD81, CD200, and ROR1 should be incorporated into diagnostic algorithms for the differential diagnosis of CLL, especially from MCL.

CD81 Expression in the Differential Diagnosis of Chronic Lymphocytic Leukemia.

Immunophenotyping has a central role in CLL. However, CLL is a very heterogenous disease, both morphologically and immunophenotypically; thus, its diagnosis may prove a challenge. We investigated CD81 ex-pression in the differential diagnosis of CLL and MCL. We retrospectively examined CD81 expression with 8 color Multiparameter Flow cytometry devices in 101 CLL and 19 MCL cases. We found negative CD81 expression in CLL cases whereas it was positive in MCL cases. Our results suggest that CD81 may be a valuable marker for the differential diagnosis of CLL. We are of the opinion that it should be definitely included in the diagnostic algorithm for CLL.

CD10-positive mantle cell lymphoma: clinicopathologic and prognostic study of 30 cases.

Mantle cell lymphoma is usually negative for CD10 which is useful in distinguishing MCL from other CD10 + B cell lymphomas. Here we assessed the clinicopathologic features of 30 cases of CD10+ MCL, the largest series to date in the English literature, and compared them with a group of 212 typical MCL cases (CD5+, CD10-negative, CD23-negative, cyclin D1+). The 30 patients with CD10+ MCL included 17 men and 13 women with a median age of 68 years. Compared with CD10-negative MCL, patients with CD10+ MCL showed a lower male predominance (p = 0.01), more often had a diffuse growth pattern (p = 0.04) and blastoid/pleomorphic morphology (p < 0.0001), and more often showed BCL6 expression (p = 0.009). In all MCL patients, CD10 expression was not associated with overall survival (OS) (p = 0.16). However, in more aggressive subsets of MCL patients including those with high Ki67 (> 60%), blastoid/pleomorphic morphology, or high MCL International Prognostic Index (MIPI), CD10 expression was associated with a worse OS (p = 0.003, 0.04, and 0.001, respectively). High Ki67 (> 60%), blastoid/pleomorphic morphology, and high MIPI were also been identified as poor prognostic factors patients with in CD10+ MCL (p = 0.001, 0.0003, and 0.01, respectively). In summary, CD10+ MCL more often has a diffuse growth pattern, blastoid/pleomorphic morphology, and BCL6 expression. In MCL patients with a high Ki-67 (> 60%), blastoid/pleomorphic morphology, or high MIPI, CD10 expression contributes to an even worse prognosis. MCL should be included in the differential diagnosis of CD10 + B cell lymphomas.

Immunophenotyping of Chronic Lymphocytic Leukemia.

Chronic Lymphocytic Leukemia (CLL) is one of the most common diagnoses made by flow cytometry laboratories. There is no consensus on which markers need to be used in flow cytometry for accurate immunophenotyping. Herein, we investigated the role of markers used in flow cytometry in the distinction between CLL and MCL. A total 339 recently diagnosed B lymphoproliferative patient cases were retrospectively studied for their immunophenotypical propoerties using flow cytometry. They included 306 CCL cases and 33 MCL cases. The positivity of CD23 was diagnostic for CLL (p < 0.001). CD22, CD79b, and FMC7 expressions were highly positive in CLL cases, but not statistically significant in making differential diagnoses between atypical CLL and MCL (p = 1.000, p = 0.431 and p = 1.000, respectively). Evaluation of CD11c, CD25, CD43, and CD38 expressions, which are included in the LPD panel but not in the matatutes scoring, revealed that CD11c, CD38, and CD43 expressions are statistically significant in the distinction of atypical CLL from MCL (p < 0.001, p < 0.001, and p < 0.001). We can say that CD11c, CD38, and CD43, which have been included in our lymphoproliferative disease panel, were more valuable than CD22, CD79b, and FMC7 in the diagnosis of CLL.

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton's tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

LRPAP1 Is a Frequent B-Cell Receptor Antigen in Mantle Cell Lymphoma (MCL)

Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens.Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens.Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1.Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36)Supported by Wilhelm-Sander-Stiftung DisclosuresNo relevant conflicts of interest to declare.

Developing Novel Therapeutic Strategies to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma

MCL accounts for 7% of all non-Hodgkin lymphoma cases and is a rare and incurable subtype of B-cell lymphoma. Unfortunately, most MCL patients experience disease progression after frontline therapy, with a median overall survival of approximately 1-2 years after relapse; therefore, novel therapies for MCL are urgently needed. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was recently approved by the FDA to treat this disease. In our prior multiple-center phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68%, with a median progression free survival (PFS) of 13.9 months, surpassing the effectiveness of other therapies. Based on our phase II clinical trial, approximately 48% of patients showed no response to ibrutinib or displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment. Furthermore, the patients who initially show lengthy, durable responses to ibrutinb often acquire resistance and relapse at a median of 17 months. Once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%; therefore, the vast majority of MCL patients who experience disease progression after ibrutinib treatment will die within 12 months. Overall, the dismal outcomes of these patients demand that novel and pioneering approaches be taken to overcome ibrutinib resistance and improve the cure rate of MCL. To understand the mechanism(s) of acquired resistance for ibrutinib in MCL cells, we have recently generated two ibrutinib-resistant Jeko-1 and PF-1 MCL cell lines. These resistant cells were generated in culture by growing these cells in increasing concentrations of ibrutinib over a period of six months. The ibrutinib-resistant MCL cell lines were at least 8-10-fold more resistant to ibrutinib in comparison to the parental cell lines. The ibrutinib-resistant MCL cell lines were also cross-resistant to another BTK inhibitor, BGB, suggesting that BTK is the specific target in the resistant cell lines. Whole exome sequencing (WES) did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. Next, we conducted proteomics analyses using reverse-phase protein array (RPPA) to examine the protein expression profile of parental and ibrutinib-resistant MCL cell lines. We discovered a significant increase the levels of PI3K/AKT/mTOR/MCL-1 compensatory pathway components in both Jeko-1 and PF-1 ibrutinib-resistant cell lines when compared with their parental cells. Next, we screened for potential therapeutic agents that can overcome ibrutinib resistance. Of the many key agents tested, we discovered that the proteasome inhibitor carfilzomib (CFZ), the Bcl-2 inhibitor ABT-199, and the mTOR inhibitors BEZ and AZD demonstrated the greatest potential to overcome ibrutinib resistance in MCL cells. Interestingly, the ibrutinib-resistant MCL cells developed sensitization to CFZ. We found that the IC50 values of CFZ in the PF-1 ibrutinib-resistant cells were significantly lower (10-fold) in comparison to the IC50 values in the PF-1 parental line, which is consistent with primary MCL cells, where MCL cells derived from ibrutinib-resistant patients are more sensitive to CFZ than ibrutinib-naïve primary MCL cells. These findings are reflected by the function of the 20S proteasome because ibrutinib-resistant MCL cells have higher 20S proteasome activity. In contrast, MCL cell lines and primary MCL cells are highly sensitive to ABT-199 (IC50 < 50 nM) regardless of ibrutinib sensitivity, and ABT-199 is strictly dependent on Bcl-2 protein expression. Furthermore, based on our RPPA data, BEZ and AZD were used to target mTOR to determine whether mTOR inhibition reverses ibrutinib resistance. Our data showed that ibrutinib-resistant MCL cells are more highly resistant to both BEZ and AZD compared with the parental lines. Taken together, our data indicate that CFZ, ABT-199, and mTOR inhibitors have the potential to overcome ibrutinib resistance, and further investigation with in vivo models is warranted to validate our findings. DisclosuresPham:Amgen, Onyx, Millennium: Research Funding. Wang:Celgene: Research Funding.

Abstract 1691: Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of MCL, not all the patients respond and relapse frequently occurs. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, that was associated with plasmacytic differentiation features, like IRF4 and Blimp-1 upregulation. As the immunomoduladory drug lenalidomide has been shown to modulate IRF4 expression in various B-cell malignancies, we assessed its activity in in vitro and in vivo settings by means of flow cytometry, western blot, antibody array, real-time PCR, immunofluorescence, in vivo imaging, and immunohistochemistry. In vitro, lenalidomide as single agent was found to exert antitumor activity in 4/11 MCL cell lines, corresponding to those cells with either primary or acquired resistance to bortezomib. Lenalidomide-treated cells showed decreased IRF4 expression, increased cytosolic amounts of p27 and caspase-dependent apoptosis. Accordingly, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a lenalidomide regimen of 10-50 mg/kg/day, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated mice (p=0.04), with several hallmarks of lenalidomide activity, like downregulation of IRF4 and its target gene MYC, decreased mitotic index, p27 cytosolic accumulation and caspase-3 processing. Importantly, the inhibition of tumor growth induced by the combination of lenalidomide with bortezomib (0.15 mg/kg, twice a week) was 37% and 66% greater than that for lenalidomide alone and vehicle arms, respectively (p=0.02). Moreover, repression of MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide therapy. Accordingly, co-treatment of mice with lenalidomide plus CPI203 synergistically reduced MYC and IRF4 expression and tumor burden, and induced caspase processing. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition. Citation Format: Alexandra Moros, Vanina Rodriguez, Ifigenia Saborit-Villarroya, Arnau Montraveta, Patricia Balsas, Peter Sandy, Antonio Martinez, Emmanuel Normant, Patricia Perez-Galan, Elias Campo, Dolors Colomer, Gael Roue. Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2014-1691

Potentiated B-Cell Antigen Receptor Signaling In Mantle Cell Lymphoma Is Associated With Overexpression Of Surface CD79B and IgM

BackgroundMantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma (NHL) and represents one of the most challenging lymphoma types to treat. The B-cell antigen Receptor (BCR) signaling pathway is increasingly implicated in the pathogenesis of some B-cell malignancies, and represents an attractive target for therapy. Recent clinical trials have shown that MCL is sensitive to small molecule inhibitors targeting BCR signaling, with response rates reported to be 68% for the BTK-inhibitor Ibrutinib, but only 40% for the PI3Kδ-inhibitor CAL-101. By contrast, chronic lymphocytic leukemia (CLL) has high response rates to both drugs. We hypothesized that basal- or BCR-induced phosphorylation of key molecules that transmit the BCR signal might differ across different NHL, and thereby help us understand therapy responses to small molecular inhibitors. MethodsSingle cell flow cytometry measurements of signaling were acquired for samples of MCL (n=34), all obtained prior to therapy. For comparison, samples from CLL (n=14), follicular lymphoma (FL, n=27), diffuse large B-cell lymphoma (DLBCL, n=12), as well as healthy donor peripheral blood (n=8) and tonsils (n=4) were included. BCR-induced signaling was obtained by stimulation with goat anti-human IgM F(ab')2 and IgG F(ab')2 fragments for 4 or 45 minutes. Phosphorylation of 14 signaling molecules was measured under basal (unstimulated) and activation-induced signaling, and CD20, CD3, CD5 and BCL2 were used for discrimination between malignant B cells and tumor infiltrating T cells. ResultsMeasurements of basal phosphorylation levels of signaling molecules displayed differences among the NHL types tested. MCL together with DLBCL had highest frequency of patients with tumor cells having elevated levels of p-p65 NF-kB. In contrast, CLL and FL did not have higher basal levels of p-p65 NF-kB. Elevated basal levels of p-Syk and p-AKT1 were most frequently found in CLL and DLBCL, and were less frequent in MCL and not found in FL. In addition, elevated basal level of p-SFK and p-STAT5 were found in a group of MCL patients. BCR-induced signaling in tumor cells showed striking differences across NHL types. For key BCR-signaling molecules including SFK, SYK and PLCg2, BCR-induced phosphorylation was significantly higher in MCL as compared to the other NHL, and was strikingly different from the low levels observed in CLL (Figure 1). Similar results were obtained for Src family kinases (SFK), ERK1/2, AKT1 and STAT5. Although BCR-induced signaling was highly potentiated in most MCL cases, there was a large variation within the MCL cohort. Hence, the fold change of BCR-induced p-PLCg2, relative to unstimulated cells, ranged from 0.28 to 3.01 in MCL tumors, as compared to 0.44 to 0.89 for healthy donor B cells. Furthermore, BCR-induced p-SYK in MCL correlated strongly with p-PLCg2 (r=0.96, p<0.0001), suggesting changes early in the signaling cascade. We therefore measured surface expression of the BCR subunits, IgM and CD79B, and found a strong association between BCR-induced p-SYK and IgM (Figure 1 r=0.65) and BCR-induced p-SYK and CD79B (r=0.64, p<0.0001). [Display omitted] ConclusionDifferences in basal phosphorylation levels of key signaling molecules were found across NHL. BCR-induced signaling was highly potentiated in MCL as compared to the other NHL, and was associated with increased expression of CD79B and IgM. Differences in phosphorylation levels of signaling molecules between different NHL, as well as heterogeneity within each entity might help us understand therapy responses to small molecular inhibitors, although this needs to be tested in follow-up studies. Disclosures:No relevant conflicts of interest to declare.

The Genetic Landscape Of Mantle Cell Lymphoma and The Epigenetic Origins Of Lineage Specific Mutations

Mantle cell lymphoma is an uncommon form of non Hodgkin lymphoma that is characterized by poor responsiveness to chemotherapy and a high rate of mortality. While translocation of CCND1 is a defining feature of the disease, the role of collaborating somatic mutations that contribute to mantle cell lymphoma remains to be better defined.In this study, we sought to better understand the patterns of mutations that occur in tumors derived from closely related stages of B cell differentiation. We began by performing exome sequencing in 55 cases of mantle cell lymphoma to broadly identify the mutational landscape of the disease. We then defined the chromatin structure of the normal counterpart B cells (naïve and germinal center B cells respectively) for mantle cell lymphoma and Burkitt lymphoma by profiling their epigenetic markers using chromatin immunoprecipitation followed by sequencing (ChIP-seq). We found that the somatic mutational profiles of mantle cell lymphoma and Burkitt lymphomas overlapped strongly with areas of open chromatin in their normal counterpart B cells, identifying B cell developmental lineage as a factor in the acquisition of somatic mutations.We identified somatic mutations affecting 311 genes in at least one tumor/germline pair among MCL cases. These variants were not present in publicly available data from normal controls including dbSNP135, the 1000 Genomes Project and available exome sequencing data from healthy individuals without lymphoma. We further required each of the identified genes to have a minimum of two rare and predicted functional variants for genes already in COSMIC, and three such variants in novel genes. The most frequently mutated genes in mantle cell lymphoma were ATM (41.9%), CCND1 (14%) and TP53 (18.6%). Other frequently mutated genes included known oncogenes and tumor suppressor genes such as RB1, SMARCA4, and APC. Our data also implicated a number of genes not previously associated with mantle cell lymphoma, including POT1, FAT4 and ROBO2. Silencing mutations (frameshift and nonsense mutations) comprised a sizeable fraction of the genetic events in ATM, MLL3, RB1 and ROBO2, suggesting that those alterations result in a loss of function in mantle cell lymphoma.To better understand the genetic differences between common non Hodgkin lymphomas, we examined exome sequencing data that we and others have generated from Burkitt lymphoma and diffuse large B cell lymphoma (DLBCL). We identified all genes that were mutated in roughly 10% of at least one lymphoma type and differentially mutated relative to at least one of the other lymphoma types (P<0.05, Fisher's exact test). We found a number of genes that were predominantly mutated in each type. Mutations in ATM, CCND1, POT1 and RB1 occurred mostly in mantle cell lymphoma. Mutations in ID3 and MYC occurred predominantly in Burkitt lymphoma. Mutations in PIM1, FRMPD1 and CREBBP occurred mostly in DLBCLs. A number of genes had overlapping patterns of mutations between two or more of the diseases, including MLL3, TP53, ARID1A and SETD2.We sought to define the relationship between the epigenetically determined chromatin state of normal B cells and the lymphomas that are thought to arise from them. We began by FACS-sorting normal naïve B cells, germinal center B cells and memory B cells from otherwise normal individuals undergoing tonsillectomy. We profiled the chromatin structure and epigenetic state of the normal B cells through chIP-seq on 6 different markers: H3K4me1, H3K4me3, H3Ac, H3K27me3, H4K36me3, and CTCF. Using these epigenetic markers, differences in open chromatin between naïve B cells and germinal center B cells were computed for genes that were differentially mutated in mantle cell lymphoma and Burkitt lymphoma. We found that difference in gene mutation frequency between mantle cell lymphoma and Burkitt lymphoma is highly associated with differences in open chromatin in their corresponding cells of origin (P=0.02, Fisher's exact test).Our work demonstrates that the sequencing of relatively uncommon tumors such as mantle cell lymphoma and Burkitt lymphoma afford not only new insights into the genetics of these tumors, but also allow us to better examine the broader effects of lineage and epigenetic alterations in cancer. This work thus provides an important starting point for understanding the genetic diversity of mantle cell lymphomas and the interplay between genetic and epigenetic alterations in cancer. Disclosures:No relevant conflicts of interest to declare.

T-Cell Frequencies In MCL Are Of Prognostic Importance In a Large Population-Based Cohort

Mantle cell lymphoma is a non-Hodgkin lymphoma with, in general, a poor prognosis. A minor subset of patients with an indolent disease course has however been recognized (1,2). The various growth patterns of MCL, i.e. mantle zone (MZ), nodular (N) or diffuse (D) is assumed to correlate to stage and to disease course. The genetic aberrations underlying the pathogenesis are well defined and correlate to high tumour cell proliferation and poor prognosis. However, the effect of the lymphoma microenvironment in disease development and sustainability is largely unknown.We have used flow cytometry to investigate the non-malignant cell composition of the lymph node microenvironment in a population-based cohort of 154 MCL cases diagnosed from January 1, 1998 to December 31, 2012. Flow cytometry analyses of lymph nodes, performed as part of the diagnostic process, were used to evaluate percentages of tumour cells, remaining non-malignant B-cells and T-cell subsets (CD3+, CD3+CD4+, CD3+CD8+). As lymph node T-cell numbers reflect a high tumor load in the lymph node we also investigated the CD4/CD8 ratio, which is not dependent on T-cell percentage. Data from 26 non-malignant lymph nodes were used for comparison. T-cell percentages are shown in Table 1. Clinical and other pathological parameters of the MCL cases, including MIPI, cell morphology, tumor growth pattern and cell proliferation were also evaluated. Indolent disease (n=15), defined here as requirement of treatment > two years from diagnosis, was associated with higher amount of CD3, CD3+CD4+ and higher CD4/CD8 ratio (p=0.0429, p=0.0211 and p= 0.0032 respectively). Higher tumor cell proliferation correlated negatively with the CD4/CD8 ratio (p= 0.0007). There was a significant difference in CD3 percentages between reactive lymph nodes and MCL irrespective of growth pattern (all p<0.001). Within the entity of MCL, CD3 percentage were higher in MZ growth pattern compared to N and D (p=0.014 and p<0.001, respectively). CD3 percentages were also higher in N compared to D growth pattern (p=0.0086). CD4/CD8 ratio decreased according to growth pattern (MZ compared to N p=0.048, MZ compared to D p=0.003). Blastoid and classical MCL variants did not differ significantly in amount of CD3, CD3+CD4+, CD3+CD8+, CD19 or CD4/CD8 ratio (all p>0.05). Furthermore, male sex negatively correlated with CD4/CD8 ratio (p=0.0268). Age was not associated to T-cell percentages, CD4/CD8 ratio or growth pattern. In survival analysis a high CD4/CD8 ratio was positively correlated with OS (p= 0.0175).Table 1T-cell percentages (% of mononuclear cells) and CD4/CD8 ratio in reactive lymph nodes and MCL with different growth patterns (marginal zone (MZ), nodular (N) and diffuse (D)) (median, range).CD3CD4CD8CD4/CD8Reactive lymph nodes n=2652.9 (17.6-75.6)39.6 (14.1-63.4)10.0 (2.4-19.2)4.2 (2.1-6.9)MCL MZ n=2530.4 (10.9-84.0)17.4 (7.4-63.2)7.9 (1.2-21.1)3.1 (0.9-7.7)MCL N n=2716.7 (2.3-71.4)9.6 (1.1-58.9)5.4 (1.1-15.2)1.8 (0.6-8.6)MCL D n=469.9 (2.0-43.7)5.5 (0.7-37.0)4 (0.4-18.1)1.6 (0.4-10.4)In conclusion, our data show that the normal lymph node microenvironment is better preserved in indolent MCL and MCL with mantle zone growth pattern. The CD4/CD8 ratio is independent of lymph node tumor burden and high CD4/CD8 ratio was found to correlate with better OS. MCL tumor cells have recently been reported to impair T-cell responses (3). Our results could reflect a disease evolution towards lower tumor cell dependency on signals from the microenvironment and/or as a lymphoma mediated suppression of immune mechanisms for tumor control in aggressive MCL.

A 7-Gene Microrna Signature Characteristic of Mantle Cell Lymphoma Reveals Focal Adhesion and Integrin Signalling, Proteasome-Mediated Degradation, and the PI3K Signalling Cascade As Important to MCL Pathogenesis

AbstractAbstract 1586 BackgroundMantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and MethodsTotal RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. ConclusionsUsing the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Baseline Serum Cytokines Profiles in Mantle Cell Lymphoma Correlates with Outcome

AbstractAbstract 1579 Background:There is a growing awareness of the biological heterogeneity of MCL, which likely translates into differences in outcomes. Cytokines play an important role in the pathogenesis of lymphoma. Recent reports suggest that cytokine profiles can identify DLBCL (ASH 2010: Abstract 991) and HL pt (ASH 2011: Abstract 429) subsets with inferior outcomes. A distinct cytokine profile for MCL has not yet been defined. We therefore examined serum cytokines levels in MCL pts to gain insight into which may have biological relevance and their association with clinical outcomes. Methods:Serum samples from MCL pts (57% newly diagnosed, 43% relapsed) were prospectively collected and linked to relevant clinical data from our MCL outcomes database. Cytokine levels were determined quantitatively utilizing a commercially available protein array system. We first used Wilcoxan tests to identify relevant cytokines by comparing the distribution of 507 serum cytokines levels in MCL cases (n=97) to serum cytokines levels from normal controls (n=20). For cytokines that showed significant differences, we report the ratio of median values (cases vs. controls). To maintain a type I error rate of.05 we adjusted for multiple comparisons (Hochberg's method). Using Cox regression model and adjusting for multiplicity, we next examined the association between serum cytokines levels and outcome (PFS) in a MCL cohort uniformly treated with R-HyperCVAD (R-HCVAD). Results:214 pts with MCL were identified of which 97 pts had available serum samples. Clinical characteristics were as follows: med age 62 yr, med MIPI score of 4, med Ki67 25% (range 5–95%) and median follow up of 35 months. Our analysis identified 22 cytokines with statistically significantly distinct levels in MCL pts (vs. normal serum controls) including (p<.05): angiopoietin-1, angiostatin, endothelin, FGFR3, FGF18, GDF3, APJ, 6Ckine, CXCR4, BDNF, Glut1, Inf γ, IL2, IL3Rα, IL4R, IL10, IL15, IL17C, Gro.a, LeptinR, MCP1 and NrCAM. In univariate analysis, we were able to identify 6 cytokines (analyzed as continuous variables) that at baseline correlated with outcome (PFS) in a cohort (n=42) of uniformly treated MCL pts with R-HCVAD in the front-line setting (p<.05). These cytokines were as follows: Activin receptor type 1, Macrophage inflammatory protein 1 β (CCL4), Neutrophil collagenase (MMP8), Neurturin, Serum amyloid A protein and Ubiquitin (Table). Conclusions:Our results are the first to demonstrate a serum cytokines signature associated with MCL and additionally suggest an association between serum cytokines levels and outcome in MCL patients treated with R-HCVAD. These results may provide new insight into the biology of MCL and add to the known heterogeneity of MCL biology. An elevated pretreatment baseline serum cytokines subset, (mostly related to tumor and inflammatory processes) was found to be associated with an increased likelihood of disease relapse and an inferior outcome in patients with MCL treated with R-HCVAD and may predict pts at higher risk of relapse in that setting.CytokineCharacteristicsActivin receptor type 1TGF-B superfamily receptor type ISerum amyloid A proteinSerum acute phase proteinMacrophage inflammatory protein 1 â (CCL4)Family of chemotactic cytokinesNeutrophil collagenase (MMP8)Breakdown of extracellular matrix in remodeling/metastasis.NeurturinGFRA2 receptor ligandUbiquitinProtein recycling/high activity in MCL cells Disclosures:Mato:Celgene: Speakers Bureau; Millennium: Speakers Bureau; Seattle Genetics: Speakers Bureau; Genentech: Speakers Bureau. Feldman:Merck: Speakers Bureau; Seattle Genetics: Speakers Bureau; Celgene: Speakers Bureau; Allos: Speakers Bureau. Goy:Milennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees; Seattle Genetics: Membership on an entity’s Board of Directors or advisory committees; Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees; J & J: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees.