BCL2 Amplification Research Articles

Unraveling the Evolutionary Paths in Relapsed Early-Stage Follicular Lymphoma Showing Complete Remission

Background: Follicular lymphoma (FL) develops through a stepwise acquisition of cooperative genetic changes with t(14;18)(q32;q21)/ IGH:: BCL2 occurring early at the pre-B stage of B-cell development. Patients with FL typically show an indolent clinical course, remitting and relapsing, and eventually developing resistance to treatments. Interestingly, the majority of transformed FL do not progress directly from the preceding FL but originate from their clonally related lymphoma precursor cells (CLP). In light of this finding, we wanted to address whether such divergent tumor evolution also occurs in the setting of relapses in early-stage FL in patients who achieved complete remission following initial treatment. Methods: We identified 13 cases of FL with an early clinical stage (stage I = 9; stage II = 4) at the initial diagnosis, who showed complete remission (mean: 5 years; range: 1-11.5 years) following local radiotherapy but subsequently relapsed (≥ 2 in 5). Paired formalin-fixed paraffin-embedded diagnostic and relapsed lymphoma biopsies were obtained in each case. BCL2 translocation status was investigated using interphase fluorescence in-situ hybridization (FISH). Genomic DNA was extracted from the tumor-rich areas in each tissue specimen and targeted next-generation sequencing was performed with a customized gene panel comprising 191 genes/ regions that are implicated in FL, diffuse large B-cell lymphoma (DLBCL), and B-cell biology using the TWIST target enrichment kits and Illumina NextSeq platform. Clonality analysis of IG gene rearrangements was performed by combining the BIOMED-2 PCR assays and Illumina MiSeq sequencing. Sequencing data analyses, variant calling, and filtering were performed using a well-established data analysis pipeline from a previous study (Cucco et al., 2018). Results: Interphase FISH showed BCL2 translocation in 6 cases, BCL2 amplification (up to 8 copies) in 1 case, and BCL2 copy number gain (3-4 copies) in 3 cases. Pathogenic variants were identified in each of the lymphoma biopsies and were typical of those frequently seen in FL and DLBCL. There was no significant difference in the mutational burden between the paired diagnostic (mean: 7.8/sample; range: 1-13) and relapsed FL (7.9/sample; range: 2-13), excluding tFL. Both the diagnostic and relapsed lesions showed similar, frequent mutations in BCL2 (33.3% vs 41.67%), CREBBP (58.3% vs 58.3%), EP300 (41.7% vs 41.7%), KMT2D (50% vs 58.3%), TNFRSF14 (66.7% vs 58.3%), and STAT6 (50% vs 50%). The clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases. In 6 cases, common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating their divergent evolution from a CLP. In 2 cases, distinct B-cell clones were involved in the diagnostic and the relapsed lymphomas, including 1 case involving 2 different BCL2 translocations, indicating independent evolution of the new lymphomas. In the remaining 5 cases, the relapsed lymphomas developed via a linear progression from the preceding FL (n = 5). Conclusion: Both divergent evolution, either from clonally related or unrelated precursors, and linear evolution processes are involved in the development of relapses in patients with an early-stage FL following complete remission, with the former being more frequent. The above findings, together with the evidence of divergent evolution of tFL in the majority of cases, challenge the current therapeutic strategy of early-stage FL, which does not target the premalignant cell population that underpins lymphoma recurrence in the majority of cases. Additionally, the routine diagnostic workup, which includes IG gene clonality and BCL2 translocation analyses, cannot address the evolutionary path in clonally related lymphomas. Our results indicate the significance of mutational profiling at diagnosis as well as at relapse, and the need to comprehensively characterize and target the evasive CLP population to prevent recurrence.

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.

Copy number aberrations in circulating tumor DNA enables prognosis prediction and molecular characterization of breast cancer.

Low-pass whole-genome sequencing (LP-WGS)-based circulating tumor DNA (ctDNA) analysis is a versatile tool for somatic copy number aberration (CNA) detection, and this study aims to explore its clinical implication in breast cancer. We analyzed LP-WGS ctDNA data from 207 metastatic breast cancer (MBC) patients to explore prognostic value of ctDNA CNA burden and validated it in 465 stage II-III triple-negative breast cancer (TNBC) patients who received neoadjuvant chemotherapy in phase III PEARLY trial (NCT02441933). The clinical implication of locus level LP-WGS ctDNA profiling was further evaluated. We found that a high baseline ctDNA CNA burden predicts poor overall survival and progression-free survival of MBC patients. The post hoc analysis of the PEARLY trial showed that a high baseline ctDNA CNA burden predicted poor disease-free survival independent from pathologic complete response (pCR), validating its robust prognostic significance. The 24-month disease-free survival rate was 96.9% and 55.9% in [pCR(+) and low I-score] and [non-pCR and high I-score] patients, respectively. The locus-level ctDNA CNA profile classified MBC patients into 5 molecular clusters and revealed targetable oncogenic CNAs. LP-WGS ctDNA and in vitro analysis identified the BCL6 amplification as a resistance factor for CDK4/6 inhibitors. We estimated ctDNA-based homologous recombination deficiency status of patients by shallowHRD algorithm, which was highest in the TNBC and correlated with platinum-based chemotherapy response. These results demonstrate LP-WGS ctDNA CNA analysis as an essential tool for prognosis prediction and molecular profiling. Particularly, ctDNA CNA burden can serve as a useful determinant for escalating or de-escalating (neo)adjuvant strategy in TNBC patients.

Abstract 1631: Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs)

Abstract GISTs are common malignant mesenchymal tumors that occur in the GI tract, with an estimated incidence rate of 1% to 2%. About 75% to 80% of patients with GIST have mutations in the KIT gene, while 5% to 10% have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Tyrosine kinase inhibitors (TKIs) offer patients an improved quality of life but increased pharmacological resistance to TKIs is often observed. BCL-2 is expressed in >80% of GISTs, and amplification of BCL-2 and BCL-xL are common features associated with disease progression. One approach to enhance GIST eradication is to concurrently inhibit oncogenic KIT signaling while actively engaging apoptotic pathways. Olverembatinib (HQP1351) is a new, third-generation TKI that targets BCR-ABL1, KIT and PDGFRA and is currently in development for relapsed or refractory chronic myeloid leukemia and GIST. Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor under development for hematologic malignancies. The purpose of this study was to evaluate whether combining a BCL-2 inhibitor, lisaftoclax, with olverembatinib enhances treatment effect on imatinib-resistant GISTs. Compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1), increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in GIST430, GIST48, and GIST48B lines. Cell-based antiproliferation studies showed additive activity of olverembatinib and lisaftoclax in imatinib-resistant GIST cells. In vivo studies using imatinib-resistant GIST430 cell-derived xenograft models further revealed that coadministration of olverembatinib at 15 mg/kg (every other day for 3 weeks) with lisaftoclax at 50 mg/kg (daily for 3 weeks) resulted in enhanced antitumor effects compared to either agent alone. Tumor growth inhibition (TGI) rates for combined treatment with olverembatinib and lisaftoclax reached 76.8%, which was superior to the TGI rate of either agent alone: 57.6% and 31.2% for olverembatinib and lisaftoclax, respectively. Regarding putative mechanisms, olverembatinib treatment was shown to downregulate BCL-xL and MCL-1 protein expression, possibly via inhibition of KIT signal pathway and downstream signal transducer and activator of transcription 3 (STAT3). Lisaftoclax triggered apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly augmented cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1), thus triggering apoptosis and subsequently enhancing antitumor effects. Our results demonstrate that olverembatinib and BCL-2 inhibitor lisaftoclax have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential for treating patients with GISTs whose disease has progressed after treatment with TKIs. Citation Format: Ping Min, Guangfeng Wang, Eric Liang, Bingxing Wu, Huidan Yu, Dajun Yang, Yifan Zhai. Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1631.

Genetic Profiling of Diffuse Large B-Cell Lymphoma: A Comparison Between Double-Expressor Lymphoma and Non-Double-Expressor Lymphoma.

Data are limited regarding the genetic profiling of diffuse large B-cell lymphoma (DLBCL) with double expression of MYC and BCL2 proteins without underlying rearrangements (double-expressor lymphoma [DEL]). This study aimed to describe the genetic profiling and determine the prognostic significance in patients with DEL and in those with non-DEL. Capture-based targeted sequencing was performed on 244 patients with de novo DLBCL, not otherwise specified. Immunohistochemistry staining was performed for evaluating the MYC and BCL2 expression. Among 244 patients, 46 patients had DEL, and 198 had non-DEL. KMT2D, CD58, EP300, PRDM1, TNFAIP3 and BCL2 gain or amplification (BCL2GA/AMP) were significantly more frequently altered in the DEL group. Alterations in the BCR/TLR (p = 0.021), B-cell development and differentiation (p = 0.004), and NF-κB (p = 0.034) pathways occurred more frequently in patients with DEL. Thirty-seven DEL patients and 132 non-DEL patients were included for survival analyses. DEL was not significantly associated with progression-free survival (PFS) (p = 0.60) and overall survival (OS) (p = 0.49). In DEL patients, after adjusting for the International Prognostic Index, BCL2 alteration (HR 2.516, 95%CI 1.027-6.161; p = 0.044) remained an independent predictor of inferior PFS. BCL2GA/AMP also predicted poor PFS, but with marginal statistical significance (HR 2.489, 95%CI 0.995-6.224; p = 0.051). There was difference in profiling of altered genes and signaling pathways between the DEL group and the non-DEL group. The presence of DEL alone should not be considered as an adverse prognostic indicator, and BCL2 alteration could define a subset of patients with poor prognosis within DEL.

Inhibitors of Bcl-2 and Bruton\u2019s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma growth in vitro and in orthotopic xenotransplantation models

Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.

Preclinical Anti-Lymphoma Activity of the BCL-2 BH4 Domain Antagonist Bda-366 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell non-Hodgkin lymphoma. MCL is genetically characterized by t(11;14) translocation, which leads to cyclin D1 overexpression. Despite the introduction of high-dose chemotherapy followed by autologous stem cell transplantation and novel agents including the Bruton's tyrosine kinase inhibitor ibrutinib, the proteasome inhibitor bortezomib and the immunomodulator lenalidomide, MCL still remains an incurable disease. BCL-2 is often amplified and/or overexpressed in MCL; high-level copy number gains of the BCL2 locus at 18q21.3 are most frequent. The novel BCL-2BH4 domain antagonist BDA-366 converts anti-apoptotic BCL-2 into the pro-apoptotic molecule (Han et al., Cancer Cell 2015). Considering the promising clinical activity of the BH3 mimetics venetoclax (Davids et al., JCO 2017), we investigated anti-lymphoma effects of BDA-366 in MCL. A total of eight MCL cell lines (Z-138, JVM-2, Granta-519, MINO, JeKo-1, REC-1, MAVER-1 and NCEB-1) were used. Z-138, Granta-519, MINO and NCEB-1 have high-level DNA amplification of BCL-2 . BDA-366 induced dose- and time-dependent phosphatidylserine externalization in MCL cells, with 72-h ED50 (effective concentration inducing 50% cell killing as measured by annexin V positivity) values ranging from 9.3 to 240 nM. 72-h IC50 values (concentration at which cell growth is inhibited by 50%) were also determined, which ranged from 5 to 30 nM. BDA-366 showed smaller differences in sensitivity than venetoclax, raising the possibility that BDA-366 may exhibit broader anti-lymphoma activity against heterogeneous MCL backgrounds. Basal levels of BCL-2 (determined by flow cytometry) did not significantly affect MCL cell susceptibility (ED50) to BDA-366 (r = -0.08, P = 0.85). The presence of high-level DNA amplification of BCL-2 was not associated with BDA-366 sensitivity either. BDA-366 did not increase total BCL-2 levels but it actively promoted the exposure of pro-apoptotic BH3 domain of BCL-2, supporting its proposed mode of action. BDA-366 did not significantly affect cell cycle distribution; S and G2/M phase cells appeared to be susceptible to BDA-366-induced apoptosis. BDA-366 induced loss of mitochondrial membrane potential, caspase-3 cleavage and BAX activation, indicating mitochondrial apoptosis. p53 knockdown (>85% knockdown efficiencies, as determined by Western blotting) desensitized p53 wild-type cell lines Z-138 and JVM-2 to BDA-366 (ED50 values of 29 nM and 10 nM in Z-138; 303 nM and 108 nM in JVM-2), implying the interference of p53 with BCL-2 signaling. BDA-366 synergized with venetoclax to induce apoptosis in venetoclax-sensitive Z-138 (averaged combination index (aCI) = 0.25) and MINO cells (aCI = 0.14), indicating that the combination may abrogate BCL-2-mediated survival signaling in MCL. The combination effects of BDA-366 with doxorubicin or AraC were additive (aCI ~ 1.0). BDA-366 induced apoptosis in primary leukemia cells from patients with B-cell chronic lymphoproliferative disorders. BDA-366 appeared to take longer time to fully induce apoptosis than ABT-199, perhaps reflecting the distinct mode of action of BDA-366 that changes BCL-2 conformation. From a clinical viewpoint, the slower pharmacodynamics in inhibiting BCL-2 and inducing MCL cell death may reduce the risk of tumor lysis syndromes in patients receiving BDA-366. Collectively, BCL-2 BH4 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.

Abstract PO-08: Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma

Abstract EZH2 (Enhancer of Zeste Homolog 2) is the catalytic component of the Polycomb Repressive Complex group 2 (PRC2), which establishes the repressive epigenetic mark histone 3 lysine 27 trimethylation (H3K27me3), resulting in silencing of gene expression. EZH2 was originally identified as a tumor suppressor since loss-of-function events were observed in myelodysplastic syndrome, acute myeloid, and T-cell leukemias. However, next-generation sequencing identified recurrent activating point mutations at tyrosine residue 641 (Y641) in 25% of germinal center diffuse large B-cell lymphoma and 15% of follicular lymphomas, suggesting that EZH2 also functions as an oncogene in a cell type-dependent manner. Using a faithful genetically engineered mouse model, we previously demonstrated that the Ezh2Y641F mutation drives formation of B-cell lymphoma as a single event and in cooperation with Bcl2 amplification or p53 loss. Unexpectedly, at the chromatin level, expression of Ezh2Y641F did not monotonically increase abundance of global H3K27me3 but rather resulted in redistribution of this mark across the genome, exhibiting loss of H3K27me3 at many loci, with direct effects on transcription. Others have shown that EZH2 mediates germinal center proliferation via repression of p21 and cooperation with Bcl6; however, given the global effect of Ezh2Y641F on chromatin, the oncogenic mechanisms of these mutations remain underexplored. These include the timing of the mutation during hematopoietic development, its role during differentiation, and the oncogenic activity of downstream targets. In order to investigate the effect of Ezh2Y641 mutations on hematopoietic development and differentiation, we used Cre alleles to drive expression of the mutant protein at different stages of hematopoietic development. Our data demonstrate that expression of Ezh2Y641 in early B cells is sufficient to drive oncogenic transformation. However, expression of Ezh2Y641 in hematopoietic stem cell (HSC) is not sufficient to drive oncogenic transformation despite the presence of mature healthy B cells. Additionally, expression of Ezh2Y641F in HSCs results in loss of self-renewal, differentiation bias towards the lymphoid lineages, and a partial block at the pre-pro B-cell stage. With regards to direct downstream targets, we identified upregulation of several HoxC cluster genes in Ezh2Y641F-mutant B cells prior to transformation. Upregulation of these genes is mediated via loss of H3K27me3 at the HoxC cluster and focal gains of H3K27 acetylation (H3K27ac). Overall, our findings underline the complicated biology and oncogenic activity of EZH2Y641 mutations in lymphoma, shed light on the effect of these mutations during hematopoietic development, and underscore some of the consequences of the paradoxical loss of H3K27me3 at certain loci. Citation Format: Samantha J. Nixon, Sarah Zimmerman, Jeremy M. Simon, George P. Souroullas. Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-08.

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.

Molecular profiling of Chinese R-CHOP treated DLBCL patients: Identifying a high-risk subgroup.

Diffuse large B-cell lymphoma (DLBCL) is a clinically aggressive and heterogenous disease. Although most patients can be cured by immunochemotherapy, 30% to 40% patient will ultimately develop relapsed or refractory disease. Here, we investigated the molecular landscapes of patients with diverse responses to R-CHOP. We performed capture-based targeted sequencing on baseline samples of 105 DLBCL patients using a panel consisting of 112 lymphoma-related genes. Subsequently, 81 treatment-naïve patients with measurable disease and followed for over 1 year were included for survival analysis. Collectively, the most commonly seen mutations included IGH fusion (69%), PIM1(33%), MYD88 (29%), BCL2 (29%), TP53 (29%), CD79B (25%) and KMT2D (24%). Patients with TP53 mutations were more likely to have primary refractory disease (87.0% vs 50.0%, P = .009). For those with TP53 disruptive mutations, 91.7% patients were in the primary refractory group. Interestingly, BCL-2 somatic hypermutation was only seen in patients without primary refractory disease (P = .014). In multivariate analysis, BCL-2 amplification (hazard ratio [HR] = 2.94, P = .022), B2M mutation (HR = 2.99, P = .017) and TP53 mutation (HR = 3.19, P < .001) were independently associated with shorter time to progression (TTP). Furthermore, TP53 mutations was correlated with worse overall survival (P = .049). Next, we investigated mutation landscape in patients with wild-type (WT) TP53 (n = 58) and found that patients harboring MYD88 L265P had significantly inferior TTP than those with WT or non-265P (P = .046). Our study reveals the mutation spectrum of treatment-naive Chinese DLBCL patients. It also confirms the clinical significance of TP53 mutations and indicates the prognostic value of MYD88 L265P in TP53 WT patients.

The Role of Autologous Stem Cell Transplantation in the Treatment of Diffuse Large B-cell Lymphoma in the Era of CAR-T Cell Therapy.

For many years now and based on the results of the PARMA trial, relapsed Diffuse Large B-cell Lymphoma (DLBCL) is treated with salvage combination cytotoxic chemotherapy (most often platinum-based) followed by high dose myeloablative chemotherapy and autologous stem cell transplantation (auto-HCT). This approach has resulted in long-term disease free survival in about half of the patients. With the incorporation of rituximab in the upfront treatment (RCHOP), more patients with DLBCL are cured but there has been a signal of inferior outcomes with auto-HCT if DLBCL relapses. Nevertheless, a careful review of the literature still shows very good outcomes with auto-HCT for DLBCL with complete remission to salvage chemotherapy. For those who do not respond well to classic salvage other approaches are reviewed here including chimeric antigen receptor (CAR) T-cell therapy and treatment with antibody-drug conjugates (ADCs) as well as bispecific T-cell engagers (BiTEs). The outcome of auto-HCT after successful treatment with ADCs or BITEs is unknown. It is also unknown if CAR-T cell therapy should be reserved for those who have failed 2 lines of chemotherapy or it should be moved earlier. Finally, we review here the effects of Myc and bcl2 amplifications or translocations to the outcome of the auto-HCT. Some attempts to improve the salvage or conditioning regimens are mentioned. We also discuss the role of allogeneic stem cell transplantation (allo-HCT) in the paradigm of treatment for relapsed DLBCL.

B-ALL/NHL 2002 GMALL Protocol As First-Line Treatment in Patients with Double-Hit Lymphoma: Favorable Results in18 Consecutive Patients from Two Italian Institutions

Introduction: In the revised 2017 World Health Organization (WHO) classification of lymphoid neoplasms, high-grade B-cell lymphoma with C-MYC and BCL2 or BCL6 rearrangements is classified as Double-Hit lymphoma (DHL), while the term Triple Hit lymphoma (THL) is employed when the three genes are rearranged (Swerdlow SH, et al 2017). Amplification of C-MYC plus BCL2 and/or BCL6 rearrangement or amplification are defined as "atypical DHL and THL" (Li S., et al 2015). We will here collectively refer to DHL and THL as DHL. Both true and atypical DHL are resistant to conventional chemo-immunotherapy and are associated with poorer outcome. The median survival is shorter than 12 months with standard regimens (Snuderl M, et al 2010). The success of intensified chemo-immunotherapy protocols in Burkitt lymphoma, encouraged many Centres to include these regimens in the treatment armamentarium of DHL. Among these protocols, the B-ALL/NHL 2002 of the GMALL multicenter group is an effective and well tolerated regimen in the treatment of Burkitt's lymphoma (Pollen M, et al 2011). We have adopted the GMALL protocol, as our guideline for the treatment of patients with true or atypical DHL. The preliminary experience with the GMALL protocol in 18 patients is here reported. Methods: We retrospectively evaluated patients with true or atypical DHL treated with the GMALL protocol at the European Institute of Oncology in Milan from 2015 and at the Mauriziano Hospital in Turin from 2012. The diagnosis was confirmed according to 2017 WHO classification and immunohistochemistry analysis and fluorescent in situ hybridization were performed in all patients. Patients were treated following the R-GMALL protocol according to their age group with less intensified doses applied to patients older than 55 years. The program was part of the treatment guidelines developed at each Center for the management of lymphoma patients and a written informed consent was obtained from all patients. Results: Eighteen patients were included in this analysis, 14 were true DH and 3 atypical DH and 1 was Burkitt like lymphoma. Five were transformed lymphomas, 4 after follicular and 1 after marginal zone lymphoma. The median age of the whole group was 64 years (range 43-77years). Eleven patients were male (61%), 15 patients (83%) had Ann-Arbor stage IV and 5 (28%) had bone marrow infiltration. Median lymphoma cells proliferation fraction assessed by Ki-67 IHC was 90% (range 70-100%). Patients received a median of 6 courses (range 2-6) of the GMALL schedule, as inpatient therapy. No patients died of infectious complications during chemotherapy. Twelve patients (67%) were in complete metabolic response (CR) at end of treatment and another patient in complete response by CT scan. Of the 5 refractory patients, four have died (3 for lymphoma progression, one due to infectious complications after salvage splenectomy plus RT), one is still alive, under salvage therapy at 7 months from end of treatment. Among 12 patients who achieved CR, 3 relapsed at 7, 10, 16 months from end of treatment, they are presently alive under salvage treatment. At a median follow up of 21.7 month, the median PFS is 21.4 months while median OS is not reached. The two-year PFS and OS rates are 42% and 66%, respectively (figure 1 A &amp; B). PFS was significantly worse in patients with bone marrow infiltration with median PFS of 7 months while not reached in the rest of patients. The median PFS was approximately 17 months for patients older than 55 years (n=13) and not reached for younger patients, with PFS at 2 years of 75%. There was no correlation with transformation, or Ki-67. Conclusion: This study indicates that intensive treatment with B-ALL/NHL 2002 GMALL protocol is effective and well tolerated in true and atypical DHL patients aged up to 77 years. In this preliminary series, the OS resulted superior to that commonly reported with standard chemo-immunotherapies, with median 17-month OS in older patients while median OS is not reached in younger patients. In fact, the results were particularly promising in patients younger than 55 years probably reflecting the proper dose schedule delivery in younger patients. Based on these preliminary results, we are continuing to use the GMALL protocol as upfront therapy in DH lymphoma patients and we are exploring the possibility of improving the therapeutic efficacy by supplementing the GMALL schedule with one of the novel non-chemotherapeutic drugs. Figure 1 Disclosures Derenzini: TG-THERAPEUTICS: Research Funding. Saglio:Celgene: Consultancy; Jansen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy.

The Grey Zone: Characterization of Aggressive High-Grade B-Cell Malignancy with TdT Expression and IGH/BCL2 and MYC Rearrangement

Accurately distinguishing between mature and immature B-cell neoplasms is crucial for determining therapy. However, overlapping morphologic, cytogenetic and immunophenotypic characteristics can have diagnostic and therapeutic implications. Morphologically, high grade B-cell lymphoma with double/triple hit (HGBL) may have a "starry sky" or blastoid appearance that can resemble B-lymphoblastic leukemia (B-LBL), Burkitt's Lymphoma (BL) or diffuse large B cell lymphomas (DLBCL), but differ from immature B-cell neoplasms with expression of CD20, CD10, MUM1, and surface immunoglobulin light chains (sIG). Conversely, B-LBL and B-ALL lymphoblasts have dim or lack expression of CD20 and sIG and express markers of immaturity such as CD34 and terminal deoxynucleotidyl transferase (TdT). In this study, we present our institutional experience of 8 patients with a "grey zone" leukemia/lymphoma: 4 patients with aggressive TdT-positive HGBL and 4 patients with B-LBL or B-ALL with a "double-hit" karyotype. We hypothesize these cases may represent a separate entity between HGBL and B-LBL/B-ALL with activating Myc rearrangements. Our cohort was compiled by reviewing cases encountered in our clinical practice from July 2015 to July 2019 that could be identified as follows: (1) de novo HGBL with MYC, BCL2, and/or BCL6 rearrangements or amplifications with concurrent aberrant TdT expression at diagnosis or in subsequent biopsies and (2) de novo B-LBL or B-ALL with a "double‐hit" karyotype. Clinical and diagnostic data were obtained by review of the medical records. The Moffitt Cancer Center Institutional Review Board approved this retrospective study. Of patients with HGBL with aberrant TdT expression, 3 were HGBL with triple hit and 1 with a Bcl6 rearrangement and Bcl2 and Myc amplifications. These patients presented with aggressive features including elevated LDH at diagnosis, high IPI score, and Ki-67 &gt;/= 80%, however without bone marrow or CNS involvement. They express a typical GCB immunophenotype, positive for CD10, CD20, and MUM1 and negative for CD34 (Table 1). These patients were treated with DA-EPOCH-R (3) and R-CHOP (1), of which 1 achieved a partial response and 3 developed progressive disease (Table 2). Three patients went on to consolidative therapy with CD19-directed CAR-T (66%) or autologous transplant (33%). The median overall survival (mOS) was 12.1 months. In the second group, 2 were considered B-ALL based on immature morphology and lack of mature B-cell markers (CD20 negative or variable and MUM1 negative), although they did not express TdT. They both showed IGH-Bcl2 and Myc rearrangements. They were treated with DA-EPOCH-R, of which 1 experienced progressive disease and 1 was unable to complete therapy. The other 2 patients were diagnosed with de novo B-LBL that was CD10 and TdT positive. Of these, 1 showed MUM1 positivity and genomic testing revealed IGH-Bcl2 and Myc rearrangements accompanied by a TP53 mutation. The other showed IGH-Bcl2 rearrangement and Myc activating mutation. One was treated with HyperCVAD with an interval CR and the other with modified DFCI died 5.1 months after diagnosis. Prognosis in this second subset was poor with a mOS of 5.3 months. Within the entire cohort, the majority (75%) were treated as DLBCL with R-CHOP-based therapy, while the remaining (25%) were treated with HyperCVAD. 25% experienced a PR or interval CR, while the remainder experienced progressive disease (PD). Overall, we observed a median PFS of 6.6 months and mOS 5.6 months with 0% undergoing allogeneic transplant in first remission, 1 (12.5%) undergoing autogeneic transplant in first remission and 2 (25%) undergoing CD19-directed CAR-T following salvage therapy. HGBL that has acquired TdT expression and B-LBL/B-ALL associated with IGH/BCL2 translocation and activating MYC rearrangements or mutations are uncommon, but represent a biologically unique group with a poor clinical outcome, likely due to accumulated somatic hypermutations. The high percentage of MUM1 expression in these cases suggests a role of dysregulated MUM1 in cooperation with Myc dysregulation. The recognition of this group may be under-appreciated due to the lack of awareness. Inclusion of evaluation of TDT in "mature" HGBL and identification of genetic alterations including BCL-2, BCL-6 and Myc in "immature" B-LBL/B-ALL will be helpful. Consolidation upon first remission with transplant or CAR-T may be of benefit. Disclosures Chavez: Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Bello:Celgene: Speakers Bureau. Bunting:Transenterix: Equity Ownership; Tilray: Equity Ownership; Crispre: Equity Ownership; Celgene: Equity Ownership. Shah:Kite/Gilead: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Celgene/Juno: Honoraria.

Correlation Between C-MYC, BCL-2, and BCL-6 Protein Expression and Gene Translocation as Biomarkers in Diagnosis and Prognosis of Diffuse Large B-cell Lymphoma.

This study investigates the protein expression of C-MYC, BCL-2, and BCL-6 in diffuse large B-cell lymphoma (DLBCL) and their relationship with genetic abnormalities. A retrospective study of 42 cases on paraffin-embedded tissue specimens diagnosed with DLBCL was performed using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The expression of C-MYC, BCL-2, BCL-6 protein, and gene abnormalities in these tissue samples was analyzed. The relationship in genetic abnormalities and Ki-67, Hans classification, gender, and age was also evaluated. It was found that the positive rate of C-MYC expression was 47.6% (20/42), the rate of C-MYC gene abnormality was 26.2% (11/42), in which gene translocation accounted for 23.8% (10/42) and gene amplification 2.4% (1/42); C-MYC protein expression was positively correlated with C-MYC gene translocation (χ2 = 11.813; P = 0.001); C-MYC gene translocation was mainly found in germinal center B cell type (χ2 = 4.029; P = 0.045). The positive rate of BCL-2 protein expression was 85.71% (36/42), the positive rate of translocation was 42.86% (18/42) and the amplification rate was 26.19% (11/42); the overexpression of BCL-2 protein was correlated with the BCL-2 translocation (χ2 = 3.407; P = 0.029). The positive rate of BCL-6 protein expression was 45.24% (19/42), the positive rate of BCL-6 translocation was 14.29% (6/42) and the positive rate of BCL-6 amplification was 7.14% (3/42); the overexpression of BCL-6 protein was significantly correlated with BCL-6 translocation (χ2 = 6.091; P = 0.014). The Ki-67 index was significantly higher in C-MYC translocation cases than in non-C-MYC translocation cases (χ2 = 4.492; P = 0.034). Taken together, our results suggest that the protein expression of C-MYC, BCL-2, and BCL-6 are positively correlated with their gene translocation. Overexpression of C-MYC, BCL-2, BCL-6 protein suggests the possibility of translocation. Therefore, immunohistochemical detection of C-MYC, BCL-2, and BCL-6 are useful in diagnosis and prognosis of DLBCL.

Clinicopathological and fluorescence in situ hibridisation analysis ofprimary testicular diffuse large B-cell lymphoma: asingle-centre case series.

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) represents arare and aggressive extranodal non-Hodgkin's lymphoma (NHL) with some specific features that differ from other NHLs. Formalin fixed, paraffin wax embedded (FFPE) samples of21 PT-DLBCLs and 30 comparative patients with DLBCL were analysed. All PT-DLBCL patients were treated with rituximab-containing regimens, intrathecal prophylaxis (10 patients), and irradiation ofthecontralateral testis (9 patients). FFPE samples were additionally analysed by immunohistochemistry (Bcl-2, c-Myc protein expression) and fluorescence in situ hybridisation (FISH) (BCL2 and MYC). The patients with PT-DLBCL (median age 48.5 years), had low frequency of B symptoms (28.6%) and were often diagnosed in I and II Ann Arbor clinical stage (66.0%). Themajority ofPT-DLBCL (80.9%) had anon-germinal centre B-cell-like immunophenotype. Immunohistochemical staining showed increased c-Myc protein expression in thePT-DLBCL group compared to thecontrol group (p=0.016). MYC rearrangement was detected in 1 of14 (7.0%), and MYC amplification in 3 of14 (21.0%) patients. One ofthe14 cases (7.0%) in thePT DLBCL group showed BCL2 rearrangement, and four of14 (28.05%) cases showed BCL2 amplification. Complete remission (CR) was achieved in 75.0% ofPT-DLBCL patients who had superior survival compared to those who did not achieve CR (median 48 vs. 21 months, p=0.012). Patients with PT-DLBCL express some immunohistochemical, biological, and clinical features that might differentiate them from nodal and extranodal DLBCL patients, indicating theneed for amore personalised treatment approach.

Abstract 45: BTM-3528 potently induces G1/G0 cell cycle arrest and is efficacious in preclinical models of diffuse large B-cell lymphoma

Abstract Aberrant regulation of cell cycle control, integration of signals from oncogenic signaling pathways, and cancer cell metabolism play a key role in the pathogenesis of multiple hematologic malignancies and solid tumors. BTM-3528 is an orally available compound that causes concentration-dependent cell cycle arrest in the G0/G1 phase of the cell cycle, rapidly decreases cellular glutathione levels, induces expression of p21, and decreases expression of Cyclin D 1/3. BTM-3528 has no significant biochemical activity against a panel of known drug targets such as kinases (including cyclin-dependent kinases), G-protein coupled receptors, ion channels, and histone deacetylases. For the described study, the activity of BTM-3528 was evaluated in in vitro and in vivo models of diffuse large B-cell lymphoma (DLBCL). Profiling of BTM-3528 across a panel of hematologic and solid tumor cancer cell lines demonstrated potent antiproliferative activity against a select set of cancer cell types without effect on normal human cells. These included multiple DLBCL cell lines notably of germinal center B-cell (GCB) subtype. The majority of the DLBCL cell lines tested were characterized by Myc and Bcl-2 gene rearrangements or amplifications. Cell proliferation was 100% inhibited with IC50s ranging from 0.14 to 0.52 μM in these DLBCL cell lines. BTM-3528 induced apoptosis with caspase 3/7 activation and decreased Mcl1 protein expression at concentrations associated with cell growth inhibition. In vivo pharmacokinetic studies showed that BTM-3528 has excellent oral bioavailability with a murine t ½ of 6 hours. BTM-3528 was tested in a human subcutaneous DLBCL xenograft model (BJAB). Post tumor implantation, animals with tumors ≥ 250 mm3 were treated with BTM-3528 at the maximum tolerated dose of 75 mg/kg once daily orally for 14 days. Significant antitumor activity was observed after 3 days of treatment followed by sustained tumor regressions. Furthermore, following discontinuation of BTM-3528 after 14 days of treatment, sustained complete tumor regressions were maintained in 50% of the treated animals. Preliminary toxicology evaluation of BTM-3528 showed no adverse effect on hematopoiesis or hepatic function. Conclusion: BTM-3528 is a novel orally bioavailable compound with a unique mechanism of action based on G1/G0 cell cycle arrest and modulation of cancer cell metabolism. It shows significant activity and a favorable toxicity profile in in vitro and in vivo models of DLBCL. These results support the clinical development potential of BTM-3528 for the treatment of DLBCL. Citation Format: Matthew Kostura, Jedd Levine, Vibha Oza, Alan Cooper, Meztli Arguello, Peter W. White, Stephane Ciblat, Anthony Regina, Edith Bellavance, Martine Brault, Arshad Siddiqui, Michael Luther. BTM-3528 potently induces G1/G0 cell cycle arrest and is efficacious in preclinical models of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 45.

Comparative analysis of primary versus relapse/refractory DLBCL identifies shifts in mutation spectrum.

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.

Noninvasive detection of clinically relevant copy number alterations in diffuse large B-cell lymphoma.

7507 Background: Somatic copy number alterations (SCNAs) are common and clinically important genomic events in lymphomas. For example, MYC and BCL2 amplifications are associated with adverse outcomes (Quesada, ASH 2016), while PD-L1 ( CD274) amplifications are associated with improved response to checkpoint inhibitors (Ansell, NEJM 2015). However, noninvasive detection of these events from circulating tumor DNA (ctDNA) remains difficult. Using CAPP-Seq, a targeted high-throughput sequencing platform, we developed a method to profile both focal and broad SCNAs from plasma. Methods: We profiled plasmas from a cohort of 75 pretreatment diffuse large B-cell lymphoma patients and 48 healthy controls. Focal SCNAs were evaluated at ultra-high depths (~10,000x), allowing for detection of lesions at ~1% ctDNA fraction. Thresholds were tuned to allow a false positive rate of 1%, which was empirically validated in an independent healthy cohort (n = 15), yielding a panel-wide false discovery rate of ~2.3% (0% in our genes of interest). Sequencing reads outside the targeted regions were separately pooled and analyzed to evaluate arm and chromosome level SCNAs. Results: We detected SCNAs in clinically relevant genes at the frequencies reported in literature, including amplifications in MYC (8.0%), BCL2 (24.0%), and BCL6 (14.7%) and deletions in TP53 (13.3%) and CDKN2A (9.3%). Remarkably, 26.7% of the cohort demonstrated amplification of both PD-L1 and PD-L2 ( PDCD1LG2). Furthermore, we discovered amplifications in PD-L2, but not PD-L1, in 13.3% of our patients. Interestingly, PD-L1 amplifications were more common in patients with relapsed lymphoma than in those with treatment-naïve disease (43.5% vs 19.2%, p = 0.02). Most PD-L1 amplifications were focal (65%) while the remainder typically involved &gt; 80% of Chr9p. Corresponding tissue profiling data is in progress and will also be presented. Conclusions: Noninvasive sampling of lymphoma ctDNA enables detection of both focal and broad SCNAs, including amplifications of MYC, BCL2, and PD-L1. The ability to noninvasively profile copy number altered regions allows for biopsy-free discovery of clinically significant structural alterations in lymphoma patients.

Sole rearrangement but not amplification of MYC is associated with a poor prognosis in patients with diffuse large B cell lymphoma and B cell lymphoma unclassifiable.

Rearrangement of MYC is associated with a poor prognosis in patients with diffuse large B cell lymphoma (DLBCL) and B cell lymphoma unclassifiable (BCLU), particularly in the setting of double hit lymphoma (DHL). However, little is known about outcomes of patients who demonstrate MYC rearrangement without evidence of BCL2 or BCL6 rearrangement (single hit) or amplification (>4 copies) of MYC. We identified 87 patients with single hit lymphoma (SHL), 22 patients with MYC-amplified lymphoma (MYC amp) as well as 127 DLBCL patients without MYC rearrangement or amplification (MYC normal) and 45 patients with DHL, all treated with either R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) or intensive induction therapy. For SHL and MYC amp patients, the 2-year progression-free survival rate (PFS) was 49% and 48% and 2-year overall survival rate (OS) was 59% and 71%, respectively. SHL patients receiving intensive induction experienced higher 2-year PFS (59% vs. 23%, P=0·006) but similar 2-year OS as compared with SHL patients receiving R-CHOP. SHL DLBCL patients treated with R-CHOP, but not intensive induction, experienced significantly lower 2-year PFS and OS (P<0·001 for both) when compared with MYC normal patients. SHL patients appear to have a poor prognosis, which may be improved with receipt of intensive induction.

Abstract 1271: The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC)

Abstract BCL6 is a transcription factor that promotes lymphomagenesis by repressing target genes involved in DNA damage sensing and response. In NSCLC patients, BCL6 is amplified in 40% of Squamous Cell Carcinomas (197/501, TCGA) and in 2.2% of Adenocarcinomas (5/230, TCGA), suggesting a potential oncogenic role for BCL6 in lung cancer. To determine whether BCL6 plays a role in sustaining NSCLC, we first measured the expression of BCL6 in a panel of 15 NSCLC cell lines and found BCL6 expression in 15/15 cell lines. Furthermore, in 7/15 cells lines (46%) we found BCL6 gene amplification. To gain insight into BCL6 function, we engineered two NSCLC cell lines with BCL6 amplification (H1299 and H838) to stably express BCL6-shRNA. BCL6 silencing resulted in 3-5 fold mRNA increase of key DNA damage response genes such as ATR, Chek1, p21 and p53. BCL6-mediated repression of DNA damage response genes has functional effects as BCL6 silencing resulted into i) G1 cell-cycle arrest as determine by flow cytometry analysis, ii) 1.5-2 fold increase in cell doubling time, iii) 40-60% reduction in colony formation. These results suggest that a major role of BCL6 in lung cancer could be to enable proliferation under genotoxic stress, a function that BCL6 exerts physiologically in B-cells during antibody generation. Under these premises, BCL6 inhibition should preferentially affect the proliferation of cells with elevated genomic instability. Thus, we measured the amount of chromosomal aberration harbored by the proliferating fractions of BCL6-shRNA cells compared to the isogenic parental cells using micronuclei assay. BCL6 silencing resulted in 30-40% reduction of micronuclei amount in the proliferating fraction of cells, supporting our hypothesis. Moreover, exposing NSCLC cells to DNA damaging agents leads to 2-3 fold increase in BCL6 expression regardless the presence of BCL6 amplification. In lymphomas, BCL6 represses genes by recruiting co-repressors to form “repressosome” complexes in gene promoters and enhancers. In particular, repression of DNA damage response genes is achieved by the recruitment of three co-repressors (SMRT, N-CoR, and BCOR) to the BTB-domain of BCL6. To determine whether the BTB-domain of BCL6 is mediating the repression of DNA damage genes in NSCLC, we employed FX1085, a small molecule that specifically interacts with the BCL6 BTB domain and prevents the formation of the repressosome complex. Exposure to FX1085 resulted in 3-10 fold increase in ATR, Chek1, p21 and p53 mRNA levels and prevented the proliferation of NSCLC cells with elevated genomic instability. Moreover, FX1085 affected the survival of 6/15 NSCLC cell lines (40%) at GI50 doses comparable to those effective in lymphoma cells, indicating that BCL6 maybe a suitable therapeutic target in NSCLC. Overall, our data indicate a role for BCL6 in sustaining NSCLC genomic instability and suggest that this protein may be a potential therapeutic target in this disease. Citation Format: Rossella Marullo, Haelee Ahn, Mariano Cardenas, Ari Melnick, Fengtian Xue, Leandro Cerchietti. The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC). [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 1271.