BCL2 Mutations Research Articles

Sequence analyses of relapsed or refractory diffuse large B-cell lymphomas unravel three genetic subgroups of patients and the GNA13 mutant as poor prognostic biomarker, results of LNH-EP1 study.

Advances in molecular profiling of newly diagnosed diffuse large B-cell lymphoma (DLBCL) have recently refine genetic subgroups. Genetic subgroups remain undetermined at the time of relapse or refractory (RR) disease. This study aims to decipher genetic subgroups and search for prognostic molecular biomarkers in patients with RR-DLBCL. From 2015 to 2021, targeted next-generation sequencing analyses of germline-matched tumor samples and fresh tissue from RR-DLBCL patients were performed. Unsupervised clustering of somatic mutations was performed and correlations with patient outcome were sought. A number of 120 patients with RR-DLBCL were included in LNH-EP1 study and a molecular tumor landscape was successfully analyzed in 87% of patients (104/120 tumor samples). The median age was 67.5 years (range 27.4-87.4), median number of previous treatments was 2 (range 1-9). The most frequently mutated genes were TP53 (n=53 mutations; 42% of samples), CREBBP (n=39; 32%), BCL2 (n=86; 31%), KMT2D (n=39; 28%) and PIM1 (n=54; 22%). Unsupervised clustering separated three genetic subgroups entitled BST (enriched in BCL2, SOCS1, and TNFRSF14 mutations); TKS (enriched in TP53, KMT2D, and STAT6 mutations); and PCM (enriched in PIM1, CD79B, and MYD88 mutations). Median overall survival (OS) was 11.0 (95% confidence interval [CI]: 8.1-12.6) months. OS was not significantly different between the three genetic subgroups. GNA13 mutant was significantly associated with an increased risk of death (hazard ratio: 6.6 [95% CI: 2.1-20.6]; p=.0011) and shorter OS (p=.0340). At the time of relapse or refractory disease, three genetic subgroups of DLBCL patients were delineated, which could help advance precision molecular medicine programs.

Somatic Mutations Alter Interleukin Signaling Pathways in Grade II Invasive Breast Cancer Patients: An Egyptian Experience.

This study aimed to investigate the impact of somatic mutations on various interleukin signaling pathways associated with grade II invasive breast cancer (BC) in Egyptian patients to broaden our understanding of their role in promoting carcinogenesis. Fifty-five grade II invasive BC patients were included in this study. Data for somatic mutations in 45 BC patients were already available from a previous study. Data for somatic mutations of 10 new BC patients were included in the current study. Somatic mutations were identified using targeted next-generation sequencing (NGS) to study their involvement in interleukin signaling pathways. For pathway analysis, we used ingenuity variant analysis (IVA) to identify the most significantly altered pathways. We identified somatic mutations in components of the interleukin-2, interleukin-6, and inter-leukin-7 signaling pathways, including mutations in JAK1, JAK2, JAK3, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Interestingly, six mutations which were likely to be novel deleterious were identified: two in the SCH1 gene, two in the IL2 gene, and one in each of the IL7R and JUN genes. According to IVA analysis, interleukin 2, interleukin 6, and interleukin 7 signaling pathways were the most altered in 34.5%, 29%, and 23.6% of our BC group, respectively. Our multigene panel sequencing analysis reveals that our BC patients have altered interleukin signaling pathways. So, these results highlight the prominent role of interleukins in the carcinogenesis process and suggest its potential role as promising candidates for personalized therapy in Egyptian patients.

Targeting Venetoclax Resistant CLL Using a Protac-Based BCL-2/BCL-XL Degrader

Treatment for chronic lymphocytic leukemia (CLL) is primarily based upon the use of small molecules targeting Bruton's tyrosine kinase (BTK; ibrutinib, acalabrutinib) or BCL-2 (venetoclax). However, resistance mechanisms including mutations in BTK or BCL-2 and adaptive switches in survival dependence to alternative BCL-2 family proteins drive disease progression on these therapies with a poor subsequent outcome. We evaluated BCL-2/BCL-xL dual targeting PROTACs aimed at overcoming venetoclax resistance in CLL. We conducted single cell RNA-seq on CLL samples at baseline and after relapse on the combination of ibrutinib and venetoclax therapy and saw the emergence of novel clusters of cells upon relapse. Cells in the relapsed clusters retained high expression of BCL-2 and showed BCL-xL expression, suggesting that novel BCL-2/BCL-xL inhibitors could overcome venetoclax resistance. Further, BH3 profiling indicated that OSU-CLL cells with the BCL-2 mutation G101V release more cytochrome C than wildtype (WT) cells upon exposure to BAD and XXA1_Y4eK peptides, suggesting increased sensitivity to BCL-2 and BCL-xL inhibition. Earlier studies of dual BCL-2/BCL-xL inhibitors were hampered by platelet toxicity. We used a BCL-2/BCL-xL PROTAC, PZ18753b, and its more soluble analog WH2544, synthesized from navitoclax linked to a VHL ligand. The VHL E3 ligase is not expressed in platelets, allowing for a therapeutic window to selectively target cancer cells. Both PZ18753b and WH2544 exposure led to key events of the mitochondrial apoptosis pathway in primary CLL cells, including BAK and BAX transformation, cytochrome C release, mitochondrial depolarization (assessed by tetramethylrhodamine methyl ester, TMRM, staining) and phosphatidylserine exposure in the outer cell membrane (assessed by Annexin V staining). The efficacy of WH2544 and PZ18753b in treating naïve CLL cells fell in between that of venetoclax and navitoclax. At 18 h of treatment, 100 nM PZ18753b was associated with near complete cell death accompanied by a 50% BCL-xL degradation (assessed by Western Blot) and inhibition of BCL-2 via the formation of a [BCL-2:PZ18753b:VCB] ternary complex (assessed by NanoBRET assay). Additionally, blocking VHL E3 ligase activity decreased the potency of the degraders PZ18753b and WH2544, evidencing that VHL activity is involved in their mechanism of action. To determine whether the BCL-2/BCL-xL degraders can overcome the venetoclax resistance-promoting mutations in BCL-2, we generated OSU-CLL cells incorporating BCL-2 mutations via CRISPR knock in. BCL-2 mutant cells (G101V, F104 and R107-110dup) exhibited decreased sensitivity to venetoclax relative to WT cells as shown by Annexin V staining. In contrast, both WT and BCL-2 mutant cells were more sensitive to PZ18753b treatment at matched concentrations. Apoptosis was preceded with rapid and potent BCL-xL degradation, and partial BCL-2 degradation in OSU-CLL cells, despite the presence of BCL-2 mutations. In conclusion, our results suggest that venetoclax refractory CLL cells retain survival dependency on BCL-2 and BCL-xL proteins to evade apoptosis. This vulnerability can be therapeutically exploited using the BCL-2/BCL-xL dual targeting PROTACs PZ18753b and WH2544 in CLL.

Crebbp Mutations Are Associated with Shorter Time to Progression in Limited-Stage Follicular Lymphoma Treated with Radiation

Background: Limited-stage follicular lymphoma (FL), defined as stage I or II disease, is generally treated with radiation therapy (RT) to involved nodes with excellent overall response rates. However, nearly half of all limited-stage FL patients will ultimately experience a relapse. Most prior research on genetic prognostication in FL has focused on patients with advanced disease undergoing systemic therapy, however, there is evidence to suggest that there are genetic differences in limited versus advanced-stage FL, for example, in the frequency of KMT2D and BCL2 mutations as well as gene expression profiles. Objective: We hypothesized that genetic characterization of pre-treatment biopsies can identify those limited-stage FL patients that are at high risk of relapse following radiation therapy. Methods: Pre-treatment formalin-fixed and paraffin-embedded tissue block samples were obtained from adult patients with limited-stage FL, grade 1-3A, treated with curative-intent RT diagnosed between 1995-2018, from 6 international sites in Canada, Europe and Australia. Hybridization-based targeted sequencing of a 71 gene panel was performed, which included genes that are known to be recurrently mutated in FL. Samples with >50X mean on target coverage were retained in the analysis. Variant calling was performed using Mutect2. Germline polymorphisms were filtered out by cross-referencing with dbSNP and gnomAD databases. Only coding, non-silent mutations detected at a variant allele frequency greater than 0.1 were considered. Patient demographics and clinical data were examined retrospectively. Genes mutated at a proportion of 10% or more of the cohort were tested for association with time to progression (TTP) and overall survival (OS). Results: One hundred and seventeen pre-treatment samples from distinct patients were accrued. The median age was 58 years (range: 26-86), and 68 (58%) were female. There were 99 (85%) patients with stage I disease, and 18 (15%) with stage II. Grade was 1 or 2 in 86% of patients. Mean maximum tumour diameter was 3.33cm (range: 0.5-8cm). Of 110 patients with FLIPI scores available, 100 patients had a low FLIPI score (91%), 9 (8%) had an intermediate score, 1 (1%) patient had a high score. Patients were staged with cross-sectional imaging, including a PET scan in 73 (63%) of cases, and bone marrow assessment was completed in 105 (90%) of patients. The most commonly mutated gene in the cohort was CREBBP (n=61; 52.1%), followed by KMT2D (n=45; 38.5%), TNFRSF14 (n=40; 34.2%), STAT6 (n=25; 21.4%), and BCL2 (n=20; 17.1%). BCL2 mutations were more common in patients with stage II (n=8; 44%) compared to stage I disease (n=12; 12%) (p-value=0.003; after FDR adjustment, p-value=0.056). The median number of mutated genes per patient was 4 (range: 0-12, mean 4.0). The median follow-up of living patients was 5.65 years (range: 0.63-18.1 years) and there were 37 progression events in our cohort. CREBBP mutations were associated with shorter TTP (log-rank p-value 0.027), with 5-year TTP estimates of 73.3% vs. 86.0% (Figure 1A). CREBBP mutations were not significantly associated with age, stage, tumor bulk or PET staging. Using Cox univariable analysis for TTP, the hazard ratio (HR) for CREBBP mutations was 2.13 (95% confidence interval (CI) 1.07-4.25; p-value=0.031) and in multivariable analysis adjusting for age and sex the HR was 2.05 (95%CI 1.03-4.10; p-value= 0.041). However, statistical significance was not met after adjusting for the false discovery rate (FDR; p-value 0.621). CREBBP mutations were not significantly associated with OS (log-rank p-value=0.76) (Figure 1B), nor transformation (log-rank p-value=0.9), but the numbers of survival (n=14) and transformation events (n=9) were small. No other genes from the panel were associated with patients' outcomes. Conclusions: In conclusion, we found an association between CREBBP mutations and shorter time to progression in limited-stage FL treated with RT. To our knowledge, this represents a novel genetic prognostic finding in limited-stage FL. Further studies with larger sample sizes are required to confirm the association. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Towards a Unified Genetic Classification System for Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell neoplasm and the most common lymphoid malignancy worldwide. DLBCL, NOS is a genetically and biologically complex entity that remains uniformly treated at diagnosis despite the growing appreciation for subgroups with molecular distinctions. There are various approaches for assigning DLBCL into one or more molecular subgroups using either gene expression and/or mutation patterns. Several genetic classification systems have been proposed, each sharing commonalities. Distinctions between the definitions of individual classes and the ability to reproducibly assign individual patient samples to subgroups using each system remains a significant barrier to their implementation, further emphasized by up to 50% of patients that remain unclassified. The disparities among the genetic features considered by each classification scheme further limits the ability to consolidate, let alone adopt, these for research and clinical studies. We aimed to develop a classification system that unifies and extends the robustness of existing classification algorithms. Methods: We assembled whole genome sequencing (WGS) data from 900 tumors from DLBCL, Burkitt (BL), follicular (FL) and other mature B-cell neoplasms to comprehensively determine the set of significantly mutated genes (SMG) and common targets of aberrant somatic hypermutation (aSHM). These were intersected with the features used by the current DLBCL classification systems and a curated set of lymphoma genes to tabulate the mutation matrix. Common structural rearrangements involving oncogenes were determined from WGS data and the copy number state of focal high-level recurrent alterations was united with simple somatic mutations. For genes containing mutation hot-spots each was considered separately. Results: Clustering all cases using non-negative matrix factorization (NMF) analysis resulted in a solution with 7 distinct genetic subgroups (Figure 1). Many of these directly resemble clusters in the existing systems with either expanded coverage or refinement of a subgroup into two. First, the BNZ cluster was highly enriched for any aberration in BCL6, 3'UTR mutations in NFKBIZ, and truncating mutations in NOTCH2. Unlike the similar BN2/C1/NOTCH2 groups defined previously, the NFKBIZ mutations were weighted more highly than those of NOTCH2 in defining the BNZ subgroup. The cluster we named MCaP resembled the MCD/C5/MYD88 group. Tumors classified in this subgroup had evidence of aSHM with frequent non-coding mutations in PIM1,CD44, GRHPR, PIM2, and BCL11A among others. Another cluster, MP3, was enriched for coding mutations or aSHM in the MYC TSS (MYC_aSHM), which were generally correspondent with IG::MYC rearrangements. MP3 tumors were characterized by TP53 mutations, including hotspots, and significantly depleted for aberrations in BCL2, BCL6 and lower mutation rates at aSHM targets SGK1, PAX5, CIITA, RHOH and others. Notably, tumors in this subgroup were enriched for IRF4 translocations, suggesting potential overlap with large B-cell lymphoma with IRF4 rearrangement. Our approach further refined both the the ST2/C4 and EZB/C3/BCL2 classes into two smaller subgroups, aSEL/aSCI and EGB/ETB, respectively. The EGB tumors were enriched for GNAI2 mutations, and MYC translocations involving the BCL6 enhancer, and were more often associated with MEF2B hotspot mutations. Notably, ETB was significantly enriched for mutations in the lysine acetyltransferase (KAT) domain in CREBBP, while EGB was significantly enriched for non-KAT domain mutations. ETB tumors were further characterized by mutations in STAT6 and FOXO1 hotspot regions, MYC rearrangements with other non-IG partners, and TNFRSF14 mutations, largely resembling the mutational profile of follicular lymphomas. Conclusions: We present here a refined classification of DLBCL genetic subgroups that combines previously described classification algorithms and extends it to allow classification of more tumors into additional subgroups that preserve the major divisions of existing systems. This represents an important advancement that will facilitate further understanding of genomic complexity in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Laboratory Mutation Analysis Performed on Tumor Biopsies from Patients with Newly-Diagnosed Germinal Center Aggressive B Cell Lymphoma

Background Comprehensive genomic analyses of tumor biopsies (bx) from patients (pts) with newly-diagnosed germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) and high grade B cell lymphoma (HGBL) have identified molecular subtypes predictive of inferior survival outcomes following receipt of first-line immunochemotherapy (PMID: 29713087, 29641966, 30523719, 30523716). These experimentally-defined molecular subtypes are characterized by somatic mutations (mut), which can be detected through clinical laboratory mutation analysis (CLMA). We sought to identify the frequency and predictive value of these mut as detected by CLMA performed at our center on tumor bx from pts with newly-diagnosed GCB DLBCL/HGBL. Methods Pts diagnosed with de novo or previously-untreated transformed indolent GCB DLBCL/HGBL defined by Hans algorithm from 2015-21 who received R-CHOP or R-EPOCH in the first-line setting whose initial tumor bx underwent CLMA performed at the University of Pennsylvania with either the lymphoma sequencing panel (LSP) or PennSeq Lymphoma panel (PSLP) were retrospectively analyzed. Immunohistochemical staining (IHC) for MYC and BCL2 expression as well as fluorescence in situ hybridization (FISH) for MYC, BCL2 and BCL6 rearrangements were performed as per institutional standard. Therapy was given at the discretion of the treating physician. Disease free survival (DFS) was defined as the interval between diagnosis of DLBCL/HGBL to DLBCL/HGBL relapse or last follow-up in remission. Overall survival (OS) was defined as the interval between diagnosis and time of death or last follow-up while alive. Data were censored on 7/1/22. Results CLMA was performed on 59 pt tumor bx with 58 successful assays (98% success rate) and a median result turnaround time (TAT) of 16 days. 5 bx were excluded due to specific request for CLMA by treating clinician (3) or diagnosing pathologist (2) and 2 additional bx were excluded due to lack of clinical follow-up, resulting in analysis of 51 bx performed at random from pts evaluable for survival (n=32 LSP, n=19 PSLP). Baseline characteristics, including mut in genes of interest (GOI) identified in the above references, are listed in the Table. Of note, BCL2, MYC, DDX3X and PTEN mutations were not included the LSP and therefore not included in statistical analyses due to small sample size. R-EPOCH was received by 15 pts (29%) and R-CHOP by 36 pts (71%). With a median follow-up of 25.2 months, the Kaplan Meier estimate of 2 year (y) DFS was 60% (95% confidence interval [CI] 41-74%) and 2y OS 80% (95% CI 64-89%). As shown in the Figure, 2y DFS was significantly lower for pts with CREBBP mut vs not (45% [95% CI 18-68%] vs 67% [95% CI 44-83%], P=0.045), but not for pts based on other mut analyzed. All progression events for pts with CREBBP mut occurred ≤12 months (mo) from diagnosis. Of 16 bx with CREBBP mut, 15 mut were predicted to result in loss of function (LOF) (PMID: 28890946, 21390126). CREBBP mut were not significantly associated with baseline characteristics except for EZH2 mut (50% with CREBBP mut vs 11% no CREBBP mut, P=0.005 by Fisher's exact). Characteristics listed in the Table which were predictive of disease relapse by 2y with P<0.10 on univariate Cox regression analysis were International Prognostic Index Score ≥3 vs <3 (hazard ratio [HR] 2.3, 95% CI 0.86-6.5, P=0.098), HGBL vs DLBCL histology (HR 5.4, 95% CI 1.8-15.7, P=0.002), double hit lymphoma vs not (HR 9.0, 95% CI 2.6-30.8, P<0.001) and CREBBP mut vs not (HR 2.6, 95% CI 0.98-7.0, P=0.054). Conclusions In this cohort of pts with newly-diagnosed GCB DLBCL/HGBL treated with first-line immunochemotherapy whose tumor bx underwent CLMA at random, mut in CREBBP resulting in predicted LOF were frequently detected, not associated with routinely-analyzed baseline clinicopathologic characteristics and predicted for significantly lower 2y DFS with progression events occurring early. These findings may be of interest in light of availability of agents which may overcome loss of histone acetyltransferase function. Given the high success rate and rapid TAT of CLMA, results from this assay could inform the management of pts with newly-diagnosed DLBCL/HGBL in real-time, and additional efforts to incorporate findings from experimental molecular assays into CLMA-based studies as well as those to standardize CLMA gene panels may ultimately benefit this pt population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery of HZ-L105, a Next Generation of Bcl-2 Inhibitor, Overcomes Bcl-2 Mutation and Exhibits Superior Antitumor Activity

Introduction: Bcl-2, a key regulator of apoptosis pathway, is over-expressed in many hematologic malignancies, which results in blocking of apoptosis and cell survival. Targeting Bcl-2 for the treatment of malignant hematological tumors has been well validated and demonstrated by FDA approval of the first generation Bcl-2 inhibitor Venetoclax for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). Although Venetoclax provides significant clinical benefits, there are reports show that after long-term monotherapy patients develop disease progression , often driven by acquisition of Bcl-2 Gly101Val (G101V), Asp103Tyr (D103Y) resistance mutations. Here, we described the discovery of a next generation Bcl-2 inhibitor HZ-L105 as with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and Venetoclax-resistant hematological cancers. Methods:Biochemical inhibition assays were conducted towards Bcl-2/Bcl-2 G101V/Bcl-2 D103Y/Bcl-xL by time-resolved fluorescence resonance energy transfer (TR-FRET);Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;Human platelet viability was measured by Cell-titer Glo assay;Cytochrome P450 inhibition assays were performed using fluorometric assay;In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;Preclinical pharmacokinetics (PK) studies were assayed in mice and dogs and parameters were determined by LC-MS. Results: In biochemical assays, HZ-L105 potently inhibited Bcl-2 WT, Bcl-2 G101V and Bcl-2 D103Y with IC50 of 0.54 nM, 6.16 nM and 5.57 nM，while Venetoclax's IC50 of 1.62 nM, 440.6 nM and 279.8 nM, respectively. Therefore, it is expected to overcome the acquisition of Bcl-2 resistance mutations. In cellular assays, HZ-L105 suppressed cell growth not only in RS4;11 but also in RS4;11-G101V stable line (IC50=0.30 nM in RS4;11 and 243.36 nM in RS4;11-G101V), which was much more superior to Venetoclax (IC50= 2.91 nM in RS4;11 and 3360.79 nM in RS4;11-G101V). HZ-L105 was also highly selective against Bcl-2, showing >1000 folds selectivity to Bcl-xL. Furthermore, platelet viability assay in vitro showed HZ-L105 induced hypotoxicity as similarly to Venetoclax, suggesting its good potential to avoid thrombocytopenia due to Bcl-xL inhibition. In the preclinical Pharmacodynamics studies, oral administration of HZ-L105 induced rapid and robust antitumor effect in RS4;11 ALL xenografts. HZ-L105 at a dose of 5 mg/kg demonstrated significantly excellent efficacy than Venetoclax at a dose 15mg/kg in RS4;11-derived xenograft mouse model. In the preclinical pharmacokinetic studies, HZ-L105 showed excellent pharmacokinetic properties in ICR mice and beagle dogs, with long T1/2 and high dose-normalized AUC. HZ-L105 did not reduced hERG tail current (% current blocked 2.87% at 30μM), indicating the compound has a low potential to induce QT prolongation. HZ-L105 has less drug-drug interaction potential, as evidenced by no inhibitory effect on CYPs, especially in CYP2C9 (IC50 >20 μM) compared with Venetoclax (IC50=1.12 μM). Besides, in 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mg/kg/day, and only leukopenia was observed similarly to Venetoclax. Conclusion: In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1 Study Evaluating the Safety, Tolerability, Pharmacokinetics, and Preliminary Antitumor Activity of Bcl-2 Inhibitor Bgb-11417 in Adult Patients with Mature B-Cell Malignancies: Preliminary Data

Background/Introduction: The efficacy of Bcl-2 inhibitors in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was established by the approval of agents such as venetoclax across all lines of therapy. However, adverse events (AEs) related to venetoclax and the development of BCL2 mutations leading to resistance may limit the utility of venetoclax in the clinic. Compared to venetoclax, BGB-11417 is a more potent (>10 fold in biochemical assays) and highly selective Bcl-2 inhibitor with the potential to achieve deeper target inhibition and clinical responses. Here, we report preliminary data from a phase 1 trial of BGB-11417 in patients with mature B-cell malignancies. Methods: BGB-11417-102 (NCT04883957) is a multicenter, phase 1, open-label, dose-finding study conducted in China. The primary objectives were to determine the safety, maximum tolerated dose (MTD) or maximum administered dose, and the recommended phase 2 dose (RP2D) of BGB-11417 monotherapy for the selected B-cell malignancy dose-finding cohorts. Patients with relapsed/refractory (R/R) B-cell malignancies received escalating doses of BGB-11417 monotherapy (80, 160, 320, or 640 mg once daily) with a weekly or daily ramp-up to the intended target dose. Dose-limiting toxicity for each dose cohort was evaluated by a Bayesian logistic regression model during dose ramp-up through day 21 at intended dose. AEs were reported per Common Terminology Criteria for AEs v5.0. Results: As of June 16, 2022, 41 patients were treated. Twenty-seven with R/R non-Hodgkin lymphoma (NHL; 13 diffuse large B-cell lymphoma, 7 follicular lymphoma, 4 marginal zone lymphoma, and 3 transformed NHL) received BGB-11417 ≤640 mg/d and 14 with R/R CLL/SLL received ≤320 mg/d. MTD has not yet been reached. The median values were as follows: age, 62 years (range 31-84); lines of prior systemic therapy, 2 (range 1-7); and follow-up, 5.7 months (range 0.7-9.5). A total of 19 patients (18 NHL and 1 CLL/SLL) discontinued the study treatment (14 progressive disease, 4 withdrawal by subject, and 1 investigator decision). At least 1 treatment-emergent AE (TEAE) was reported by 97.6% of patients and grade ≥3 AEs were reported by 51.2%. The most common TEAEs (reported in ≥10% of patients) across all dose levels are listed in the Table. Dose-limiting toxicity events were reported in 7.3% of patients (grade 3 febrile neutropenia and grade 3 platelet count decrease at 80 mg/d dose level each; grade 3 bone pain at 160 mg/d dose level). Serious TEAEs were reported in 9.8% of patients, and TEAEs leading to drug interruption were reported in 12.2%. No patients reported TEAEs leading to death or treatment discontinuation. Laboratory tumor lysis syndrome (TLS) events were reported in 4.9% of patients, and all were controlled without dose modification. No clinical TLS events were reported. Of the 31 patients available for tumor assessment (23 NHL and 8 CLL/SLL), 4 patients with NHL and 5 patients with CLL/SLL achieved responses (NHL: 3 partial responses [PR] at 160 mg/d dose level and 1 PR at 320 mg/d dose level; CLL/SLL: 2 complete responses [CRs]/CR with incomplete count recovery, 1 PR at 80 mg/d dose level, and 2 PR at 160 mg/d dose level). One patient with CLL had a CLL count of less than 10-4 in both blood and bone marrow aspirate after 4.5 months of treatment with 80 mg/d dose, as measured by European Research Initiative in CLL flow cytometry assay with 10-4 sensitivity. Conclusion: These results demonstrated that BGB-11417 is effective and well tolerated at all tested doses, with no increase in AEs with increasing dose. Dose escalation is still ongoing because an MTD has not yet been reached in any dose-escalation cohort as of the data cutoff date, with patients being recruited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1 Study with the Novel B-Cell Lymphoma 2 (Bcl-2) Inhibitor Bgb-11417 As Monotherapy or in Combination with Zanubrutinib (ZANU) in Patients (Pts) with CLL/SLL: Preliminary Data

Background/introduction: The effectiveness of Bcl-2 inhibitors as a treatment for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was established by the approval of venetoclax in pts with CLL/SLL across all lines of therapy. However, the related adverse events (AEs) and emergence of BCL2 mutations, resulting in resistance, can limit the utility of venetoclax. BGB-11417 is a highly selective Bcl-2 inhibitor with potency >10 times that of venetoclax in biochemical assays. BGB-11417 monotherapy is tolerable, with no maximum tolerated dose (MTD) reached after dose escalation through all planned doses to 640 mg once daily (QD) in pts with non-Hodgkin lymphoma (EHA 2022. Abstract P687). The combination of Bcl-2 and Bruton tyrosine kinase (BTK) inhibitors is tolerable with synergistic activity in CLL and mantle cell lymphoma (MCL) (J Clin Oncol 2019;37:2722-9; N Engl J Med 2019;380:2095-103; EHA 2020. Abstract S158; N Engl J Med 2018;378:1211-23). ZANU, a next-generation BTK inhibitor, has shown favorable activity and safety in pts with CLL/SLL (EHA 2021. Abstract LB1900) and Waldenström macroglobulinemia (Blood. 2020;136(18):2038-2050). BGB-11417-101 is an ongoing first-in-human phase 1/1b dose-escalation/expansion study (NCT04277637). Pts with various B-cell malignancies were enrolled; data from CLL/SLL cohorts are presented here. Methods: In separate monotherapy and combination therapy cohorts, pts received escalating doses of BGB-11417 (40, 80, 160, 320, or 640 mg QD) with a ramp-up to the intended target dose to minimize risk of tumor lysis syndrome (TLS). In combination therapy cohorts, pts received ZANU (320 mg QD or 160 mg twice daily) beginning 8-12 weeks before BGB-11417. Dose-limiting toxicity for each cohort was evaluated by a Bayesian logistic regression model during dose ramp-up through day 21 at the intended dose. AEs were reported per Common Terminology Criteria for AEs v5.0. Minimal residual disease (MRD) was assessed by a European Research Initiative on CLL flow cytometry assay. Results: As of May 15 2022, 50 pts with CLL received treatment: 6 monotherapy (all relapsed/refractory [R/R]) and 44 combination (22 R/R; 22 treatment naïve [TN]). The monotherapy CLL cohort received BGB-11417 doses up to 160 mg. Based on emerging safety data from other cohorts, pts in combination cohorts with R/R CLL received BGB-11417 up to 640 mg and pts with TN CLL received up to 320 mg (data include 8 pts in ZANU pretreatment not yet dosed with BGB-11417). MTD has not yet been reached for any CLL cohort, with dose escalation ongoing. Median follow-up was 11.5 mo (range 8.5-18.3) for monotherapy and 5.8 mo (range 0.2-10.5) for combination. Treatment-emergent AEs (TEAEs) across all doses are listed in the Table. With monotherapy, cytopenias were the most common TEAEs (≥50%), with 33% grade ≥3. With combination, contusion, neutropenia, and low-grade gastrointestinal toxicity were the most common TEAEs (≥22.7%); neutropenia was the most common grade ≥3 TEAE (11.4%) with 5 pts. No pts discontinued monotherapy treatment, and 1 pt discontinued combination treatment (disease progression; Richter transformation). Only 1 high-risk pt with CLL on monotherapy had laboratory TLS that resolved with no intervention (overall laboratory TLS ≤2%). No clinical TLS was reported. Diarrhea was mostly grade 1 and grade ≥3 was not seen. Although efficacy data are early, most pts with CLL/SLL had notable reductions in absolute lymphocyte count (ALC) with responses seen at doses as low as 1 mg (Figure), consistent with improved potency of BGB-11417 vs venetoclax. Four responses (66%, partial response [PR] or better) and 32 responses (72.7%, PR with lymphocytosis or better) were observed with mono- and combination therapy, respectively. MRD data are early: among 4 MRD evaluable pts at 160 mg, 3 pts (2 monotherapy; 1 combination) had a peripheral blood CLL count <10-4 at 24 weeks after BGB-11417 initiation. Conclusion: These preliminary data show BGB-11417, alone or in combination with ZANU, was well-tolerated in most patients. Grade ≥3 neutropenia was uncommon and manageable. Efficacy is supported by the rapid reduction in ALC during ramp-up, and early response data are promising. TLS rates are low; the prophylactic measures and ramp-up schedule seem to adequately mitigate TLS across all dose levels tested. Mature MRD data are forthcoming, and venetoclax-treated CLL/SLL cohorts will soon be open for enrollment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic and predictive biomarkers for response to neoadjuvant chemoradiation in esophageal adenocarcinoma

BackgroundEsophageal adenocarcinoma is a lethal disease. For locally advanced patients, neoadjuvant chemoradiotherapy followed by surgery is the standard of care. Risk stratification relies heavily on clinicopathologic features, particularly pathologic response, which is inadequate, therefore establishing the need for new and reliable biomarkers for risk stratification.MethodsThirty four patients with locally advanced esophageal adenocarcinoma were analyzed, of which 21 received a CROSS regimen with carboplatin, paclitaxel, and radiation. Capture-based targeted sequencing was performed on the paired baseline and post-treatment samples. Differentially mutated gene analysis between responders and non-responders of treatment was performed to determine predictors of response. A univariate Cox proportional hazard regression was used to examine associations between gene mutation status and overall survival.ResultsA 3-gene signature, based on mutations in EPHA5, BCL6, and ERBB2, was identified that robustly predicts response to the CROSS regimen. For this model, sensitivity was 84.6% and specificity was 100%. Independently, a 9 gene signature was created using APC, MAP3K6, ETS1, CSF3R, PDGFRB, GATA2, ARID1A, PML, and FGF6, which significantly stratifies patients into risk categories, prognosticating for improved relapse-free (p = 4.73E-03) and overall survival (p = 3.325E-06). The sensitivity for this model was 73.33% and the specificity was 94.74%.ConclusionWe have identified a 3-gene signature (EPHA5, BCL6, and ERBB2) that is predictive of response to neoadjuvant chemoradiotherapy and a separate prognostic 9-gene classifier that predicts survival outcomes. These panels provide significant potential for personalized management of locally advanced esophageal cancer.

EBV+/HHV-8+/HIV+ high grade T Cell Lymphoma in the stomach

Abstract Introduction/Objective T-cell Lymphoma is overly aggressive. T cells overgrow triggered by aberrant T-cell signaling and transform into a lymphoma. Virus infection contributes to the transformation, for example, EBV (Epstein Barr Virus) virus is associated with Extranodal NK/T-cell lymphoma, Angioimmunoblastic T-cell lymphoma, and peripheral T-cell lymphoma, HHV-8 is associated with Primary Effusion Lymphoma, Kaposi sarcoma, and very rarely associated with T cell lymphoma. The HIV (Human Immunodeficiency Virus) virus is associated with Burkitt lymphoma, Plasmablastic lymphoma et al. Among these lymphomas, only Primary Effusion lymphoma is associated with all three virus infections and is a B cell lymphoma. T cell lymphoma with all three infections has not been reported. Since most patients are diagnosed with advanced disease. So early diagnosis of this disease is especially important. Methods/Case Report An HIV+ 38-year-old male with no significant medical history complained of weakness, dizziness, and presenting with lower extremities swelling, and anemia for the last 2 weeks. The patient had a history of an unsafe sexual encounter with male sex workers but denies iv. drug use and blood transfusion. The screen before endoscopy reveals active, acute Hepatitis B Virus infection. Endoscopy findings show gastritis. The biopsy specimen shows mucosa is diffusely infiltrated by atypical plasmablastic cells. Immunostaining reveals they are positive for CD3, CD7, CD30, MUM1, CD43, and HHV8, but negative for Cd2, Cd4, Cd5, Cd8, Cd10, Cd21, Cd23, Cd25, Cd34, Cd56, Cd138, CD19, CD20, CD79a, PAX5, CD45, CD57, P53, CyclinD1, BCL6, BCL2, ALK, c-MYC, TdT, Granzyme B, TIA, S-100, Pancytokeratin. In situ hybridization for EBER is positive, but negative for Kappa and Lambda. The proliferative index (Ki-67) is high (99%). The morphologic and immunophenotypic features are consistent with an EBV CD30+ high-grade lymphoma. The sequencing from OnkoSightTM Advanced Chronic Lymphoid NGS Report shows a BCL-6 and EP300 mutation that has not been reported in a publicly available database (COSMIC; gnomAD). Results (if a Case Study enter NA) N/A. Conclusion To our knowledge, this is the first reported case of EBV+/HHV-8+/HIV+ high grade T Cell Lymphoma in the stomach. This type of lymphoma is extremely aggressive, and the early diagnosis is particularly important for prognosis.

Whole-exome sequencing analysis identifies distinct mutational profile and novel prognostic biomarkers in primary gastrointestinal diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma, and about 10% of DLBCL cases primarily occur in the gastrointestinal tract. Previous reports have revealed that primary gastrointestinal-DLBCL (pGI-DLBCL) harbors different genetic mutations from other nodal or extranodal DLBCL. However, the exonic mutation profile of pGI-DLBCL has not been fully addressed.MethodsWe performed whole-exome sequencing of matched tumor tissues and blood samples from 53 pGI-DLBCL patients. The exonic mutation profiles were screened, and the correlations between genetic mutations and clinicopathological characteristics were analyzed.ResultsA total of 6,588 protein-altering events were found and the five most frequent mutated genes in our pGI-DLBCL cohort were IGLL5 (47%), TP53 (42%), BTG2 (28%), P2RY8 (26%) and PCLO (23%). Compared to the common DLBCL, significantly less or absence of MYD88 (0%), EZH2 (0%), BCL2 (2%) or CD79B (8%) mutations were identified in pGI-DLBCL. The recurrent potential driver genes were mainly enriched in pathways related to signal transduction, infectious disease and immune regulation. In addition, HBV infection had an impact on the mutational signature in pGI-DLBCL, as positive HBsAg was significantly associated with the TP53 and LRP1B mutations, two established tumor suppressor genes in many human cancers. Moreover, IGLL5 and LRP1B mutations were significantly correlated with patient overall survival and could serve as two novel prognostic biomarkers in pGI-DLBCL.ConclusionsOur study provides a comprehensive view of the exonic mutation profile of the largest pGI-DLBCL cohort to date. The results could facilitate the clinical development of novel therapeutic and prognostic biomarkers for pGI-DLBCL.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

Abstract A28: BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and the activated B cell-like subtype (ABC-DLBCL) is the most aggressive form and harbors frequent mutations of immune signaling pathways that culminate in constitutive activation of CARD11-MALT1-BCL10 (CBM) complex and downstream NF-κB pathway. CBM complexes form large macromolecular structures due to signal-induced polymerization of the BCL10 subunit, which is affected by recurrent somatic mutations in ABC-DLBCLs. Through biochemical, structural and functional dissection of these mutations, we find that BCL10 mutations fall into two functionally distinct classes: missense mutations of the BCL10 CARD domain (hotspot R58Q) and truncation of its C-terminal tail (hotspot E140X). To explore the functional consequences of BCL10 mutations, we established reporter systems to evaluate their impact on MALT1 and NF-𝜅B activities which are BCL10 downstream signaling cascades. We found that almost all mutants induced aberrantly strong NF-𝜅B and MALT1 activities in lymphoma cells as compared to WT BCL10, indicating the gain-of-function effect of BCL10 mutations. By performing immunohistochemistry staining of p65 in a set of tumor tissue microarray from DLBCL patients (n=298), we revealed that BCL10 mutant tumors have significantly (Mann-Whitney p&amp;lt;0.0001) increased p65 nuclear staining score compared to BCL10 WT tumors, suggesting enhanced NF-𝜅B activity. To investigate the biochemical impact of BCL10 mutants on CBM complex formation, we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 proteins and found that both BCL10R58Q and BCL10E140X manifested faster and more spontaneous polarization compared to BCL10WT. Surprisingly, through mapping the BCL10-MALT1 interaction, we found that truncating mutation (E140X) abrogated a novel protein interaction motif through which MALT1 inhibits BCL10 polymerization, thus unleashing spontaneous CBM filament formation and inducing addiction to MALT1 activity. In marked contrast, the CARD missense mutation (R58Q) on BCL10 filament interface not only does not disrupt but enhances filament formation and it also alters CBM complex kinetics forming glutamine network structures that stabilize BCL10 filaments, but this still may require the upstream signal to activate MALT1. Importantly, we found that BCL10 mutant cells were less dependent on upstream CARD11 activation in MALT1 activation, NF-𝜅B signaling and cell growth assays performed in ABC-DLBCL lines. Furthermore, in vitro and in vivo xenograft studies revealed that BCL10 mutant lymphomas are resistant to BTK inhibitors, whereas BCL10 truncating (E140X) but not missense CARD (R58Q) mutants were hypersensitive to MALT1 protease inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL and further precision can be achieved by tailoring therapy (e.g. MALT1 inhibitors that are currently being tested in clinical trials) according to specific biochemical effects of distinct mutation classes. Citation Format: Min Xia, Liron David, Matthew R Teater, Johana Gutierrez, Xiang Wang, Cem Meydan, Andrew Lytle, Graham Slack, David Scott, Ozlem Onder, Kojo Elenitoba-Johnson, Nahuel Zamponi, Leandro Cerchietti, Tianbao Lu, Ulrike Philippar, Lorena Fontan, Hao Wu, Ari Melnick. BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A28.

P11.17.B Molecular characterization of primary central nervous system lymphoma vs non-CNS lymphoma and correlation between mutational profile and treatment response

Abstract Background Primary central nervous system lymphoma (PCNSL) is a unique type of non-Hodgkin lymphoma restricted to the central nervous system. Most cases are diffuse large B-cell lymphomas, but prognosis is far less favorable compared to extracerebral cases. Many mutations found in PCNSL converge on the NF-κB signaling pathway, leading to downstream activation of B-cell receptor (BCR) and NF-κB pathway. The differences in mutation frequency when compared to non-CNS lymphoma and how mutational profiles affect treatment response are not well understood. We compared the genomic profile of PCNSL and non-CNS lymphoma, evaluated the association between specific molecular alterations and survival, and analyzed the association between various mutations and response to therapy. Materials and Methods A total of 60 patients with PCNSL, 8 with secondary CNS lymphoma, and 202 with non-CNS B-cell lymphomas underwent molecular profiling at Caris Life Sciences (Phoenix, AZ). Analyses included next-generation sequencing of DNA (592 Genes, NextSeq or WES, NovaSeq) and RNA (WTS, NovaSeq). X2/Fisher’s-exact U tests were used for comparison, and significance was determined as p-value adjusted for multiple comparison by the Benjamini-Hochberg method (q&amp;lt;0.05). Overall survival (OS) was calculated from the start of temozolomide (TMZ) or tissue collection to last contact using insurance claims data. Results When compared to non-CNS lymphomas, PCNSL tumors showed significantly higher mutation rates in MYD88 (70% vs. 7%), PIM-1 (58% vs. 7%), CD79B (42% vs. 3%) and higher rates of TMB-High (cutoff 10mut/MB; 28% vs. 10%) (q&amp;lt;0.01). In addition, mutations in CARD11 (12% vs. 4%) and IRF4 (8% vs. 1%) trended to be more prevalent in PCNSL (p&amp;lt;0.05). In contrast, mutations in KMT2D (35% vs. 16%), EZH2 (13% vs. 2%), CREBBP (22% vs. 7%), TNFRSF14 (10% vs. 0) and BCL2 (19% vs. 2%) trended to be more prevalent in non-CNS lymphomas compared to PCNSL (p&amp;lt;0.05). In patients with PCNSL, there was no difference in survival in those with or without MYD88, PIM1 or CD79B mutations. No between-group differences were observed in the small cohort of patients with secondary CNS lymphoma (n=8). In the subgroup in whom these data were available, 25% of tumors were MGMT methylated, 71% had a “high or intermediate” tumor mutational burden, 8% were MSI high, and 54% were PD-L1 expression positive when tested by immunohistochemistry. Patients with PCNSL had worse overall survival (OS) than non-CNS lymphomas (30 vs 81 months, p&amp;lt;0.001). Conclusion MYD88, PIM-1, and CD79B mutations are more frequent in PCNSL compared to non-CNS lymphomas, but none of these mutations affected OS. Only 25% of PCNSL patients are MGMT methylated, but a majority have expression of PD-L1 suggesting a benefit from PD-1-targeted therapy. Response and survival in patients with mutation-guided therapy will be presented.

Targeting Acute Myeloid Leukemia with Venetoclax; Biomarkers for Sensitivity and Rationale for Venetoclax-Based Combination Therapies.

Simple SummaryVenetoclax has proven to be a promising therapy for newly diagnosed, relapsed and refractory AML patients ineligible for induction chemotherapy. Current ongoing clinical trials are evaluating its effectivity as frontline therapy for all acute myeloid leukemia (AML) patients. However, response rates vary wildly, depending on patient characteristics and mutational profiles. This review elaborates on the efficacy and safety of venetoclax compared to conventional chemotherapy for treatment of AML patients, comparing the response rates, overall survival and adverse events. Moreover, it gives an overview of genetic and epigenetic AML cell characteristics that give enhanced or decreased response to venetoclax and offers insights into the pathogenesis of venetoclax sensitivity and resistance. Additionally, it suggests possible treatment combinations predicted to be successful based on identified mechanisms influencing venetoclax sensitivity of AML cells.Venetoclax is a BCL-2 inhibitor that effectively improves clinical outcomes in newly diagnosed, relapsed and refractory acute myeloid leukemia (AML) patients, with complete response rates (with and without complete blood count recovery) ranging between 34–90% and 21–33%, respectively. Here, we aim to give an overview of the efficacy of venetoclax-based therapy for AML patients, as compared to standard chemotherapy, and on factors and mechanisms involved in venetoclax sensitivity and resistance in AML (stem) cells, with the aim to obtain a perspective of response biomarkers and combination therapies that could enhance the sensitivity of AML cells to venetoclax. The presence of molecular aberrancies can predict responses to venetoclax, with a higher response in NPM1-, IDH1/2-, TET2- and relapsed or refractory RUNX1-mutated AML. Decreased sensitivity to venetoclax was observed in patients harboring FLT3-ITD, TP53, K/NRAS or PTPN11 mutations. Moreover, resistance to venetoclax was observed in AML with a monocytic phenotype and patients pre-treated with hypomethylating agents. Resistance to venetoclax can arise due to mutations in BCL-2 or pro-apoptotic proteins, an increased dependency on MCL-1, and usage of additional/alternative sources for energy metabolism, such as glycolysis and fatty acid metabolism. Clinical studies are testing combination therapies that may circumvent resistance, including venetoclax combined with FLT3- and MCL-1 inhibitors, to enhance venetoclax-induced cell death. Other treatments that can potentially synergize with venetoclax, including MEK1/2 and mitochondrial complex inhibitors, need to be evaluated in a clinical setting.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

P615: BCL2 RESISTANCE MUTATIONS IN A REAL-WORLD COHORT OF PATIENTS WITH VENETOCLAX-TREATED CHRONIC LYMPHOCYTIC LEUKAEMIA

Background: The Bcl-2 inhibitor venetoclax has transformed the therapeutic landscape of therapy refractory and treatment naïve chronic lymphocytic leukaemia (CLL). Despite the remarkable response rates in both patient groups, a subset of CLL patients receiving venetoclax treatment experience disease progression, frequently associated with pathogenic variants of the BCL2 gene. Apart from the most common hotspot G101V and D103Y mutations, pathogenic variants affecting several different loci of the coding region of BCL2 have been described, however data and international recommendations on the sensitive detection and monitoring of these mutations are still absent to date. Aims: Our aim was to develop a droplet digital PCR (ddPCR) based assay for the detection of BCL2 G101V and D103Y mutations and to reveal further resistance mutations emerging at relapse by performing targeted ultra-deep next-generation sequencing (NGS). Methods: Peripheral blood samples from 71 patients treated with venetoclax-rituximab or first line venetoclax-obinutuzumab combination were collected from 11 Hungarian oncohematological centres. Genomic DNA was extracted from peripheral blood mononuclear cells and leukemic cell purity was assessed by flow cytometry. Fractional abundance of the most common hotspot BCL2 mutations G101V and D103Y was assessed using the QX200 digital droplet PCR system. Following ddPCR analyses, targeted ultradeep NGS was performed on samples obtained at relapse using a custom-designed panel covering the whole coding regions of BCL2, BTK, PLCG2 and TP53 genes. Fractional abundance of BCL2 mutations identified by NGS was assessed retrospectively in serial peripheral blood samples by ddPCR using custom-designed, allele-specific assays. Results: Lower quantitative limit of detection of the BCL2 ddPCR assays was tested and could ubiquitously be established as 0.01%. With a median follow up of 14 months, BCL2 G101V and D103Y mutations were detected in 8.5% (6/71) and 9.9% (7/71) of the patients, respectively, with 90% (9/10) of them experiencing relapse during the follow-up period. Three patients harbored both resistance mutations as detected by ddPCR. BCL2 G101V and/or D103Y were observed in 43.5% (10/23) of the cases showing signs of disease progression. In four cases, emergence of the mutations predated the first clinical signs of relapse with a median 4 months. In samples obtained at relapse, targeted ultradeep NGS uncovered two additional BCL2variants (V156D and A113G), which were successfully backtracked and detected by ddPCR. Both V156D and A113G were detected in patients previously found to harbor G101V or D103Y, with no further BCL2 resistance mutations identified in G101V and D103Y wild type cases. Summary/Conclusion: In a real-world cohort of CLL patients receiving venetoclax in combination therapy, comprehensive and sensitive screening of BCL2 mutations can identify molecular mechanisms of venetoclax resistance in nearly half of the patients experiencing relapse. In patients harboring multiple BCL2 mutations, convergent evolution of the CLL subclones may contribute to the driver mechanisms of resistance, justifying the comprehensive approach for the detection of these variants. In secondary venetoclax resistant cases displaying wild type BCL2, further molecular screening methods are required to reveal alternative genetic or non-genetic reasons for disease progression.

P670: ABSENCE OF BTK, BCL2, AND PLCG2 MUTATIONS IN RELAPSING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER FIRST-LINE TREATMENT WITH FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V)

Background: CAPTIVATE (PCYC-1142; NCT02910583) is a multicenter phase 2 study of first-line I+V in patients (pts) with CLL in 2 cohorts: Minimal Residual Disease (MRD) and Fixed Duration (FD). We previously reported that fixed-duration I+V provides deep, durable responses (Ghia et al, ASCO 2021; Wierda et al, J Clin Oncol 2021). Clinically-relevant mutations in BTK, PLCG2, and BCL2 have previously been reported in pts with progressive disease (PD) following single agent BTK or BCL2 inhibitor therapies. Such resistance-associated mutations may occur with increasing frequency after prolonged drug exposure. Fixed duration I+V is hypothesized to decrease the likelihood of developing resistance mutations and facilitate extended clinical benefit. Aims: We sought to ascertain presence or absence of resistance mutations at the time of PD in samples from pts treated with fixed-duration I+V in CAPTIVATE. Baseline genomic risk features and potentially prognostic mutations in CLL-related genes of interest were also evaluated in pts with and without subsequent PD. Methods: The analysis was conducted in 191 pts aged ≤70 y with previously untreated CLL who completed fixed-duration I+V, defined as 3 x 28d cycles of I then 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally); this included pts from the FD cohort, and pts from the MRD cohort with confirmed undetectable MRD who received an additional cycle of I+V and were randomized to placebo. Samples from pts at the time of PD were evaluated for resistance mutations by targeted next-generation sequencing (NGS) using a custom-designed high-read-depth 100-gene panel (limit of detection 1% VAF, negative predictive value ≥95% assuming population prevalences <20%, for BTK, PLCG2, or BCL2 variants) in CD19-enriched peripheral blood (PB) or bone marrow. Mutations in additional genes of interest (Table) were evaluated by NGS using the Personalis ACE panel in baseline PB samples. Genomic risk features (Table) were also assessed at baseline. Mutations and genomic risk features evident at baseline were compared across PD and non-PD pts. Results: Baseline mutations in genes of interest and genomic risk features in 190 pts who completed fixed-duration I+V and with available baseline data are shown in the Table. Rates of mutations were similar between pts with (n=30) and without (n=160) subsequent PD. Of 30 pts with PD events (occurring between 16 and 53 mos after start of treatment), 22 (73%) had baseline mutations in genes of interest and 27 (90%) had ≥1 baseline genomic risk feature. Pts with PD generally had more baseline genomic risk features than pts without PD (median 3 vs 2; Wilcoxon P=0.02; analysis restricted to FD cohort pts to avoid bias). To date, 26 of 30 PD patient samples have been evaluated for mutations; no resistance-associated variants in BTK, PLCG2, or BCL2 were detected. Image:Summary/Conclusion: Earlier work has demonstrated a promising safety profile and efficacy of fixed-duration I+V in first-line CLL. Importantly, the current results show that no resistance-associated mutations in BTK, PLCG2, or BCL2 were identified in the pts with PD evaluated to date after first-line, fixed-duration I+V. The absence of such mutations at PD suggests that fixed-duration I+V treatment may offer the possibility of effective subsequent retreatment options with I and/or V and extend clinical benefit.

P669: FIXED-DURATION (FD) IBRUTINIB + VENETOCLAX FOR FIRST-LINE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)/SMALL LYMPHOCYTIC LYMPHOMA (SLL): 3-YEAR FOLLOW-UP FROM THE PHASE 2 CAPTIVATE STUDY FD COHORT

Background: CAPTIVATE (PCYC-1142) is a multicenter phase 2 study of first-line ibrutinib + venetoclax in CLL. The primary analysis evaluating FD treatment with ibrutinib + venetoclax was previously presented (Ghia et al., ASCO 2021). Aims: To present 3-year follow-up results from the FD cohort of CAPTIVATE. Methods: Patients aged ≤70 years with previously untreated CLL/SLL received 3 cycles of ibrutinib then 12 cycles of ibrutinib + venetoclax (ibrutinib 420 mg/day orally; venetoclax ramp-up to 400 mg/day orally). Responses were investigator assessed per iwCLL 2008 criteria. Undetectable minimal residual disease (uMRD; <10-4) was measured by 8-color flow cytometry. Serious AEs (SAEs) deemed related to ibrutinib reported >30 days after last dose of study drug were collected. Results: 159 patients were enrolled (median age 60 years), including patients with high-risk features of del(17p)/TP53 mutation (17%), unmutated IGHV (uIGHV; 56%), and complex karyotype (19%). 147 (92%) and 149 (94%) patients completed treatment with ibrutinib and venetoclax, respectively. With 1 year of additional follow-up since primary analysis, median time on study was 39 months (range 1-41). ORR was 96% and was consistent (96%-97%) in patients with high-risk features (Table). The primary endpoint of complete response (CR) including CR with incomplete bone marrow recovery (CRi) rate in patients without del(17p) (n=136) increased nominally from 56% (95% CI, 48-64) to 58% (95% CI 50-66); in all patients, CR rate increased from 55% (95% CI 48-63) to 57% (95% CI 50-65). In patients achieving CR, 93% had durable responses lasting ≥12 months post-treatment. Of patients with uMRD in peripheral blood at 3 months post-treatment, 66/85 (78%) evaluable patients maintained uMRD through 12 months post-treatment. At 36 months, PFS was 88% (95% CI 82‒92) and OS was 98% (95% CI 94-99); similar rates were seen in patients with high-risk features (Table). All patients are off treatment; no new SAEs of any kind have occurred since the primary analysis. Available data on relevant mutations in BTK, PLCɣ2, or BCL-2 at time of PD will be presented. As of January 2022, 12 patients were retreated with single-agent ibrutinib after progressive disease (treatment duration range 3-29 months); of evaluated patients, 7/9 had partial responses and 2/9 had stable disease. Image:Summary/Conclusion: Fixed duration ibrutinib + venetoclax continues to provide deep, durable responses and clinically meaningful PFS, including in patients with high-risk disease features, representing an all-oral, once-daily, chemotherapy-free FD regimen for previously untreated patients with CLL/SLL. With an additional 1 year of follow-up, no OS events or SAEs occurred. Manageable safety profile is unchanged as previously reported. To date, successful single-agent ibrutinib retreatment responses are observed.