BCL2 Mutations Research Articles

Abstract 3102: Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia

Abstract The BH3-mimetic venetoclax is highly cytotoxic for chronic lymphocytic leukemia (CLL) cells. Nonetheless, some patients (pts) fail to clear minimal residual disease (MRD) and develop progressive disease (PD) on venetoclax-based therapy (V-Rx). Acquisition of de novo BCL2 mutations that reduce venetoclax-binding to BCL2 has been observed in CLL cells of pts who develop PD on V-Rx (Blombery et al., 2019; Blombery et al., 2020; Tausch et al., 2019). However, such BCL2 mutations generally are either subclonal or not invariably detected in CLL cells of pts who develop resistance to venetoclax (Blombery et al., 2019; Guieze et al., 2019). Moreover, CLL cells of pts with PD on V-Rx appear even more resistant to the cytotoxic effects of venetoclax when cultured under conditions mimicking stroma (Blombery et al., 2019; Thijssen et al., 2015), arguing that factors other than BCL2 mutations account for venetoclax resistance. We find that high-level expression of ROR1 and Wnt5a-induced ROR1-signaling causes enrichment in cancer-stemness gene expression in CLL. We examined whether this was associated with resistance to V-Rx. We performed flow cytometry and transcriptome analyses on CLL cells of pts prior to V-Rx (pre-Rx) and on CLL cells of the same pts after they developed PD on V-Rx after a median of 2 years of therapy. At PD, we identified de novo BCL2 mutations that were subclonal in 66% of these cases. Pre-Rx CLL cells of all 6 pts expressed high levels of ROR1, which became even higher on the CLL cells of the same pts after they developed PD on V-Rx. We found that that transcriptomes of CLL cells of pts with PD had even greater enrichment in cancer-stemness gene expression than pre-Rx CLL cells of these same pts. To examine whether expression of ROR1 could influence cancer stemness and resistance to venetoclax, we transfected the CLL-cell line MEC1 with ROR1 to generate MEC1-ROR1 cells. We found that MEC1-ROR1 had greater enrichment in cancer-stemness gene expression than MEC1 cells and had increased resistance to venetoclax in vitro. Moreover, mutant forms of BCL2 identified in CLL cells of pts with PD on V-Rx, and reducing venetoclax-binding to BCL2, were significantly more effective in protecting MEC1-ROR1 cells than MEC1 cells from the cytotoxic effects of venetoclax in vitro. We find that treatment of primary CLL cells with Wnt5a to induce ROR1-signaling could enhance their resistance to the cytotoxic effects of venetoclax in vitro; such induced-resistance could be inhibited by an anti-ROR1 mAb (cirmtuzumab), which can block Wnt5a-induced ROR1-signaling. Collectively these studies demonstrate that Wnt5a-induced ROR1-signaling in CLL promotes cancer dedifferentiation/stemness and increases resistance to Ven. Strategies that inhibit ROR1-signaling with agents such as cirmtuzumab may enhance the cytotoxic activity of venetoclax and/or mitigate risk of developing resistance to venetoclax therapy. Citation Format: Emanuela M. Ghia, Laura Z. Rassenti, Michael Y. Choi, Miguel Quijada-Alamo, Elvin Chu, George F. Widhopf, II, Thomas J. Kipps. Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3102.

Characterization of the Immunogenomic Landscape of Follicular Lymphoma

Background: Follicular Lymphoma (FL) is an indolent, but incurable form of non-Hodgkin lymphoma (NHL), with variable clinical behavior. The immune environment plays a major role in dictating the biology of FL. For example, gene expression profiling experiments have demonstrated that recurring “immune response” gene signatures in diagnostic FL specimens were more accurate in predicting clinical outcomes than gene expression patterns in FL cells themselves. Furthermore, immune cells, including T cells, are often found infiltrating or surrounding malignant follicles in FL, and our group has identified the presence of highly clonal T cell populations in most FL samples, suggesting that FL cells express antigens that drive the expansion of lymphoma-specific T cells. In addition, inactivating mutations in B2 microglobulin are relatively common in FL, indicating that down-regulation of antigen expression may occur as a result of host immune surveillance. Lastly, FL is responsive to immunotherapies, such as PD-1 antibody blockade. Importantly, the FL antigens that promote spontaneous immune recognition remain unknown. Emerging data has revealed a critical relationship between the genetic landscape and the immunogenicity of cancer. Somatic mutations in malignant cells encode for altered proteins (i.e. neoantigens) that are “sensed” as foreign by T cells, and the mutational “load” of a given tumor has direct implications for the host immune response.Methods: To address the hypothesis that somatic mutations generate neo-antigens which are the targets of T cell recognition in patients with FL, whole exome sequencing (WES) was performed on FACS-purified FL B cells (CD10+CD19+ cells) and matched T cells from 7 FL biopsy samples. HLA A, B and C haplotypes were predicted using PolySolver, OptiType, Phlat and Seq2hla software with >90% concordance. HLA binding affinity was predicted for all possible 8-11-mer peptides generated from each somatic mutation and corresponding wild-type peptides using NetMHCpan v2.8. These tiled peptides were analyzed for their predicted binding affinities (IC50 nM) to HLA class I molecules. An IC50 <150 nM was considered a predicted strong binder, ≥150 but ≤500 nM an intermediate to weak binder, and >500 nM a non-binder.Results: A median number of 71 somatic mutations were identified in each sample (range, 8-135). Non-synonymous single nucleotide variants (SNV) were cataloged, and in silico analysis was utilized to predict potential high-affinity epitopes capable of binding patient-specific HLA molecules. We achieved an average of 90% proportion of nucleotides > Phred quality score 30 of total bases, according to the base calling accuracy (range, 89.02%-92.06%). G>A/C>T transitions were the most common nucleotide substitutions observed. Consistent with published reports, recurrent somatic mutations were identified in EZH2, CREBBP, IGLL5, MUC21, PCLO, and PIM1. We also identified recurrent mutations in BCL2 and MLL2 (frameshift insertions) known to dominate the mutational landscape of FL. Mutations in HLA class II alleles were common, occurring in 3/7 samples. In silico analysis was next employed to identify putative neo-antigens. A total of 139 candidate neo-antigenic peptides were predicted across all patients. On average, 19 neo-antigens were predicted for each patient (range, 3-41). No public neo-antigens were identified, suggesting that the neo-antigens arising in FL are patient-specific. This observation is consistent with what has been described for other cancer subtypes. Functional validation of lead candidate neo-antigen peptides is ongoing.Conclusion: While overall numbers of somatic mutations are modest in FL, putative neo-antigens were identified in all patient samples analyzed. Our findings suggest that immune therapies incorporating neo-antigen-specific approaches, such as personalized vaccines, should be explored in FL patients. DisclosuresNo relevant conflicts of interest to declare.

High Grade B Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements Depict a High Complexity on the Cytogenetic, but Not on the Molecular Genetic Level and Show MYC Mutations As Prognostic Marker

Background: In the revised WHO classification of 2016, cases of large B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements were categorized as “HGBL (high grade B cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements)”, except for cases that fulfill the criteria for a follicular or lymphoblastic lymphoma. A detailed molecular analysis of these cases and comparison of accompanying aberrations has not been available to date.Aims: Evaluation of associated genetic alterations and prognosis in HGBL cases with rearrangement of (i) MYC + BCL2 , (ii) MYC + BCL6 or (iii) MYC + BCL2+BCL6.Patient cohorts and methods: The total cohort comprised 70 cases with MYC and BCL2 and/or BCL6 rearrangements (39 male, 31 female). Cases were selected by detection of the rearrangements by FISH (fluorescence in situ hybridization), cases diagnosed as CLL, follicular or lymphoblastic lymphoma were excluded. Median age was 67 years (range: 44-87 years). All patients were investigated using chromosome banding analysis (CBA) and FISH using probes for MYC , IGH-BCL2 and BCL6 . Additionally, mutation analyses of ATM , BCL2, BIRC3, BRAF, BTK, CBL, CSF3R, CSNK1A1, CXCR4, EGR2, ETNK1, FBXW7, FLT3, ID3, IDH1, IDH2, KIT, KLF2, KRAS, MAP2K1, MYC, MYD88, NFKBIE, NOTCH2, PHF6, PLCG2, POT1, PTPN11, SAMHD1, SETBP1, SF3B1, SRSF2, STAT3, STAT5B, TET2, TP53, U2AF1, UBR5, WT1, XPO1 and ZRSR2 was performed. Variants of unknown significance were excluded from statistical analysis.Results: We identified 33/70 cases with rearrangement of MYC + BCL2 , 10/70 cases with MYC + BCL6 and 27/70 cases with MYC + BCL2 + BCL6 (47%, 14% and 39%, respectively). In the total cohort, CBA revealed a complex karyotype (>3 abnormalities) in 59/70 (84%) of patients, 10/70 (14%) harbored an aberrant but non-complex karyotype, whereas in 2/70 (3%) of cases, CBA analyses failed due to insufficient cell proliferation. Regarding the subgroups, a complex karyotype was observed in 82%, 80% and 89% of cases, respectively (for MYC + BCL2 , MYC + BCL6 and MYC + BCL2 + BCL6 rearrangement). If only aberrations additional to MYC / BCL2 / BCL6 rearrangements were calculated, >3 aberrations were detected in 64% of cases (67%, 70% and 60%, respectively). The most frequent additional aberrations included 13q deletions, +12, gain of 1q and 6q deletions. Median number of additional cytogenetic aberrations was n=8 (range: 0-36). Despite the high cytogenetic complexity, the frequency of TP53 deletions was relatively low (6% in the total cohort), three of them harboured a concomitant TP53 mutation in the other allele. Mutation analyses of 40 genes revealed a median number of n=1 mutations in the total cohort with no differences between the three subgroups. The range of the number of molecular mutations was 0-3 (0 mutations: 31%; 1 mutation: 44%; 2 mutations: 20%; 3 mutations: 5%). Interestingly, out of the 40 genes analysed, mutations were almost exclusively detected in BCL2 (40%), MYC (21%) and TP53 (21%) in the total cohort. Other recurrent mutations were observed for ID3 and PHF6 only (3%, respectively). Cases with MYC+BCL6 rearrangement showed a higher - although not statistically significant - frequency of MYC mutations compared to the other cases (40% vs. 18%), while BCL2 mutations were rarely detected in this subgroup (10% vs. 45%, p=0.033). Of note, mutations in TP53 were found to show a trend towards a lower frequency in cases with a “triple hit” (MYC+BCL2+BCL6 ) in comparison to the “double hit” cases (MYC+BCL2 and MYC+BCL6 ) (11% vs. 28%).Median overall survival (OS) in the total cohort was 61 months. Cases with MYC + BCL2 showed a shorter OS compared to cases with MYC+BCL2+BCL6 rearrangements, although this was not statistically significant (37 vs.61 months). Unfortunately, OS could not be determined for cases with MYC+BCL6 due to the low number of cases. Mutations in MYC, but not in BCL2 or TP53 , showed a negative impact on OS in the total cohort (16 vs. 61 months, p=0.046).Conclusion: 1) Cases with rearrangements in MYC , BCL2 and/or BCL6 depict a high complexity on the cytogenetic, but not on the molecular genetic level. 2) Molecular mutations are almost exclusively detected in BCL2 , MYC and TP53. 3) Cases with MYC+BCL6 rearrangements differ from the other subgroups by showing a higher frequency of MYC mutations and a lower frequency of mutations in BCL2 . 4) Mutations in MYC negatively impact on OS. 5) Thus, our data support the current WHO classification and complement it on a molecular level. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Prognostic value of BCL2 and TP53 genetic alterations for diffuse large B-cell lymphoma treated with R-CHOP.

e19549 Background: Unlike BCL2 translocation ( BCL2TR), limited data about the prognostic significance of BCL2 mutations ( BCL2MUT) and BCL2 copy number variations in diffuse large B-cell lymphoma (DLBCL) is available, especially in the era of immunochemotherapy. Predicting outcomes at the time of diagnosis is of extreme importance. Methods: In this study, we performed probe capture-based high resolution sequencing on 191 Chinese patients with de novo DLBCL, attempting to comprehensively describe BCL2 genetic alterations and several other genes. We also examined correlation between genetic alterations and outcomes in 164 patients treated with R-CHOP. BCL2, MYC and BCL6 protein was determined by immunochemistry. Results: Of all the 191 patients, BCL2 gain/amplificatio n ( BCL2GA/AMP) and BCL2MUT occurred in 9.4% and 8.9% of patients, respectively, and only 4.2% of cases harbored BCL2TR. As a result, BCL2 alteration, comprised of the above three genetic alterations, was observed in 18.3% of patients. Compared to patients with the absence of BCL2 alteration, the 5-year progression-free survival (PFS) (13.7% vs 40.8%, P= 0.003) and overall survival (OS) (34.0% vs 70.9%, P= 0.036) were significantly inferior in cases that harbored BCL2 alteration. Importantly, patients who harbored BCL2GA/AMP also had remarkably inferior PFS (5-year PFS, 11.1% vs 38.3%, P&lt; 0.001) and OS (5-year OS, 22.1% vs 69.6%, P= 0.009) compared to those without BCL2GA/AMP. By contrast, neither BCL2MUT nor BCL2TR were significantly prognostic for survival. Multivariate analysis showed that the presence of BCL2 alterations, especially BCL2GA/AMP, TP53 mutation and International Prognostic Index (IPI) were significantly associated with inferior PFS and OS. Novel prognostic models were constructed based on 3 risk factors, including BCL2 alteration (Model 1) or BCL2GA/AMP (Model 2), TP53 mutation and IPI, stratifying patients into three risk groups with different outcomes. According to Model 1, the 5-year PFS were 65.9%, 25.9%, and 12.9% ( P&lt; 0.001), and 5-year OS were 89.4%, 65.0% and 16.4% ( P&lt; 0.001), respectively, for the low-risk, intermediate-risk and high-risk groups. In Model 2, the 5-year PFS was 56.9% for the low-risk, 24.1% for the intermediate-risk and 14.3% for the high-risk group ( P&lt; 0.001), and 5-year OS were 86.6%, 60.9% and 16.9% for the three risk groups ( P&lt; 0.001), respectively. Conclusions: This study demonstrated that, in patients treated with R-CHOP, the presence of BCL2 alteration, especially BCL2GA/AMP, and TP53 mutation were significantly associated with inferior outcomes independent of IPI. The novel prognostic models we proposed could aid in individual risk prediction for DLBCL patients treated with R-CHOP. Further validation of this prognostic model is warranted.

How I treat chronic lymphocytic leukemia after venetoclax

AbstractVenetoclax-based regimens have expanded the therapeutic options for patients with chronic lymphocytic leukemia (CLL), frequently achieving remissions with undetectable measurable residual disease and facilitating time-limited treatment without chemotherapy. Although response rates are high and durable disease control is common, longer-term follow-up of patients with relapsed and refractory disease, especially in the presence of TP53 aberrations, demonstrates frequent disease resistance and progression. Although the understanding of venetoclax resistance remains incomplete, progressive disease is typified by oligoclonal leukemic populations with distinct resistance mechanisms, including BCL2 mutations, upregulation of alternative BCL2 family proteins, and genomic instability. Although most commonly observed in heavily pretreated patients with disease refractory to fludarabine and harboring complex karyotype, Richter transformation presents a distinct and challenging manifestation of venetoclax resistance. For patients with progressive CLL after venetoclax, treatment options include B-cell receptor pathway inhibitors, allogeneic stem cell transplantation, chimeric antigen receptor T cells, and venetoclax retreatment for those with disease relapsing after time-limited therapy. However, data to inform clinical decisions for these patients are limited. We review the biology of venetoclax resistance and outline an approach to the common clinical scenarios encountered after venetoclax-based therapy that will increasingly confront practicing clinicians.

Identification of novel, clonally stable, somatic mutations targeting transcription factors PAX5 and NKX2-3, the epigenetic regulator LRIF1, and BRAF in a case of atypical B-cell chronic lymphocytic leukemia harboring a t(14;18)(q32;q21).

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia

BCL-6 mutations are accumulated during B-cell transit through the germinal center (GC) and provide a histogenetic marker for B-cell tumors. On the basis of a comprehensive analysis of 308 B-cell neoplasms, we (1) expand the spectrum of tumors associated with BCL-6 mutations; (2) corroborate the notion that mutations cluster with GC and post-GC B-cell neoplasms; and (3) identify heterogeneous mutation frequency among B-lineage diffuse large cell lymphoma (B-DLCL) subsets. Mutations are virtually absent in acute lymphoblastic leukemia (P <.001) and mantle cell lymphoma (P <.05), whereas they occur frequently in GC or post-GC neoplasms, including lymphoplasmacytoid lymphoma, follicular lymphoma, MALT lymphomas, B-DLCL and Burkitt lymphoma. Among B-DLCL, mutations occur frequently in systemic nodal B-DLCL, primary extranodal B-DLCL, CD5(+) B-DLCL, CD30(+) B-DLCL, and primary splenic B-DLCL, suggesting a similar histogenesis of these B-DLCL subsets. Conversely, mutations are rare in primary mediastinal B-DLCL with sclerosis (10.0%; P <.01), supporting a distinct histogenesis for this lymphoma. Longitudinal follow-up of B-DLCL transformed from follicular lymphoma shows that they BCL-6 mutations may accumulate during histologic progression. Mutations also occur in some B-cell chronic lymphocytic leukemias, small lymphocytic lymphomas, and hairy cell leukemias, consistent with the hypothesis that a fraction of these lymphoproliferations are related to GC-like cells. Finally, the molecular pattern of 193 mutational events reinforces the hypothesis that mutations of BCL-6 and immunoglobulin genes are caused by similar mechanisms. (Blood. 2000;95:651-659)

The Molecular and Phenotypic Profile of Primary Central Nervous System Lymphoma Identifies Distinct Categories of the Disease and Is Consistent With Histogenetic Derivation From Germinal Center–Related B Cells

AbstractPrimary central nervous system lymphoma (PCNSL) is a major cause of morbidity and mortality among human immunodeficiency virus (HIV)-infected individuals. The precise histogenetic derivation and the molecular pathogenesis of PCNSL is poorly understood. In an attempt to clarify the histogenesis and pathogenesis of these lymphomas, 49 PCNSL (26 acquired immunodeficiency syndrome [AIDS]-related and 23 AIDS-unrelated) were analyzed for multiple biologic markers, which are known to bear histogenetic and pathogenetic significance for mature B-cell neoplasms. PCNSL associated frequently (50.0%) with mutations of BCL-6 5′ noncoding regions, which are regarded as a marker of B-cell transition through the germinal center (GC). Expression of BCL-6 protein, which is restricted to GC B cells throughout physiologic B-cell maturation, was detected in 100% AIDS-unrelated PCNSL and in 56.2% AIDS-related cases. Notably, among AIDS-related PCNSL, expression of BCL-6 was mutually exclusive with expression of Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP)-1 and, with few exceptions, also of BCL-2. All but one PCNSL expressed hMSH2, which among mature B cells selectively stains GC B cells. These data suggest that PCNSL may be frequently related to GC B cells and may be segregated into two major biologic categories based on the expression pattern of BCL-6, LMP-1, and BCL-2. BCL-6+/LMP-1−/BCL-2− PCNSL occur both in the presence and in the absence of HIV infection and consistently display a large noncleaved cell morphology. Conversely, BCL-6−/LMP-1+/BCL-2+ PCNSL are restricted to HIV-infected hosts and are represented by lymphomas with immunoblastic features. These data are relevant for the pathogenesis and histogenesis of PCNSL and may be helpful to segregate distinct biologic and prognostic categories of these lymphomas.© 1998 by The American Society of Hematology.

Frequent Mutation of the 5′ Noncoding Region of the BCL-6 Gene in Acquired Immunodeficiency Syndrome-Related Non-Hodgkin's Lymphomas

AbstractAcquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHL), a major source of morbidity and mortality among human immunodeficiency virus (HIV)-infected individuals, are derived from B cells and are classified into two major categories, Burkitt's lymphoma (BL) and diffuse large cell lymphoma (DLCL). Anaplastic large cell lymphoma (ALCL) and body-cavity-based lymphoma (BCBL) represent less frequent AIDS-NHL types. The molecular pathogenesis of AIDS-NHL is characterized by distinct genetic pathways, including chromosomal rearrangements of c-MYC and BCL-6 in AIDS-BL and AIDS-DLCL, respectively. In addition to gross rearrangements, recent evidence has suggested that BCL-6 may also be affected by mutations of the gene 5′ noncoding regions. Here we have investigated the distribution of BCL-6 mutations in a panel representative of all the AIDS-NHL subtypes. Forty-three AIDS-NHL were analyzed for mutations in the first exon-first intron boundary region of BCL-6. Mutations were detected in all categories of AIDS-NHL (25 of 43 cases; 58%), including 12 of 20 AIDS-BL, 10 of 15 AIDS-DLCL, two of three AIDS-ALCL, and one of five of AIDS-BCBL. BCL-6 mutations occurred independent of BCL-6 rearrangements and presence of other genetic lesions frequently associated with AIDS-NHL. These results indicate that mutations of BCL-6 5′ noncoding regions represent the most common genetic alteration presently detectable in AIDS-NHL. The frequency of these mutations, as well as their location in the proximity of BCL-6 regulatory sequences, suggest that they may play a role in AIDS-related lymphomagenesis.

Identification of Recurrent Mutations in the microRNA-Binding Sites of B-Cell Lymphoma-Associated Genes in Follicular Lymphoma.

Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).

Characterizing Specificities of Chronic Lymphoid Leukemia Harboring a BCL2 rearrangement

Characterizing Specificities of Chronic Lymphoid Leukemia Harboring a BCL2 rearrangement

BCL10 Gain-of-Function Mutations Aberrantly Induce Canonical and Non-Canonical NF-Kb Activation and Resistance to Ibrutinib in ABC-DLBCL

ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment. Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function. BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion. To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms. As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants. This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients. Disclosures Fontan: Johnson &amp; Johnson: Current Employment. Melnick:Epizyme: Consultancy; Jubilant: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding.

Neoantigens Are Typically Associated with Intact HLA Class I Presentation in Early-Stage Follicular Lymphoma

Neoantigens Are Typically Associated with Intact HLA Class I Presentation in Early-Stage Follicular Lymphoma

Integrative Analysis of the Genomic and Transcriptomic Landscape of Relapsed/Refractory Multiple Myeloma Patients Treated With Venetoclax in Combination With Bortezomib and Dexamethasone: Biomarker Analyses From the Phase 3 BELLINI Study

Integrative Analysis of the Genomic and Transcriptomic Landscape of Relapsed/Refractory Multiple Myeloma Patients Treated With Venetoclax in Combination With Bortezomib and Dexamethasone: Biomarker Analyses From the Phase 3 BELLINI Study

Ultra-Deep Targeted Sequencing Reveals Cancer-Associated, Prognostically Significant Mutations in Circulating Cell-Free DNA in Treatment-Naive Patients with Follicular Lymphoma

Background: Follicular lymphoma is the second most frequent non-Hodgkin lymphoma (NHL) accounting for 20-25% of NHL cases in western countries. Although it has an indolent character, progressive disease or relapse occurs within first two years following therapy initiation in ~20% of FL cases. Several somatic mutations were identified in genes of epigenetic regulation or other biological processes by sequencing of FL tumors. Current diagnostic and prognostic evaluations include invasive methodologies that may be less effective and more risky for the FL patients. Therefore, there is urgent need for development of non-invasive methods to improve diagnosis as well as risk stratification. Accumulating evidence has shown that circulating cell-free DNA (cfDNA) includes tumor-derived mutations in several cancer types; however, this possibility has not been comprehensively investigated in FL patients. Here we evaluated the potential diagnostic and prognostic value of cfDNA in FL cases by addressing the proportion by which cfDNA samples contains cancer-associated and prognosis-related mutations. Methods: Twenty FL cases with available clinic data were included in this study. Thirteen of these cases were symptomatic who were later treated with R-chemo whereas rest of the cases were asymptomatic who were in watchful-waiting. Plasma cfDNA, granulocyte DNA, and FFPE tumor tissue DNA samples were obtained from treatment-naive FL cases. A custom gene panel including exons and exon-intron boundaries of 110 FL-associated genes was constructed based on previously published studies for ultra-deep targeted sequencing. Paired-end sequencing of the captured regions were performed using a HiSeq system in Novogene, which generated 150 bp NGS reads. Targeted genomic regions were covered with &amp;gt; 1500X average effective sequencing depth for identification of somatic variants with low variant allele fractions (VAF). Variants present in cfDNA and tumor tissue DNA but not in patient-matched granulocyte DNA were identified with the GATK pipeline including the MuTect2 variant caller. Somatic variants associated with hematopoietic and lymphoid tissues in the COSMIC database were chosen for further analyses. The final high-confident list of variants was determined by visual investigation and through additional filtering of each variant using Integrative Genomics Viewer. Selected variants were cross-validated with Sanger sequencing. Survival analyses were performed with Survival and Survminer R packages. Results: Ultra-deep targeted sequencing revealed 91 somatic variants (71 missense, 12 nonsense, 4 indel, 4 splice site) in 31 genes included in the panel. Consistent with previous reports, the most frequently mutated genes were CREBBP (40%), BCL2 (30%), STAT6 (25%), EZH2 (20%), and CARD11 (20%). In symptomatic cases, 41.5% of the variants was present in both cfDNA and tumor tissue DNA, whereas 52.3% and 6.2% of them was present only in tumor tissue DNA or in cfDNA samples, respectively. In asymptomatic cases, 11.5% of the detected variants was present in both cfDNA and tumor tissue DNA, while 84.6% and 3.8% of them were in only tumor tissue DNA or cfDNA samples. Mutations previously reported to be associated with FL pathogenesis (e.g. KMT2D R2417*) were in the list of common variants observed in both cfDNA and tumor tissue DNA. We observed high Ki67 staining, elevated LDH levels, presence of BCL2 or CCND3 mutations to be significantly associated with progression-free survival (Figure 1A, B). Importantly, survival analysis by stratifying patients based on BCL2 mutations present only in cfDNA also predicted poor prognosis (Figure 1C). One of the FL patient who had progressive disease contained histological transformation-associated gene (i.e. B2M and BTG1) mutations only in the cfDNA but not in tumor tissue DNA sample. Finally, we cross-validated the selected somatic variants with VAF &amp;gt;20% using Sanger sequencing, which showed 100% consistency with NGS results. Conclusions: Tumor tissue-derived mutations can be detected in most FL patients albeit to a lesser extent than those in DLBCL. Plasma cfDNA genotyping may be useful for improving diagnosis and prognosis especially in symptomatic FL patients. Given that some somatic mutations associated with disease progression are detected only in plasma cfDNA samples, cfDNA genotyping may be useful for choosing appropriate therapy for high-risk FL patients. Disclosures No relevant conflicts of interest to declare.

Evaluation of the Different Stratification Models for POD24 Prediction in Patients with Follicular Lymphoma

Evaluation of the Different Stratification Models for POD24 Prediction in Patients with Follicular Lymphoma

Expert Variant Curation Combined with in-Silico analysis for Clinical Interpretation of BCL2 variants in Resistance to BCL2 Inhibitors in Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma

Venetoclax is an oral, highly selective, BCL2 inhibitor approved by the FDA for use in chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia. Despite favorable responses, multiple biological mechanisms lead to treatment resistance. One such mechanism includes somatic mutations in the BCL2 gene. Multiple lines of evidence suggest that hot-spot mutations in BCL2 such as Gly101Val induce treatment resistance by disrupting the binding of BCL2 to the BCL2 inhibitors such as venetoclax. Further, widespread use of high-throughput NGS technologies has identified multiple BCL2 mutations and additional concurrent molecular alterations at various variant allele frequencies in patients with progression while undergoing venetoclax therapy. In order to understand and determine the clinical significance of each of these mutations, careful expert curation and integration into somatic variant annotation AMP/ASCO/CAP guidelines is needed. Further, curation of those somatic variants that may not have sufficient functional evidence in literature may benefit from additional tools such as in silico analysis. To address these issues, we have undertaken an effort to integrate the contributions of a multidisciplinary expert panel (clinical laboratory diagnosticians, oncologists, biomedical informaticians and lab-based researchers) for curation of BCL2 variants in hematological malignancies under the umbrella of ClinGen, an NIH/NHGRI funded consortium to establish standards and centralized resources for assessing the clinical significance of gene variants. Within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) ((https://www.clinicalgenome.org/working-groups/somatic/), the somatic hematological malignancy taskforce has identified 56 peer-reviewed publications on BCL2 inhibitors (Jan 2014 to June 2020). The functional evidence contained within these publications was curated using CIViC (Clinical Interpretation of Variants in Cancer, civicdb.org), an open access, crowdsourced aggregation of expert curated evidence. Only a fraction of the somatic variants identified in BCL2 has established functional evidence on variant induced disruption to Venetoclax inhibition. Curation of these remaining variants of unknown significance (VUS) only have in silico functional assays to provide evidence on their potential resistance to Venetoclax. The current curation guidelines do not consider in silico prediction as a strong line of evidence for the interpretation of somatic sequence variants, however this recommendation is meant to interpret generalized in silico predictors and not robust computational models of specific protein function. The latter are more comparable to an experimental functional assay, and provide curators with more trustworthy computational assessments of disruption to protein specific functions. In order to assess their potential to integrate and supplement experimental evidence, the interaction of Ventoclax with several drug resistant BCL2 variants was simulated using AutoDock Vina (J Comput Chem. 2010;31(2):455-61). Facilitated by the SNP2SIM workflow (BMC Bioinformatics. 2019;20(1):171), the relative impact on binding energy was compared to the wildtype system. The in silico binding assay accurately predicted resistance (Fig 1), and demonstrates the utility of applying these methods to the large number of VUS in BCL2. In conclusion, the evidence-based expert curation of BCL2 variants provides a standardized approach for reporting and interpretation across all labs. For those variants (Tier 3) with limited published evidence, computational models that can predict specific changes to functional protein interactions can provide additional tools to the expert curators. Development and incorporation of these tools into curation guidelines requires the refinement of the predictive models through focused validation studies. Disclosures No relevant conflicts of interest to declare.

Abstract PO-38: Genomic subtypes correlate with the risk of central nervous system (CNS) recurrence in diffuse large B-cell lymphoma (DLBCL)

Abstract Clinicopathologic features are poor at predicting the risk of CNS relapse after initial treatment in DLBCL, or at guiding CNS prophylactic therapy. However, mutational profiles may correlate with primary extranodal disease, including primary CNS lymphoma (Ollila T, Curr Treat Options Oncol 2018). We hypothesized that specific genomic profiles in DLBCL would also correlate with CNS relapse or with systemic-only (non-CNS) relapse. We examined 26 DLBCLs with CNS-only (n=13) or systemic-only relapse (n=13) using a 592 gene next-generation sequencing (NGS) assay (Illumina NextSeq, average coverage depth &amp;gt;750x; Caris Life Sciences) performed on formalin-fixed tissue. We also determined cell of origin (COO) by IHC; MYC/BCL2/BCL6 rearrangements by FISH, and karyotype by cytogenetics. We used the novel LymphGen genomic DLBCL classifier (Wright GW, Cancer Cell 2020) to group tumors into the MCD, ST2, EZB, BN2, and A53 subtypes. We then constructed a simplified hierarchical classifier (hc) usable with common clinical multigene NGS panels to identify 3 relevant subtypes: (1) hc-MCD subtype (defined as MYD88L265P or &amp;gt;2 mutations in CD79B, PIM1, ETV6, BTG1, TBL1XR1, or PRDM1), (2) hc-TP53 subtype characterized by TP53 mutations +/- complex karyotype, and (3) hc-GCB subtype with ≥2 mutations in BCL2, CREBBP, EZH2, KMT2D, TNFRSF14, GNA13, MEF2B, or PTEN. We compared prevalence of these subtypes between our groups and a reference dataset of unselected DLBCL tumors combining data from Chapuy (Nat Medicine 2018; n=135; dbGaP phs000450) and Reddy (Cell 2018; n=1001; EGA: EGAS00001002606). There was no difference between DLBCL with CNS-only or systemic recurrence in any standard clinicopathologic characteristics, including COO, FISH, or karyotype. The LymphGen MCD subtype was significantly more frequent in DLBCL with CNS-only relapse compared with reference datasets (39% vs. 7%, P=.001), but not in DLBCL with systemic relapse (P=.05). Conversely, the ST2 subtype was more frequent in DLBCL with systemic relapse (23% vs. 3%, P=.010) and completely absent in CNS relapse (0%). The A53 or EZB subtypes did not significantly differ between any groups. The simplified NGS classifier identified 96% of LymphGen MCD cases and 100% of A53 cases. The hc-MCD subtype was also significantly associated with CNS relapse (46% vs. 18%, P=.018) but not with systemic relapse (P=.26), whereas hc-TP53 and hc-GCB subtypes did not differ from the reference dataset (P=.15 and P=.70, respectively). Our data demonstrate that the MCD DLBCL subtype is specifically associated with the risk of CNS relapse. Full molecular classification of DLBCL is not yet incorporated into current clinical practice, as it requires integration of single nucleotide, structural chromosomal variants, and copy alterations. We show that a meaningful molecular predictor signature for the MCD subtype can be obtained from clinically validated multigene NGS assays. If validated, this signature may prove useful for selecting patients for CNS-directed prophylactic therapy. Citation Format: Thomas A. Ollila, Habibe Kurt, Jozal Waroich, John Vatkevich, Ashlee Sturtevant, Nimesh Patel, Diana O. Treaba, Patrycja M. Dubielecka, Adam J. Olszewski. Genomic subtypes correlate with the risk of central nervous system (CNS) recurrence in diffuse large B-cell lymphoma (DLBCL) [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-38.

Abstract IA44: ctDNA in indolent lymphoid cancers treated with targeted therapies

Abstract The use of circulating tumor DNA (ctDNA) in diagnosing and monitoring treatment responses in hematologic malignancies has multiple advantages: (1) ctDNA is noninvasive, allowing for frequent, serial, real-time tracking of multiple tumor clones; (2) ctDNA is highly sensitive for clone-specific genomic aberrations, and may rival traditional minimal residual disease (MRD) methodologies in detecting low-volume disease; and (3) ctDNA “integrates” the tumor genome across multiple body compartments, allowing the detection of emerging resistant clones that would otherwise be missed by single-site biopsies. Since 2014, our group has evaluated the use of ctDNA in patients with hematologic cancers initially as an investigational tool, and more recently as an accredited (ISO15189) hybridization-based NGS panel that can perform sequence variant detection, genome-wide copy number calling, and structural variant detection from ctDNA. In this talk, we will discuss different clinical scenarios and the evolution of ctDNA at our center. Our initial proof-of-concept study was conducted in patients receiving BTK inhibitor (BTKi) and/or BCL2 inhibitor (BCL2i) therapy for chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Clinical challenges in this population include response assessment being confounded by BTKi redistribution lymphocytosis, differential compartmental responses to different classes of novel agents, and emergence of resistant clones in disparate disease sites. In our study of CLL, serial samples from 32 patients treated with either ibrutinib (BTKi, n=22) or venetoclax (BCL2i, n=10) were analyzed, including 3 patients who developed RS. Using a bespoke TS panel of 7 commonly mutated genes in CLL (SF3B1, NOTCH1, ATM, TP53, MYD88, KRAS, and BIRC3), somatic mutations comprising 0.1% to 90% of total ctDNA were detected in 25 of 32 (78%) patients. Serial monitoring of MAF was concordant with disease response as determined by CT scans and, importantly, ctDNA was able to “look beyond” the artificial rise in circulating tumor cells caused by BTKi, showing an early and consistent fall in MAF. ctDNA was sensitive to the detection of disease clones in occult body compartments—of 88 serial timepoints assessed by both ctDNA and MNL DNA after the initiation of novel therapy, mutations were found to be detectable only in ctDNA in 18 (20%) timepoints. In 30 timepoints where MRD testing was performed by both ctDNA and high-resolution flow cytometry, there was 100% concordance in results. In a second study, serial ctDNA analyses were performed in a cohort of 24 patients with MCL receiving combination ibrutinib and venetoclax therapy. Using TS and a panel of 42 genes known to be recurrently mutated in MCL, trackable ctDNA mutations were detected in 71% of patients, with baseline MAF ranging from 0.4-60%. Similar to our experience with CLL, dynamic changes in ctDNA closely mirrored tumor responses as determined using traditional staging methods of CT, PET, endoscopy, and bone marrow examination. In one patient, ctDNA started rising several weeks before relapse was evident by traditional MRD methods. A further patient had progression in an occult site (pleura), with acquisition of new CNAs on chromosome 6, including 6p loss. In this patient, these new CNAs were readily identified by LC-WGS of plasma. Finally, in a patient with primary refractory disease, an acquired loss of chromosome 9p was identified in plasma 8 weeks before disease progression was evident on imaging. We have since optimized and validated ctDNA testing for routine use into our diagnostic laboratory NGS workflow and we will discuss recent cases of BTK and BCL2 mutations being readily detectable in patients in early stages of novel agent resistance, facilitating early transition to next-line therapy. In aggressive lymphomas, we will demonstrate the value of ctDNA in detecting CD274(PD-L1)/PDCD1LG2 (PD-L2) copy number abnormalities and structural variants in patients with lymphomas in inaccessible tissue sites, guiding the rational use of checkpoint inhibitors. Other important structural variants that are able to be detected in ctDNA through this methodology are IGH-MYC and IGH-BCL2 translocations, thus allowing rapid categorization of patients with diffuse large B-cell lymphoma as “double-hit” lymphoma. Taken together, these studies underscore the value of ctDNA as a noninvasive, highly sensitive tool for assessment of genomic landscape and tracking clonal dynamics across multiple body compartments in patients with hematologic malignancies. Citation Format: Constantine S. Tam, Piers Blombery. ctDNA in indolent lymphoid cancers treated with targeted therapies [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 IA44.

Abstract PO-18: Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome

Abstract Diffuse large B-cell lymphoma (DLBCL) is characterized by deregulation of numerous signaling pathways that direct cell proliferation and survival during B-cell maturation in the germinal center reaction. The subtype of germinal center B-cell DLBCL (GCB-DLBCL) demonstrates addiction to tonic B-cell receptor (BCR) signaling in part through the PI3K-AKT pathway. However, a comprehensive catalogue of the somatic genetic alterations leading to this deregulation is currently lacking and represents a limitation in advancing precision medicine options. In this study, we present a comprehensive analysis in 347 DLBCL biopsies uniformly treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), describing recurrent mutations, copy number alterations, and clinical parameters that are associated with AKT activation (pAKT) as determined by immunohistochemistry. We observed a significantly enhanced pAKT population including players in the Gα13 signaling (GNA13, P2RY8, ARHGEF1, RHOA, and S1PR2) and PI3K activation pathways (NFATC1, USP7, PTEN, INPP4B). Following this correlation, we observed a co-occurrence of BCL2 protein overexpression and mutation in Gα13 signaling associated with advanced clinical stage (P = .008) in 183 GCB-DLBCL cases, as well as activation of cell migration and cell-cell adhesion pathways, indicating their contribution to lymphoma dissemination. This co-occurrence was shown to be associated with shorter time to progression in GCB-DLBCL (P = .005). We furthermore uncovered recurrent deletions of INPP4B, a lipid phosphatase involved in AKT deactivation, to be associated with increased pAKT in GCB-DLBCL. We observed that INPP4B loss is similarly associated with shorter time to progression (P = .036) in GCB-DLBCL, and demonstrated altered AKT phosphorylation in a GCB-DLBCL-derived cell line with a monoallelic INPP4B deletion. We present here a useful comprehensive tool, through a positive correlation to enhanced pAKT staining, to uncover novel prognostic indicators related to genetic alteration status in DLBCL. Our findings provide novel insight into somatic genomic alterations underlying prosurvival signaling of GC-derived lymphomas, and present new avenues for therapeutic targeting. Citation Format: Shannon Healy, Daisuke Ennishi, Elena Viganò, Aixiang Jiang, Anja Mottok, Stacy Hung, Olga Kutovaya, Gerben Duns, Bruce Woolcock, David Scott, Christian Steidl. Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome [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-18.