BCL2 Mutations Research Articles

Testicular large B-cell lymphoma is genetically similar to PCNSL and distinct from nodal DLBCL.

Testicular large B-cell lymphoma (TLBCL) is an infrequent and aggressive lymphoma arising in an immune-privileged site and has recently been recognized as a distinct entity from diffuse large B-cell lymphoma (DLBCL). We describe the genetic features of TLBCL and compare them with published series of nodal DLBCL and primary large B-cell lymphomas of the CNS (PCNSL). We collected 61 patients with TLBCL. We performed targeted next-generation sequencing, copy number arrays, and fluorescent in situ hybridization to assess chromosomal rearrangements in 40 cases with available material. Seventy percent of the cases showed localized stages. BCL6 rearrangements were detected in 36% of cases, and no concomitant BCL2 and MYC rearrangements were found. TLBCL had fewer copy number alterations (p < 0.04) but more somatic variants (p < 0.02) than nodal DLBCL and had more frequent 18q21.32-q23 (BCL2) gains and 6q and 9p21.3 (CDKN2A/B) deletions. PIM1, MYD88 L265P , CD79B, TBL1XR1, MEF2B, CIITA, EP300, and ETV6 mutations were more frequent in TLBCL, and BCL10 mutations in nodal DLBCL. There were no major genetic differences between TLBCL and PCNSL. Localized or disseminated TLBCL displayed similar genomic profiles. Using LymphGen, the majority of cases were classified as MCD. However, we observed a subgroup of patients classified as BN2, both in localized and disseminated TLBCL, suggesting a degree of genetic heterogeneity in the TLBCL genetic profile. TLBCL has a distinctive genetic profile similar to PCNSL, supporting its recognition as a separate entity from DLBCL and might provide information to devise targeted therapeutic approaches.

Different Shades of Desmoid-Type Fibromatosis (DTF): Detection of Noval Mutations in the Clinicopathologic Analysis of 32 Cases.

Desmoid-type fibromatosis (DTF) is a locally aggressive myofibroblastic/fibroblastic neoplasm with a high risk of local recurrence. It has a variety of histologic features that might confuse diagnosis, especially when detected during core needle biopsy. The Wnt/β-catenin pathway is strongly linked to the pathogenesis of DT fibromatosis. This study examined 33 desmoid-type fibromatoses (DTFs) from 32 patients, analyzing its clinical characteristics, histologic patterns, occurrence rates, relationship with clinical outcomes, immunohistochemical and molecular findings. The DTFs exhibit a range of 1 to 7 histologic patterns per tumor, including conventional, hypercellular, myxoid, hyalinized/hypocellular, staghorn/hemangiopericytomatous blood vessels pattern, nodular fasciitis-like, and keloid-like morphology. No substantial association was found between the existence of different histologic patterns and the clinical outcome. All thirty-three (100%) samples of DTF had a variable percentage of cells that were nuclear positive for β-catenin. An NGS analysis detected novel non-CTNNB1 mutations in two DTFs, including BCL10, MPL, and RBM10 gene mutations. This study reveals a diverse morphology of DTFs that could result in misdiagnosis. Therefore, surgical pathologists must comprehend this thoroughly. Also, the importance of the newly identified non-CTNNB1 gene mutations is still unclear. More research and analyses are needed to completely grasp the clinical implications of these mutations.

Specific Mutation Predict Relapse/Refractory Diffuse Large B-Cell Lymphoma.

The application of rituximab has significantly enhanced the overall survival rates in patients with diffuse large B-cell lymphoma (DLBCL). Regrettably, a significant number of patients still progress to relapse/refractory DLBCL (rrDLBCL). Herein, we employed targeted sequencing of 55 genes to investigate if gene mutations could predict the progression to rrDLBCL. Additionally, we compared the mutation profiles at the time of DLBCL diagnosis with those found in rrDLBCL cases. Our findings highlighted significantly elevated mutation frequencies of TP53, MEF2B and CD58 in diagnostic biopsies from patients who progressed to relapse or refractory disease, with CD58 mutations exclusively observed in the rrDLBCL group. In assessing the predictive power of mutation profiles for treatment responses in primary DLBCL patients, we found that the frequency of CARD11 mutations was substantially higher in non-response group as compared with those who responded to immunochemotherapy. In addition, we revealed mutations in HIST2H2AB, BCL2, NRXN3, FOXO1, HIST1H1C, LYN and TBL1XR1 genes were only detected in initial diagnostic biopsies, mutations in the EBF1 gene were solely detected in the rrDLBCL patients. Collectively, this study elucidates some of the genetic mechanisms contributing to the progression of rrDLBCL and suggests that the presence of CD58 mutations might serve as a powerful predictive marker for relapse/refractory outcomes in primary DLBCL patients.

Genomic analysis reveals molecular characterization of CD30+ and CD30− extranodal natural killer/T-cell lymphomas (ENKTLs)

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is prevalent in the Asian population; however, little is known about its molecular characteristics. In this study, we examined the CD30 expression in ENKTLs and then performed whole exome sequencing on ten CD30+ ENKTL and CD30− ENKTL paired samples. CD30 was positive in 55.74% of the ENKTLs. Single nucleotide and insertion/deletion polymorphism analyses revealed that 53.41% of the somatic mutations in CD30+ ENKTLs were shared with CD30− ENKTLs, including mutations in SERPINA9, MEGF6, MUC6, and KDM5A. Frequently mutated genes were primarily associated with cell proliferation and migration, the tumor microenvironment, energy and metabolism, epigenetic modulators, vascular remodeling, and neurological function. PI3K-AKT, cAMP, cGMP-PKG, and AMPK pathways were enriched in both CD30+ and CD30− ENKTLs. Copy number variation analysis identified a unique set of genes in CD30+ ENKTLs, including T-cell receptor genes (TRBV6-1 and TRBV8), cell cycle-related genes (MYC and CCND3), immune-related genes (GPS2, IFNA14, TTC38, and CTSV), and a large number of ubiquitination-related genes (USP32, TRIM23, TRIM2, DUSP7, and UBE2QL1). BCL10 mutation was identified in 6/10 CD30+ ENKTLs and 7/10 CD30− ENKTLs. Immunohistochemical analysis revealed that the expression pattern of BCL10 in normal lymphoid tissues was similar to that of BCL2; however, its expression in ENKTL cells was significantly higher (67.92% vs. 16.98%), implying the potential application of BCL10 inhibitors for treating ENKTLs. These results provide new insights into the genetic characteristics of CD30+ and CD30− ENKTLs, and could facilitate the clinical development of novel therapies for ENKTL.

Genomic analysis of BAX and Bcl-2 gene mutations in human papilloma virus-associated squamous cell carcinoma of the cervix

PurposeThe aim of the current study was to molecularly determine Bcl-2 and BAX gene mutations in HPV-associated squamous cell carcinoma of the cervix.MethodsFormalin-fixed, paraffin-embedded tissue blocks, all consisting of squamous cell carcinoma of the cervix, were used for this study. The nucleic acid amplification technique and various steps for DNA sequencing, including DNA extraction and polymerase chain reaction, were used.ResultsMutations were detected in the Bcl-2 gene of patients with squamous cell carcinoma of the cervix in the 10–860 bp region, while BAX gene mutations were detected in the 10–320 bp region. The nucleotide mutations in the Bcl-2 gene were A > G (50%), C > T (33.33%), and G > A > T (16.67%), while the BAX gene mutations were A > (16.67%), T > (16.67%), G > (16.67%), A > C (16.67%), T > G (16.67%), and T > C (16.67%). The mutations in the BAX gene were Indel (50%), Transversion (33.4%), and Transition (16.6%), while only the Transition mutation (100%) was detected in the Bcl-2 gene. The functional mutations in the BAX gene were only missense mutations (100%), but in the Bcl-2 gene, the functional mutations were missense (50%) and silent (50%) mutations.ConclusionOur findings revealed genomic mutations of different types and frequencies in the BAX and Bcl-2 genes in squamous cell carcinoma of the cervix, which should encourage further research to better understand these mutations and exploit them for clinical use.

Plasma circulating tumour DNA is a better source for diagnosis and mutational analysis of IVLBCL than tissue DNA.

Diagnosis of intravascular large B-cell lymphoma (IVLBCL) is a challenge due to its heterogeneous clinical presentation and lack of specific markers. This retrospective study investigated the utility of circulating tumour DNA (ctDNA) sequencing for diagnosing IVLBCL and analysing its mutation landscape. A cohort of 34 IVLBCL patients enrolled and underwent plasma ctDNA targeted sequencing. The median plasma ctDNA concentration was 135.0 ng/mL, significantly higher than that in diffuse large B-cell lymphoma (DLBCL) controls. Correlations were found between ctDNA concentration and disease severity indicators, LDH and SF. Mutation analysis revealed frequent mutations in B-cell receptor and NF-κB signalling pathways, including MYD88 (56%), CD79B (44%), TNFAIP3 (38%) and IRF4 (29%). CNS involvement was significantly related with BCL6 and CD58 mutation. Patients with complicated hemophagocytic lymphohistiocytosis had significantly higher mutation rates in B2M. Comparison with DLBCL subtypes showed distinctive mutation profiles in IVLBCL. Moreover, plasma ctDNA detected more mutations with higher variant allele fraction than tissue DNA, suggesting its superiority in sensitivity and accessibility. Dynamic monitoring of ctDNA during treatment correlated with therapeutic responses. This study revealed the role of ctDNA in IVLBCL diagnosis, mutation analysis, and treatment monitoring, offering a promising avenue for improving patient diagnosis in this rare lymphoma subtype.

Transformation of t(14;18)-negative follicular lymphoma to plasmablastic lymphoma: a case report with analysis of genetic evolution

BackgroundFollicular lymphoma (FL) is characterized by t(14;18)(q32;q21) involving the IGH and BCL2 genes. However, 10–15% of FLs lack the BCL2 rearrangement. These BCL2-rearrangement−negative FLs are clinically, pathologically, and genetically heterogeneous. The biological behavior and histological transformation of such FLs are not adequately characterized. Here, we report the first case of t(14;18)-negative FL that rapidly progressed to plasmablastic lymphoma (PBL).Case presentationA previously healthy 51-year-old man presented with leg swelling. Computed tomography (CT) showed enlarged lymph nodes (LNs) throughout the body, including both inguinal areas. Needle biopsy of an inguinal LN suggested low-grade B-cell non-Hodgkin lymphoma. Excisional biopsy of a neck LN showed proliferation of centrocytic and centroblastic cells with follicular and diffuse growth patterns. Immunohistochemical analysis showed that the cells were positive for CD20, BCL6, CD10, and CD23. BCL2 staining was negative in the follicles and weak to moderately positive in the interfollicular areas. BCL2 fluorescence in situ hybridization result was negative. Targeted next-generation sequencing (NGS) revealed mutations in the TNFRSF14, CREBBP, STAT6, BCL6, CD79B, CD79A, and KLHL6 genes, without evidence of BCL2 or BCL6 rearrangement. The pathologic and genetic features were consistent with t(14;18)-negative FL. Two months after one cycle of bendamustine and rituximab chemotherapy, the patient developed left flank pain. Positron emission tomography/CT showed new development of a large hypermetabolic mass in the retroperitoneum. Needle biopsy of the retroperitoneal mass demonstrated diffuse proliferation of large plasmablastic cells, which were negative for the B-cell markers, BCL2, BCL6, and CD10; they were positive for MUM-1, CD138, CD38, and C-MYC. The pathologic findings were consistent with PBL. The clonal relationship between the initial FL and subsequent PBL was analyzed via targeted NGS. The tumors shared the same CREBBP, STAT6, BCL6, and CD79B mutations, strongly suggesting that the PBL had transformed from a FL clone. The PBL also harbored BRAF V600E mutation and IGH::MYC fusion in addition to IGH::IRF4 fusion.ConclusionsWe propose that transformation or divergent clonal evolution of FL into PBL can occur when relevant genetic mutations are present. This study broadens the spectrum of histological transformation of t(14;18)-negative FL and emphasizes its biological and clinical heterogeneity.

The genomic, transcriptomic, and immunological profile of patients with recurrent/refractory NSCLC.

8039 Background: Lung cancer is the leading cause of cancer-related deaths worldwide, and often presents at advanced stages, with &gt; 50% of patients presenting as stage III or IV. Patients with recurrent stage III NSCLC (R/R) previously treated with definitive chemoradiation and immunotherapy consolidation may have a difference in overall survival compared to de novo stage IV (DN). In this study, we aim to compare the molecular and immune landscapes of these two patient populations to identify potential genomic patterns and biomarkers of resistance. Methods: 3728 NSCLC specimens underwent sequencing of DNA (592-gene panel or whole exome) or RNA (whole transcriptome) or immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Patients were classified as R/R (n = 26) if they received Durvalumab and chemoradiation within 12 months (m) before tissue collection and treated with Pembrolizumab within 6 m after tissue collection. Patients treated with Pembrolizumab within 6 months after tissue collection and had no prior (or post) treatment with Durvalumab and chemoradiation at any point in time were classified as DN (n = 3702). Tumor microenvironment (TME) cell fractions were estimated from bulk RNA sequencing using QuanTIseq. Significance was determined using chi-square, Mann Whitney U and adjusted for multiple comparisons where applicable (q &lt; 0.05). Results: TP53 (91 vs 68%) and KEAP1 (30 vs. 14%, both p &lt; 0.05, q &gt; 0.05) mutation prevalence was higher in R/R, whereas a decreased prevalence of KRAS mutations was noted in this group (13 vs. 33%) as compared to the DN group. Compared to DN patients, R/R patients were associated with an increased prevalence of mutations in BCL2 (4.3 vs. 0%, q &lt; 0.05), BCL9 (4.3 vs. 0.2%, q &lt; 0.05), HNF1A (4.3 vs. 0.2%), TNFAIP3 (4.3 vs. 0.2%), AKT1 (4.3 vs. 0.4%), MAP3K1 (4.3 vs. 0.5%), and SPEN (4.3 vs. 0.6%; all p &lt; 0.05, q &gt; 0.05). Further, copy number amplification of NUTM1 (4.5 vs. 0%), SMO (4.3 vs. 0.5%), CHIC2 (4.5 vs. 0.2%; all q &lt; 0.05), PIK3CA (9 vs. 1.2%) and FLCN (4.5 vs. 0.3%; both p &lt; 0.05, q &gt; 0.05) was more common in R/R compared to DN. R/R TME was associated with decreased B cell (2.9 vs 4.4%, q &lt; 0.05) and regulatory T cell fractions (1.3 vs. 2.6%, p &lt; 0.05, q &gt; 0.05). Conclusions: R/R patient tumors had distinct molecular alterations and immune landscapes, including an increased prevalence of biomarkers associated with immunotherapy resistance ( TP53 and KEAP1 mutations) and lower tumor B- and T reg- cell fractions, which may suggest decreased likelihood of response to immunotherapy compared to patients with DN NSCLC. The RR population had a lower-than-expected percentage of KRASmutations. These distinct molecular differences highlight the need for NGS testing in the recurrent setting, despite initial NGS testing at diagnosis. Molecularly targeted interventions may be options in the future for patients with R/R Stage III NSCLC.

Indirubin In Vitro Apoptotic Effect on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) primarily affects the elderly, with its etiology largely unknown. It is hypothesized that hematopoietic stem cells may acquire mutations over time, such as the BCL-2 mutation, leading to disruptions in the apoptotic process. Dangui Luhui Wan, a mixture of 11 herbs used in Chinese Medicine, has shown antitumor activities across various cancer cell types. Indirubin-3'-monoxime (I3M), derived from Dangui Luhui Wan, functions as a selective inhibitor of cyclin-dependent kinases (CDKs) and can induce apoptosis in cells. Objectives: The objective of this study was to evaluate the effectiveness of I3M against CLL cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) from 14 patients were treated with I3M at concentrations ranging from 0.1 μM to 80 μM over periods of 24, 48, and 72 hours. The optimal dose was determined using Annexin V and MTT assays. The expression of apoptotic genes Bcl-2/Bax and CDK1/2 was assessed using real-time PCR. Results: The results indicated that a 20 µM concentration of I3M exhibited the highest cytotoxicity after 48 hours compared to controls (P = 0.005). Post-treatment, a decrease in Bcl-2 gene expression was observed, while changes in the Bax gene were not significant. However, an increase in the Bax/Bcl-2 gene ratio was noted, suggesting involvement of the mitochondrial pathway in I3M's apoptotic mechanism. Notably, I3M inhibited the expression of the CDK2 gene but did not affect CDK1 gene expression. Conclusions: I3M appears to exert anti-tumor effects by inducing apoptosis and inhibiting the CDK2 gene. Further research is required to elucidate the precise mechanism of action of I3M in CLL and potentially other tumor cell lines.

Discovery of the Clinical Candidate Sonrotoclax (BGB-11417), a Highly Potent and Selective Inhibitor for Both WT and G101V Mutant Bcl-2.

The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax's limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.

Incorporating mutational heterogeneity to identify genes that are enriched for synonymous mutations in cancer

BackgroundSynonymous mutations, which change the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations might be enriched in cancer has been explored in several recent studies. However, none of these studies control for all three types of mutational heterogeneity (patient, histology, and gene) that are known to affect the accurate identification of non-synonymous cancer-associated genes. Our goal is to adopt the current standard for non-synonymous mutations in an investigation of synonymous mutations.ResultsHere, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify cancer-associated genes that are enriched for synonymous mutations based on a non-coding background model that takes into account the mutational heterogeneity across these levels. Using MutSigCVsyn, we first analyzed 2572 cancer whole-genome samples from the Pan-cancer Analysis of Whole Genomes (PCAWG) to identify non-synonymous cancer drivers as a quality control. Indicative of the algorithm accuracy we find that 58.6% of these candidate genes were also found in Cancer Census Gene (CGC) list, and 66.2% were found within the PCAWG cancer driver list. We then applied it to identify 30 putative cancer-associated genes that are enriched for synonymous mutations within the same samples. One of the promising gene candidates is the B cell lymphoma 2 (BCL-2) gene. BCL-2 regulates apoptosis by antagonizing the action of proapoptotic BCL-2 family member proteins. The synonymous mutations in BCL2 are enriched in its anti-apoptotic domain and likely play a role in cancer cell proliferation.ConclusionOur study introduces MutSigCVsyn, an algorithm that accounts for mutational heterogeneity at patient, histology, and gene levels, to identify cancer-associated genes that are enriched for synonymous mutations using whole genome sequencing data. We identified 30 putative candidate genes that will benefit from future experimental studies on the role of synonymous mutations in cancer biology.

Overcoming Resistance in Chronic Lymphocytic Leukemia-Maybe Less Is More?

Acquired mutations in BTK, PLCG2, and BCL2 are associated with resistance to continuous targeted agent therapy in chronic lymphocytic leukemia (CLL). Here, we discuss new evidence that limiting the duration of CLL therapy may prevent the evolution of such resistance mutations, potentially facilitating effective retreatment strategies. See related article by Jain et al., p. 498.

Pre-Clinical Study on the Dual BCL2/BCL-XL Inhibitor AZD0466 for the Treatment of Chronic Lymphocytic Leukemia

Targeting BCL2 with venetoclax has proven successful for treatment of chronic lymphocytic leukemia (CLL), but resistance develops in a subset of patients. Among other mechanisms, mutations in BCL2 and increased BCL-XL expression confer venetoclax refractoriness in patient subgroups. It is therefore important to identify alternative treatment options targeting BCL-XL and BCL2 for combating venetoclax resistance. AZD4320 was developed as a dual BCL-2/BCL-XL inhibitor. AZD0466 is a dendrimer-drug conjugate of AZD4320, designed to optimize drug release and improve drug tolerability. Hence, we used the nano drug AZD0466 for in vivo experiments and AZD4320 for in vitro and ex vivo experiments. Firstly, we studied the efficacy of AZD0466 in vivo in the Eµ-TCL1 murine CLL adoptive transfer model. Due to prevalence of primary resistance, murine tumors with the best ex vivo response to AZD4320 were selected and 10 million cells were transferred by i.v. in syngeneic recipient mice. Upon reaching a tumor load of 10% CD19+ CD5+ cells in peripheral blood, mice were randomized and treated with 30mg/kg ibrutinib in drinking water, 34mg/kg or 70mg/kg of AZD0466 once weekly (QW) by i.v, or the vehicle. Treatments were continued until reaching a predefined humane endpoint. AZD0466 at 70mg/kg led to a significant increase in survival (median 72 days vs. 38 days, P=0.0009) similar to ibrutinib (67 days, P=0.0026) when compared to vehicle (Fig. 1). Analysis of blood parameters at euthanasia did not reveal significant difference in platelet counts between AZD0466, ibrutinib and vehicle treatments. To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. AZD0466 and vehicle treatments showed similar platelet and RBC counts measured 2 or 3 days after the 2 nd and 4 th dosing but, a significant increase in platelet and RBC counts were observed with the combination treatments. A significant decrease in WBC count after 2 weeks, and in bone marrow tumor load after 4 weeks was observed with combined AZD0466 and acalabrutinib treatment compared to the single drugs. Though spleen weights did not differ after 4 weeks between vehicle and AZD0466 monotherapy, a strong decrease in spleen weight and tumor cell number in spleen was observed with the combination of AZD0466 with ibrutinib or acalabrutinib compared to vehicle. To validate the findings in human CLL, we performed dynamic BH3 profiling on primary CLL samples with mutated (N=5) and unmutated IGHV (N=7). Exposure of CLL cells to 1µM acalabrutinib for 24 hours significantly increased BCL2 dependency in all CLL samples with unmutated IGHV and in a subset of mutated IGHV samples. These tumors also showed an increased response to venetoclax and AZD4320 upon exposure to acalabrutinib. To study the efficacy of the dual BCL2/BCL-XL inhibitor in the context of venetoclax resistance, we stably expressed the BCL2 mutations A113G, D103E, D103V, D103Y, G101V, R129L, V156D and wildtype (WT) in the venetoclax sensitive mantle cell lymphoma cell line MAVER-1. Response to AZD4320 or venetoclax was analyzed using Annexin V/7AAD staining and flow cytometry after 48 hours of treatment. The BCL2 mutants showed varying degrees of resistance to venetoclax. MAVER-1 expressing WT had an IC50 of 17.7nM while D103E, D103V, D103Y, G101V showed the strongest resistance to venetoclax with 5, 13.8, 4.5 and 7.3 fold increase in IC50 compared to WT. Of note, compared to WT (IC50 43.9nM), D103E, D103V and D103Y were sensitive to AZD4320 with a fold change in IC50 of 0.6, 1.8 and 1.3, respectively. G101V mutant cells showed a 5 fold increase in IC50 to AZD4320 compared to WT (Fig. 2). Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method. In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.

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

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

Distinct Circulating Genomic Features of Classical Hodgkin Lymphoma of Older Adults

Introduction: Despite significant progress in curing younger patients with classical Hodgkin lymphoma (cHL), older patients continue to have substantially inferior outcomes. The bimodal distribution of age at cHL diagnosis has suggested distinct underlying pathogenesis in older adults. For example, among older patients where EBV+ disease is more prevalent, EBV+ status is associated with worse outcomes, unlike younger patients where it confers more favorable prognosis (Keegan, JCO 2005). The reasons for this disparity remain enigmatic. To address these challenges, we used circulating tumor DNA (ctDNA) profiling, enabling noninvasive genotyping, risk stratification, and classification into distinct molecular subtypes (Spina, Blood 2018; Alig, ASH 2022). We leveraged several non-invasive techniques to systematically characterize the genomic peculiarities of elderly cHL patients distinguishing them from younger counterparts, with special attention to EBV status. Methods: We comprehensively profiled newly diagnosed cHL patients from 3 prospective clinical trials: LYSA-PVAB (NCT02414568) for subjects ≥60 years (Ghesquières, ASH 2019), AHL2011 (NCT01358747) for patients aged 16-59 years with advanced cHL, and BIO-LYMPH (NCT04417803) for patients ≥18 years. We used age ≥60 as a threshold for stratification of older versus younger patients, a common cutoff in clinical trials. We identified somatic single nucleotide variants using CAPP-Seq (Newman et al, Nature Biotech 2016); and analyzed copy number alterations with CANARy (Chabon, Nature 2020). Patients were categorized into high vs low pretreatment ctDNA levels using a predefined threshold (2.5 log hGE/mL; Kurtz, JCO 2018). We determined EBV status using EBER and/or LMP1 staining of FFPE tumor tissue or plasma EBV levels (&amp;gt;32 copies/mL) by VirCAPP-Seq (Garofalo, ASH 2022). Results: A total of 215 newly diagnosed cHL patients were evaluable, with a median age of 51 years, and 35% being older (≥60 years). In this cohort, 82% had advanced stage disease, 68% had high IPS (≥3), and 57% had extra nodal involvement. At 4-year follow-up, OS and PFS were 84% and 74%, respectively. At presentation, older patients (≥60y) had more advanced stage (93% vs 73%, p&amp;lt;0.001), higher IPS (78% vs 61%, p&amp;lt;0.001), and greater EBV+ prevalence (63% vs 36%, p&amp;lt;0.001). Older patients also experienced significantly more relapses (39% vs 17%, p&amp;lt;0.001). and mortality (29% vs 6%, p&amp;lt;0.001). The median pre-treatment ctDNA level was 982 hGE/mL, but not significantly different in older vs younger patients (p=0.28). Higher ctDNA levels were positively associated with risk-associated features including B symptoms (p=0.01), IPS (p&amp;lt;0.001), bulky disease (p=0.04), extra nodal disease (p=0.02) and correlated with TMTV (R=0.47, p&amp;lt;.001). Older patients had significantly fewer somatic mutations than younger counterparts (Median 107 vs 192 SNVs within targeted genomic regions, p&amp;lt;0.001; Fig. 1A top); among older patients, EBV+ cases had even fewer mutations than EBV- counterparts (Median 83 vs 142 SNVs, p=0.05). Older cHL cases harbored significantly more BCL2 mutations and recurrent copy number anomalies, but had significantly fewer mutations in STAT6, ITPKB, B2M, GNA13, PAX5 and XPO1 among others(all p&amp;lt;0.05 Fig. 1A bottom). The H2 genomic subtype prevalence, characterized by genomic instability as well as mutations in TP53, KMT2D, and BCL2 (Alig et al, ICML 2023), was higher among older patients (56%) than younger patients (21%) (p=0.01). Older H2 cHL showed no significant difference in EBV+ status compared to H1 counterparts (p=0.9). In older patients, multivariate Cox regression (including EBV status, ctDNA, and IPS) identified EBV+ status (HR=2.2, p=0.02) as an independent predictor of worse PFS and DFS outcomes (HR=2.5, p=0.02 and HR=2.7, p=0.04). When older cHL patients were stratified by baseline ctDNA level and EBV status, high-risk cases (EBV+ and ctDNA High) had particularly poor outcomes in terms of both PFS and DFS ( Fig. 1B). Conclusion: Older cHL patients exhibit unique genomic characteristics compared to younger counterparts, including more prevalent H2 tumor genetic subtype and EBV positivity. Identifying of a high-risk subgroup of older cHL underscores the importance of ctDNA levels and EBV status. We envision future trials of personalized biomarker driven treatment strategies could therapeutically target this risk, including by PD1 blockade.

Truncating SPEN Mutations Highlight BN2-Subtype DLBCL with Aggressive Biology and Features of Immune Evasion

Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations. Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 &amp; SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry. Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p&amp;lt;0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p&amp;lt;0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p&amp;lt;0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p&amp;lt;0.01, respectively). Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.

Development of Syngeneic Murine Cell Lines of Germinal Center-Derived B-Cell Lymphomas

Background. Germinal center (GC)-derived B-cell lymphomas are clinically and biologically heterogeneous. Next generation sequencing has enabled identification of genetic aberrations involved in lymphoma pathogenesis, which may provide prognostic predictions and potential therapeutic targets. However, the mechanisms driving pathogenesis are still not fully understood, especially in the context of an intact tumor microenvironment, in part because there are no available syngeneic models of lymphoma faithfully representing the pathological features of human disease. Therefore, there is a critical need for lymphoma models which can be studied in immunocompetent mice. Methods. We established eight murine cell lines derived from spontaneous B-cell lymphomas with GC origin arising in genetically engineered mouse models (GEMM). These cell lines were developed by passing an original tumor from a GEMM through several passages of Rag1-/- and wild type mice, which enabled selection of more fit/aggressive clones that grow in vitro. We also tested multiple culture conditions and characterized the lines via flow cytometry, VDJ and targeted sequencing, karyotyping, and evaluation of tumor development and its microenvironment in vivo. Results. This resource describes eight cell lines driven by recurrent genetic mutations found in diffuse large B-cell lymphoma and follicular lymphoma patients. The first cell line is defined by (1) BCL2 overexpression (hereafter B2). Four cell lines couple BCL2 overexpression with epigenetic dysregulation, defined by (2) BCL2 and gain-of-function (GOF) mutation of EZH2 Y641F (hereafter EZB), (3) EZB and MYC overexpression (hereafter EBM), (4) BCL2 with knockout of KMT2D (hereafter KB), and (5) BCL2 with knockout of SETD2 (hereafter S2B). Finally, three cell lines are defined by mutations to the Toll-like and B-cell receptors pathways, defined by (6) BCL2 and GOF mutations in Myd88 L252P and CD79b Y195H (hereafter MBC79), (7) BCL2, Myd88 L252P and GOF mutation in Card11 L251P (hereafter MBC11), and (8) BCL2 and Card11 L251P (hereafter BC11). Doubling time of these lines ranges from 30-72 hours, with EZB and EBM on the fast end and S2B on the slow end. By flow cytometry, we show that B2, MBC79, and MBC11 lose B-cell surface markers and are very plasmacytic (&amp;gt;95% B220- CD19- CD138+). In contrast, the EZB, EBM, KB, BC11, and S2B are mostly characterized by GC B-cell markers (B220+ CD19+ CD138- FAS+ CD38- GL7+). By VDJ sequencing, we found that EZB, BC11, and S2B are very monoclonal, with one rearrangement arising from the original tumor and taking over to represent &amp;gt;95% of clones. In contrast, the MCB11 and MBC79 lines are very polyclonal, with the top 10 rearrangements being equally represented, and tending to shift between passages. Thus far, S2B and EZB have been shown to grow in immunocompetent mice, representing novel syngeneic lymphoma models. While mice injected with S2B can survive for up to 30 days, mice injected with EZB die significantly earlier. We established two EZB lines from early (P1) and late (P6) passages of a tumor arising from the same original mouse. Notably, we show that mice injected with P1 survive 15-20 days, whereas mice injected with P6 survive 10-15 days. Although both P1 and P6 grow at similar rates in vitro, P1 develops high grade follicular lymphomas in C57BL6 recipients, whereas P6 develops DLBCL, suggesting additional passaging drives selection for more aggressive disease. Imaging of the tumors reveals a lack of follicular pattern. Notably, P6 tumors display less infiltration of CD4 and CD8 T-cells and low abundance or absence of FDC meshwork, as compared to P1. Imaging of mice engrafted with luciferase transduced P1 and P6 shows dissemination into spleen, lymph nodes, liver, and kidney, with more advanced disease in P6 injected mice. Conclusion. While conventional preclinical tumor mouse models are often used for drug sensitivity and treatment prediction studies, they are time-consuming and expensive. These cell lines serve as a preclinical tool to test novel therapeutic strategies and develop a deeper understanding of the molecular mechanisms driving lymphomagenesis, therapeutic response, and resistance in genetically defined subtypes of GC-derived B-cell lymphomas. Most importantly, this resource meets a critical need in the field for syngeneic models of lymphoma to study disease progression and precision therapy in immunocompetent mice.

Enriched Signalling Pathways in Venetoclax-Relapsed Chronic Lymphocytic Leukemia (CLL) Cells and Targeting Using a Protac-Based Bcl-2/Bcl-Xl Degrader

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. Unfortunately, some patients develop treatment resistance after receiving these agents in combination or sequentially. We aimed to evaluate whether CLL cells collected upon venetoclax and ibrutinib relapse retain survival dependency on alternative targets of the BCR pathway and Bcl-2 family, and to propose new vulnerabilities based on gene expression changes. Using single cell RNA sequencing (sc-RNA-seq), we analyzed paired samples from a CLL patient collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. The proportion of cells in each CLL cluster was vastly altered at relapse. Cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse and had a high BCR score, including high expression of MAP3K8 FOSB8 and NFKB1 genes. Moreover, at relapse, it contained more CLL cells expressing transcripts from the Bcl-2 family of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. We also integrated sc-RNA-seq data from three primary CLL samples collected at relapse after venetoclax and ibrutinib treatment. The four clusters of CLL cells identified were enriched for BCR, interleukin-mediated, ErbB and the Fc epsilon receptor I signalling pathways. Importantly, most of the relapsed CLL cells which expressed high levels of Mcl-1 or Bcl-xL also expressed Bcl-2, suggesting that these resistant CLL cells became dependent on various anti-apoptotic proteins for survival. These observations led us to test Bcl-2/Bcl-xL targeting PROTACs PZ18753b and WH25244 in venetoclax-resistant CLL. These PROTACs were derived from navitoclax (Bcl-2/Bcl-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. We used three models of venetoclax-resistant CLL: (1) OSU-CLL cells expressing mutant Bcl-2 (G101V, F104L, R107-110dup or A113G), (2) treatment-naïve primary CLL cells co-cultured with NK.Tert stromal cells for 24h (CLL+stroma), and (3) primary CLL cells collected at venetoclax relapse. CLL cells were treated with venetoclax, navitoclax, PZ18753b, WH25244, or negative controls (NC) lacking VHL recruitment, PZ18753b-NC or WH25244-NC. Protein degradation was evaluated by Western Blot; viability, by Cell Titer Glo; and apoptosis, by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. WH25244 was superior to venetoclax at targeting OSU-CLL cells expressing either wildtype or mutant Bcl-2, upon 72h treatment. Cell death by WH25244 was preceded by degradation of Bcl-xL and partial degradation of Bcl-2, despite presence of Bcl-2 mutations. In treatment-naïve primary CLL cells, Bcl-xL degradation by PZ18753b and WH25244 proceeded in a dose-, time- and VHL-dependent manner. The potency of these PROTACs fell in between that of venetoclax and navitoclax, with mitochondrial apoptosis occurring at low nanomolar concentrations known to spare human platelets. Stepwise, activation of BAK and BAX, and cytochrome C release were evident at 14h of treatment, followed by phosphatidyl serine exposure at 18h of treatment. A rightward shift in the dose-response curves of PZ18753b (16-fold) and WH25244 (8-fold) was noted for CLL+stroma cells relative to paired CLL cells treated in suspension. This reduction in potency was associated with (1) uptake of the PROTACs by NK.Tert cells, since Bcl-xL degradation was noted in stromal cells yet without evident toxicity, and (2) up to 2.5-fold increased levels of Bcl-xL in CLL+stroma, which shifted the dose-dependent degradation of Bcl-xL. Finally, 2 out of 3 venetoclax-relapsed primary CLL samples showed more active BAK and cytochrome C release upon treatment with WH25244 than venetoclax, evaluated at 10 nM. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies.

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques - BH3 profiling and high-throughput kinase activity mapping - we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Absence of BTK, BCL2, and PLCG2 Mutations in Chronic Lymphocytic Leukemia Relapsing after First-Line Treatment with Fixed-Duration Ibrutinib plus Venetoclax.

Mutations in BTK, PLCG2, and BCL2 have been reported in patients with progressive disease (PD) on continuous single-agent BTK or BCL2 inhibitor treatment. We tested for these mutations in samples from patients with PD after completion of first-line treatment with fixed-duration ibrutinib plus venetoclax for chronic lymphocytic leukemia (CLL) in the phase II CAPTIVATE study. A total of 191 patients completed fixed-duration ibrutinib plus venetoclax (three cycles of ibrutinib then 12-13 cycles of ibrutinib plus venetoclax). Genomic risk features [del(11q), del(13q), del(17p), trisomy 12, complex karyotype, unmutated IGHV, TP53 mutated] and mutations in genes recurrently mutated in CLL (ATM, BIRC3, BRAF, CHD2, EZH2, FBXW7, MYD88, NOTCH1, POT1, RPS15, SF3B1, XPO1) were assessed at baseline in patients with and without PD at data cutoff; gene variants and resistance-associated mutations in BTK, PLCG2, or BCL2 were evaluated at PD. Of 191 patients completing fixed-duration ibrutinib plus venetoclax, with median follow-up of 38.9 months, 29 (15%) developed PD. No baseline risk feature or gene mutation was significantly associated with development of PD. No previously reported resistance-associated mutations in BTK, PLCG2, or BCL2 were detected at PD in 25 patients with available samples. Of the 29 patients with PD, 19 have required retreatment (single-agent ibrutinib, n = 16, or ibrutinib plus venetoclax, n = 3); 17 achieved partial response or better, 1 achieved stable disease, and 1 is pending response assessment. First-line fixed-duration combination treatment with ibrutinib plus venetoclax may mitigate development of resistance mechanisms associated with continuous single-agent targeted therapies, allowing for effective retreatment. See related commentary by Al-Sawaf and Davids, p. 471.