Concurrent BCL2 Research Articles

Double-hit primary central nervous system lymphoma with histogenetically proven bone marrow infiltration: a case report and a review of the literature.

Double-hit lymphoma (DHL) formerly referred to high-grade B-cell lymphoma with concurrent MYC and BCL2 or BCL6 rearrangements, however, the updated 2022 World Health Organization Classification (5th edition online) excludes those with MYC and BCL 6 rearrangements from the high-grade category. DHL confined to the central nervous system (CNS), known as double-hit primary CNS lymphoma (DH-PCNSL), is rare with poorly understood clinical features. Here, we report a case of a 64-year-old man with multiple brain tumors diagnosed with DH-PCNSL who showed bone marrow (BM) infiltration early in the clinical course. The histological diagnosis was high-grade B-cell lymphoma with MYC and BCL6 rearrangements. Fluorodeoxyglucose positron emission tomography (FDG-PET) revealed no abnormal accumulation except in the CNS. The patient received whole-brain radiotherapy following the failure of high-dose methotrexate. After completion of radiotherapy, the patient developed thrombocytopenia, and BM biopsy showed infiltration of DHL cells, which were not detected by repeated FDG-PET. This is the first report of DH-PCNSL where identical gene rearrangements were confirmed in both the resected CNS tumor and BM tissue. Patients with DH-PCNSL require careful follow-up because they may be at a potential risk of BM infiltration, which may be undetectable by FDG-PET, particularly early in the disease course.

Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Molecular characterization of a rare case of high-grade B-cell lymphoma with MYC, BCL2, BCL6, and CCND1 rearrangements

Quadruple-hit lymphomas are extremely rare non-Hodgkin lymphomas with a reported dismal prognosis in the few reported cases. A “quadruple hit” has been defined by the presence of concurrent MYC, BCL2, BCL6, and CCND1 chromosomal rearrangements. We report a new case of a quadruple hit lymphoma in a 73-year-old Hispanic man who presented with an enlarging left-sided neck mass. Computed tomography showed a 1.9-cm mass in left the tonsil with bulky cervical lymphadenopathy. The presence of all four chromosomal rearrangements can reportedly occur with disease progression in both diffuse large B-cell lymphomas and mantle cell lymphomas. Further characterization of the tumor by next-generation sequencing may be of benefit to delineate between these two possibilities. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and next-generation sequencing were used to confirm and classify the diagnosis. Histologic sections of the cervical lymph node demonstrated an atypical lymphoid infiltrate with large and pleomorphic cells, which were positive for CD20, CD10, BCL1 (Cyclin D1), BCL2, BCL6, and cMYC and negative for CD5 and SOX11 on immunohistochemistry with a Ki-67 proliferative index of 70%. FISH demonstrated MYC, BCL2, BCL6, and CCND1 rearrangements and the diagnosis of high-grade B-cell lymphoma with MYC, BCL2, BCL6, and CCND1 was rendered. Our patient was treated with dose adjusted etoposide, doxorubicin, cyclophosphamide, prednisone, and rituximab chemotherapy and has been in remission for 20 months.

Performance of MYC, BCL2, and BCL6 break-apart FISH in small biopsies with large B-cell lymphoma: a retrospective Cytopathology Hematopathology Interinstitutional Consortium study.

Fluorescence in situ hybridization (FISH) is an essential ancillary study used to identify clinically aggressive subsets of large B-cell lymphomas that have MYC, BCL2, or BCL6 rearrangements. Small-volume biopsies such as fine needle aspiration biopsy (FNAB) and core needle biopsy (CNB) are increasingly used to diagnose lymphoma and obtain material for ancillary studies such as FISH. However, the performance of FISH in small biopsies has not been thoroughly evaluated or compared to surgical biopsies. We describe the results of MYC, BCL2, and BCL6 FISH in a series of 222 biopsy specimens, including FNAB with cell blocks, CNBs, and surgical excisional or incisional biopsies from 208 unique patients aggregated from 6 academic medical centers. A subset of patients had FNAB followed by a surgical biopsy (either CNB or excisional biopsy) obtained from the same or contiguous anatomic site as part of the same clinical workup; FISH results were compared for these paired specimens. FISH had a low hybridization failure rate of around 1% across all specimen types. FISH identified concurrent MYC and BCL2 rearrangements in 20 of 197 (10%) specimens and concurrent MYC and BCL6 rearrangements in 3 of 182 (1.6%) specimens. The paired FNAB and surgical biopsy specimens did not show any discrepancies for MYC or BCL2 FISH; of the 17 patients with 34 paired cytology and surgical specimens, only 2 of the 49 FISH probes compared (4% of all comparisons) showed any discrepancy and both were at the BCL6 locus. One discrepancy was due to necrosis of the CNB specimen causing a false negative BCL6 FISH result when compared to the FNAB cell block that demonstrated a BCL6 rearrangement. FISH showed a similar hybridization failure rate in all biopsy types. Ultimately, MYC, BCL2, or BCL6 FISH showed 96% concordance when compared across paired cytology and surgical specimens, suggesting FNAB with cell block is equivalent to other biopsy alternatives for evaluation of DLBCL or HGBCL FISH testing.

High-grade B-cell lymphoma with a quadruple-hit genetic profile including concurrent MYC, BCL2, BCL6, and CCND1 gene rearrangements.

Several reports of concurrent MYC, BCL2, BCL6, and CCND1 rearrangements in high-grade B-cell lymphoma (HGBL) have been recently described. Herein, we aimed to delineate the scope of this entity through a review of HGBL with a "quadruple-hit" genetic profile identified at our institution. We performed a retrospective review (2015-2023) at our institution of B-cell lymphoma (BCL) cases that were evaluated with concurrent MYC, BCL2, and BCL6 break-apart and IGH::MYC and IGH::CCND1 dual-color dual-fusion fluorescence in situ hybridization studies. Of 203 cases meeting inclusion criteria, 2 (1%) with a quadruple-hit genetic profile were identified. Case 1 represented a 59-year-old female with widespread lymphadenopathy and a diagnosis of HGBL who exhibited primary refractoriness to dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R) chemotherapy. Case 2 represented a 58-year-old male with mediastinal and abdominal lymphadenopathy and a diagnosis of large BCL who died from disease after 1 cycle of DA-EPOCH-R chemotherapy. Similarly, a literature review of 7 previously reported cases of HGBL with a quadruple-hit profile also demonstrated aggressive disease behavior. Our study adds 2 new cases to the rarely encountered quadruple-hit HGBL, and a brief meta-analysis of the 9 available cases indicates aggressive disease behavior conferred by this constellation of genetic events.

A MYC-rearrangement is a negative prognostic factor in stage II, but not in stage I diffuse large B-cell lymphoma

MYC oncogene rearrangements (MYC-R) negatively affect survival in patients with Ann Arbor stage III–IV diffuse large B-cell lymphoma (DLBCL), but their impact in limited stage (LS) I–II is unclear. Therefore, we assessed the impact of MYC-R on progression-free survival (PFS) and overall survival (OS) in LS DLBCL patients at the population level. We identified 1,434 LS DLBCL patients with known MYC-R status diagnosed between 2014 and 2020, who received R-CHOP(-like) regimens using the Netherlands Cancer Registry, with survival follow-up until February 2022. Stage I patients with (n = 83, 11%) and without (n = 650, 89%) a MYC-R had similar 2-years PFS (89% and 93%, p = 0.63) and OS (both 95%, p = 0.22). Conversely, stage II DLBCL patients with a MYC-R (n = 90, 13%) had inferior survival outcomes compared to stage II patients without a MYC-R (n = 611, 87%) (PFS 70% vs. 89%, p = 0.001; OS 79% vs. 94%, p < 0.0001). Both single MYC-R (single hit, n = 36) and concurrent BCL2 and/or BCL6 rearrangements (double/triple hit, n = 39) were associated with increased mortality and relapse risk. In conclusion, in stage II DLBCL a MYC-R is negatively associated with survival. In stage I DLBCL, however, survival outcomes are excellent irrespective of MYC-R status. This challenges the diagnostic assessment of MYC-R in stage I DLBCL patients.

Genetic Alterations of Follicular Lymphoma Can Predict Response to Very Low Dose Radiotherapy

Introduction Follicular lymphoma (FL) is typically indolent, but relapses and transformation to higher grade disease are common. FL is often radiosensitive and can show complete response (CR) even to very low dose radiotherapy (VLDRT, 4Gy). There are no known genetic markers of FL radiosensitivity. We sought to identify molecular signatures of FL radiosensitivity to aid in patient selection for VLDRT. Methods We analyzed our institutional database and identified 110 FL patients with 113 tumors treated with radiotherapy (RT) and preexisting MSK-IMPACT, a targeted exon sequencing panel. For each patient, we collected key clinicodemographic information including pre- and post-RT treatment history, prior large cell lymphoma, and Ann Arbor stage at RT. For treated tumors, we obtained tumor site, grade, diameter, maximum SUV, BCL2 translocation (trans) status through fluorescence in situ hybridization, RT dose and fractionation, and the treatment strategy ( i.e. treating all sites of disease vs a subsite of disease). Tumor site was dichotomized by pelvic tumors vs all others, as this was the most frequently altered site. The presence of mutant (mut) genes compared to wild type (WT) were associated with RT response, measured by PET scan within 6 months of RT, using logistic regression. Log-rank testing and Cox proportional hazards models were used to analyze local progression free survival (LPFS), censored at start of unplanned therapy, with 2-year (2y) survival reported. Multivariate modeling was used to adjust gene associations with outcomes, controlling for the clinicodemographic and tumor characteristics mentioned above. Results The patient and tumor characteristics are summarized in table 1. We found CREBBP to be the most frequently altered gene (66% of tumors). We identified 5 signatures of altered genes, most of which showed altered CREBBP and relatively long LPFS (e.g. one signature showed CREBBP mut, TNFRSF14 mut and IRF8/STAT6 mut with a 2y LPFS 78%); however, one signature with concurrent KMT2D mut and altered BCL2 including trans and mut had significantly shorter LPFS than all other signatures (2y LPFS 47%, p &amp;lt; 0.01) and lower odds of CR relative to the other signatures (p= 0.03). CREBBP mut was the only alteration associated with increased odds of CR (Odds ratio: 2.39 (95% CI: 1.06-5.37, p = 0.04), and this effect remained significant after adjusting for pelvic disease site (p=0.04). Mutations of BCL2, IRF8, and KMT2D were associated with LPFS using log-rank testing (p&amp;lt;0.01, p=0.02, and p=0.04, respectively). BCL2 mut or concurrent BCL2 mut and BCL2 trans (mut/trans) had shorter LPFS for the overall cohort (p&amp;lt;0.01 for both), while for VLDRT, only BCL2 mut/trans was associated with shorter LPFS (p=0.02). In the VLDRT cohort, we found that CREBBP histone acetyltransferase (HAT) mut was associated with improved LPFS compared to WT (n=39, 2y LPFS 74% vs 52%, HR:0.41 (95% CI: 0.18-0.93, p=0.03)), but this was not observed for tumors receiving &amp;gt;4Gy (n=44, HR: 1.27 (95% CI: 0.32-5.08), p=0.74)). We observed that either BCL2 mut, BCL2 trans, or BCL2 mut/trans and CREBBP HAT WT had a 2y LPFS of 13% whereas BCL2 mut, BCL2 trans or BCL2 mut/trans and CREBBP HAT mut had a 2y LPFS of 83% (p&amp;lt;0.01), and this relationship persisted on multivariate analysis (Figure 1). Conclusions Incorporating genetic signatures associated with radiosensitivity, and specifically alterations involving BCL2 and CREBBP, may independently improve patient selection for RT. CREBBP HAT domain mutations are potentially targetable and may have important implications for augmenting radiosensitivity to VLDRT.

The significance of concurrent MYC and BCL2 expression in Egyptian patients with diffuse large B-cell NHL

This study aimed to investigate the frequency of MYC/BCL2 co-expression using immunohistochemistry (IHC) in Egyptian patients with diffuse large B-cell lymphoma (DLBCL) and its impact on treatment outcomes, overall survival (OS), and disease-free survival (DFS). A total of 100 adult patients with newly diagnosed DLBCL were included in the study and treated with the R-CHOP regimen. The cases were classified into germinal center B-cell (GCB) and non-germinal center B-cell (non-GCB) subtypes based on IHC. Our results showed that a subset of the studied cases didn’t achieve complete remission (CR) after chemotherapy (22%). Co-expression of MYC and BCL2 was observed in 20% of the cases, with most of them belonging to the non-GCB subtype. The CR rate was significantly lower in the double-expressor (DE) group (45%), while no significant difference was observed in CR between the GCB and non-GCB cases. The DE group was associated with advanced stage, extra-nodal involvement, and high-risk R-International Prognostic Index (R-IPI). Both the non-DE and GCB groups demonstrated better DFS, while the non-DE group only showed better OS. Both non-DE status and GCB subtype were identified as independent factors affecting DFS. In conclusion, the concurrent expression of MYC and BCL2 in DLBCL indicates an inferior outcome and lower chemotherapy response. The current chemotherapeutic strategy has not effectively improved the outcomes of this aggressive lymphoma subtype. Therefore, the identification of these patients is crucial for selecting appropriate treatment options.

Inverse correlation of concurrent BCL2, and C MYC expressions and CD30 ‎positivity as regard the Prognostic significance in diffuse large B-cell ‎lymphoma

Inverse correlation of concurrent BCL2, and C MYC expressions and CD30 ‎positivity as regard the Prognostic significance in diffuse large B-cell ‎lymphoma

Follicular lymphoma and diffuse large B-cell lymphoma with BCL2 and IRF4 rearrangements in adult patients

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) with concurrent BCL2 and IRF4 rearrangements are rare. It is unclear whether such cases should be classified as large B- cell lymphoma with IRF4 rearrangement or FL/DLBCL-not otherwise specified. We identified 5 adult patients (FL, N=3 and FL/DLBCL, N=2) with concurrent BCL2 and IRF4 rearrangements. The median age at presentation was 77 years, and three patients presented with advanced stage disease. Both nodal and extranodal sites were involved and involvement was not limited to head and neck region. With a median follow-up of 18 months, 1 patient died and 4 patients were alive, including 3 who received chemotherapy and 1 who was observed. The neoplasms were histologically heterogeneous, including grade 2 and 3 FL and DLBCL. Four cases coexpressed CD10, BCL6, BCL2 and MUM1/IRF4. The Ki67 labelling index ranged from 20% to 95%. In 4 patients, the percentage of cells with BCL2 rearrangement was equal to or slightly greater than the cells harboring IRF4 rearrangement. Two cases underwent next generation sequencing tailored for lymphoid neoplasms. Both lacked mutations involving IRF4 and NF-kB pathway genes that are frequently detected in large B-cell lymphoma with IRF4 rearrangement, and one case showed DLBCL-EZH2 type mutations, including KMT2D and BCL2 mutations, similar to 2 previously reported DLBCL with BCL2 and IRF4 rearrangements. Adults with FL and FL/DLBCL with BCL2 and IRF4 rearrangements display clinicopathologic and mutational features more akin to FL and DLBCL and should not be characterized as large B-cell lymphoma with IRF4 rearrangement.

Cytotoxicity of the CD3×CD20 bispecific antibody epcoritamab in CLL is increased by concurrent BTK or BCL-2 targeting

AbstractChronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti–CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.

Impact of Double Expression of MYC and BCL-2 on Outcomes in Primary CNS Lymphoma: A UK Multicentre Analysis

Introduction Primary central nervous system lymphoma (PCNSL) is a rare B cell lymphoma with an aggressive course. Relapses remain common even with intensive immuno-chemotherapy treatment options such as MATRix and consolidation with autologous stem cell transplant (ASCT), and prognostic tools are limited. Although, double expressor status (co-expression of both MYC and BCL-2) is well established in systemic de novo DLBCL, its prognostic impact in PCNSL has been understudied. Here we evaluated the prognostic value of BCL-2, BCL-6, and MYC expression in a retrospective cohort of treated PCNSL patients diagnosed at 7 academic centres in the UK. Methods We performed this retrospective analysis on histologically-confirmed PCNSL patients diagnosed consecutively between 1st May 2015 and 31st May 2020. Treatment with a high-dose methotrexate-based chemotherapy regimen was a mandatory inclusion criterion. BCL-2 and MYC immunohistochemistry (IHC) protein expression data were collated from local diagnostic histology reports and double expressor (DE) status was defined as per WHO criteria (MYC: surface expression > 40% of cells and BCL-2: >50% of cells). Outcomes included response rates, progression-free survival (PFS) and overall survival (OS). Where available, diagnostic fluorescence in situ hybridisation (FISH) data was collected for BCL-2, BCL-6 and MYC loci translocations. Cox-regression for PFS and OS were used to determine baseline predictors of response and survival and to construct multivariable models for factors associated with outcome. Results In total, 242 patients were analysed. Key baseline patient characteristics and IHC data are summarised in Table 1. Median age was 65 years (IQR 56-71), 60% were male and 64% of patients had an ECOG performance status (PS) of 0-1. One hundred and seventy-four (72%) patients received treatment with MATRix chemotherapy; these patients were younger (median age 62 vs. 73 years, p< 0.01), had better baseline PS (PS ≤ 1: 75% vs. 37%, p< 0.01) and higher rates of consolidation with BCNU-thiotepa ASCT (58% vs. 7%, p< 0.01). Double expressor (DE) status was assessable in 169/242 (69%) patients. Reasons for absent DE status were missing IHC expression data (BCL-2: n=28; MYC: n=44) or IHC expression data not reported in line with WHO recommended criteria (BCL-2: n=45; MYC: n=7). In the DE-assessable cohort (n = 169 patients), 40% of patients had DE PCNSL, higher than estimates of DE prevalence in systemic DLBCL (reported rates between 20 and 30%). As only 40 cases (17%) underwent FISH assessment for concurrent MYC and BCL-2 loci translocation (double hit status), correlation between double hit and DE status was not possible. DE status was not associated with patient age at diagnosis, baseline PS or treatment received. With a median follow up of 1.7 years, 34% of patients relapsed and 45% died. Although single MYC or BCL-2 expression alone had no significant association with OS or PFS in the entire cohort, DE status was associated with significantly poorer PFS, and earlier relapse (median 0.86 years vs. 2.77 years; HR 1.61 (1.07 - 2.41); p=0.021) (Fig. 1). In a subgroup analysis restricted to patients treated with MATRix chemotherapy (n = 116 patients), there was a trend towards inferior PFS and OS, although this did not reach significance in a univariable analysis. However, the adverse prognostic impact of DE status on PFS was maintained in a multivariable analysis modelling age, baseline PS, treatment received, ASCT consolidation status and DE status both for the entire cohort (HR 1.75 (1.15 - 2.66); p<0.01) and the MATRix treated subgroup (HR 1.91 (1.08 - 3.36); p=0.03). A trend towards an association between DE status and OS was noted but did not reach significance in either a univariable or multivariable analysis. We observed no significant difference in PFS or OS between DE-PCNSL and non-DE-PCNSL patients who received MATRix and consolidation with ASCT, perhaps indicating that the adverse impact of DE status is modified by this treatment strategy, although the sample size was small (n=66). Conclusions In a large, retrospective cohort of PCNSL patients treated at 7 UK academic treatment centres, double expression of MYC and BCL-2 by IHC was associated with inferior PFS even in patients treated with MATRix chemotherapy. Validation of this finding in independent cohorts is warranted. ___ JO, CPF and KC share senior authorship Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1280: TARGETED INHIBITION OF DHODH IS SYNERGISTIC WITH BCL2 BLOCKADE IN LYMPHOMA WITH CONCURRENT MYC AND BCL2 REARRANGEMENT

Background: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, can be cured in approximately two-thirds of patients after initial therapy. The remaining one-third of patients have inferior survival outcomes due to relapse or refractoriness despite salvage chemotherapy with or without stem cell transplantation. The dual dysregulation of MYC and BCL2 is one of the most important pathogenic mechanisms. Thus, new therapies are needed to improve the prognosis of patients, and combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine and has been shown to be a potential therapeutic target for multiple diseases. Recent data have demonstrated that DHODH inhibitors can target MYC in some tumors. Aims: To develop and assess new therapeutics, we investigated the effects of the DHODH inhibitor brequinar in DHL lymphoma cell lines and the synthetic effects of brequinar and venetoclax. Methods: We first investigated the activity of DHODH inhibition in genetically defined double-hit DLBCL(DHL) cell lines. We then investigated whether c-MYC could be repressed by brequinar in DHL cells. Then we tried to uncover the synergistic mechanism of brequinar and venetoclax. To understand the genome-wide effects and target genes of brequinar and the combination with venetoclax, we performed an RNA-seq of SU-DHL4 cells treated with brequinar, venetoclax, or the combination of both agents versus DMSO-treated cells. At last, we confirmed the synergistic effect of DHODH inhibition and BCL2 blockade in vivo. Results: DHODH inhibition could repress DLBCL cell lines with MYC and BCL2 abnormalities. The protein level of c-MYC was upregulated 6 hours after brequinar treatment and then downregulated gradually with prolonged treatment time. The gene levels of c-MYC decreased over time. The synergistic mechanism of brequinar and venetoclax is the complementarity of different pathways. RNA-seq of SU-DHL4 cells treated with different agents further implied the possible synergistic mechanism of the two agents. Finally, in vivo study confirmed targeted inhibition of DHODH is synergistic with BCL2 blockade in lymphoma with concurrent MYC and BCL2 rearrangement Summary/Conclusion: Our study demonstrates that combined blockade of DHODH and BCL2 could synergistically inhibit DLBCL with concurrent MYC and BCL2 abnormalities through multiple pathways. Thus, it can be reasonably expected that a clinical-grade DHODH inhibitor combined with venetoclax might improve outcomes for patients with DHL or double-expression lymphoma and other cancers with high expression of MYC and BCL2 or MYC and MCL-1. Our findings provide new insights into the molecular basis of this effect and offer opportunities to design new targeted treatments for lymphoma.

Parsaclisib in Combination with R-CHOP for Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma: Preliminary Results of a Phase 1/1b Study

Parsaclisib in Combination with R-CHOP for Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma: Preliminary Results of a Phase 1/1b Study

Heterogeneous modulation of Bcl-2 family members and drug efflux mediate MCL-1 inhibitor resistance in multiple myeloma

AbstractAntiapoptotic Bcl-2 family members have recently (re)emerged as key drug targets in cancer, with a tissue- and tumor-specific activity profile of available BH3 mimetics. In multiple myeloma, MCL-1 has been described as a major gatekeeper of apoptosis. This discovery has led to the rapid establishment of clinical trials evaluating the impact of various MCL-1 inhibitors. However, our understanding about the clinical impact and optimal use of MCL-1 inhibitors is still limited. We therefore explored mechanisms of acquired MCL-1 inhibitor resistance and optimization strategies in myeloma. Our findings indicated heterogeneous paths to resistance involving baseline Bcl-2 family alterations of proapoptotic (BAK, BAX, and BIM) and antiapoptotic (Bcl-2 and MCL-1) proteins. These manifestations depend on the BH3 profile of parental cells that guide the enhanced formation of Bcl-2:BIM and/or the dynamic (ie, treatment-induced) formation of Bcl-xL:BIM and Bcl-xL:BAK complexes. Accordingly, an unbiased high-throughput drug-screening approach (n = 528) indicated alternative BH3 mimetics as top combination partners for MCL-1 inhibitors in sensitive and resistant cells (Bcl-xL>Bcl-2 inhibition), whereas established drug classes were mainly antagonistic (eg, antimitotic agents). We also revealed reduced activity of MCL-1 inhibitors in the presence of stromal support as a drug-class effect that was overcome by concurrent Bcl-xL or Bcl-2 inhibition. Finally, we demonstrated heterogeneous Bcl-2 family deregulation and MCL-1 inhibitor cross-resistance in carfilzomib-resistant cells, a phenomenon linked to the MDR1-driven drug efflux of MCL-1 inhibitors. The implications of our findings for clinical practice emphasize the need for patient-adapted treatment protocols, with the tracking of tumor- and/or clone-specific adaptations in response to MCL-1 inhibition.

Incidence of Central Nervous System (CNS) Relapse in MYC Rearranged Diffuse Large B-Cell Lymphoma: Does CNS Prophylaxis Improve Outcome?

Introduction:Central nervous system (CNS) relapse in diffuse large B-cell lymphoma (DLBCL) is rare and associated with poor outcome. The presence of MYC rearrangement (MYC -R)with or without BCL2 and/or BCL6 rearrangement (high-grade B-cell lymphomas, or HGBCL, per 2016 WHO revision) has been associated with inferior survival with traditional R-CHOP chemotherapy and a higher incidence of CNS relapse. The incidence of CNS relapse and patient outcome in patients with HGBCL and MYC -R with increasing use of aggressive CNS directed chemotherapy or CNS prophylaxis is unknown.Methods:In this single-center retrospective cohort study we compared the incidence of CNS relapse in HGBCL with at least MYC -R (MYC- R group) with DLBCL not otherwise specified (DLBCL NOS, non- MYC -R group). Differences between groups were analyzed using Chi-square, Fisher's exact and t-Student tests. Statistical significance was set at P<0.05. Kaplan Meier method was used to calculate progression-free survival (PFS) and overall survival (OS). Log-rank test was used to determine differences in survival. Statistical analyses were performed using SPSS.24 software.Results:Of 205 patients, 72 (35%) were MYC -R and 133 (65%) were non- MYC -RDLBCL. The median age at diagnosis was 62 (range 18-70 years), and 126 patients were male (61%). The MYC -R (HGBCL) group included 58 (80%) patients with concurrent BCL2 and/or BCL6 rearrangement. Among MYC -R patients, 42 (58%) had a high-intermediate or high international prognostic score (IPI) score, 61 (of 68; 90%) were of germinal center B-cell (GCB) type, and 56 (of 69; 81%) presented with advanced stage disease. Among non- MYC -R patients, 49 (of 121; 40%) had high-intermediate or high IPI, and 90 (of 132; 68%) had advanced stage disease. For treatment, 28 (of 66; 42%) MYC -R and 31 (of 120; 25%) non- MYC -R patients received CNS prophylaxis either as intrathecal methotrexate or cytarabine or as systemic therapy with high-dose methotrexate. One MYC -R patient had CNS involvement at presentation. Chemotherapy among MYC -R patients included R-CHOP (23 patients), R-HyperCVAD (8), DA-R-EPOCH (27), R-CHOEP (4) and other diverse regimens including clinical trials (6). Among non- MYC -R patients, regimens included R-CHOP (102), R-HyperCVAD (3), DA-REPOCH (9), and R-CHOEP (13). CNS relapse was seen in 4 patients in the MYC -R group (5.6%) and 1 patient (0.7%) in the non- MYC -R group (P=0.053). Three of the 4 MYC -R patients with CNS relapse had received CNS prophylaxis. The non- MYC -R patient who had CNS relapse initially presented with CNS involvement and had been treated with high dose methotrexate. Twenty-six (26) MYC -R patients and 24 non- MYC -Rpatients had primary refractory disease. Median PFS was 15.9 months in MYC- R vs. not reached in non- MYC- R (HR 2.835; 95% CI 2.291 to 6.757, P<0.0001), and median OS was 30 months in MYC- R vs. not reached in non- MYC- R (HR 4.179; 95% CI 2.945 to 10.55, P<0.0001).Conclusion:The incidence of CNS relapse among both MYC -R (HGBCL) and non- MYC -R (DLBCL NOS) cohorts was overall low and may have been mitigated with the use of prophylaxis. Although, CNS relapse occurred despite CNS prophylaxis in both groups. Both the PFS and OS among MYC -R patients were significantly worse compared to non- MYC -R patients despite the use of more aggressive therapy in a majority of MYC- R patients. Larger studies are needed to confirm these findings and determine optimal strategies for CNS prophylaxis and therapy in HGBCL and DLBCL NOS. [Display omitted] DisclosuresReddy:BMS: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Gilead: Speakers Bureau.

Tumor Immune Microenvironment Components and Checkpoint Molecules in Anaplastic Variant of Diffuse Large B-Cell Lymphoma.

BackgroundAnaplastic diffuse large B-cell lymphoma(A-DLBCL) is a rare morphological subtype characterized by the presence of polygonal, bizarre-shaped tumor cells. Our previous research found that A-DLBCL displays many genetic alterations and biological features that differ greatly from those of ordinary DLBCL. However, the status of tumor immune microenvironment components and checkpoint molecules in A-DLBCL remains unclear.MethodsThirty A-DLBCL patients were enrolled to study tumor immune microenvironment components and checkpoint molecules and their associations with clinicopathological features and prognosis.ResultsPatients with A-DLBCL presented higher expression of PD-L1 (40% vs 10%, P=0.004) than patients with ordinary DLBCL. FISH analysis showed that extra copies of PD-L1 were more frequent in A-DLBCL cases than in ordinary DLBCL cases (23.3% vs 4.0%, P=0.001). The numbers of PD-1+ TILs (tumor infiltrating lymphocytes) and CD8+T cells were significantly lower in A-DLBCL versus ordinary DLBCL. In contrast, the numbers of GATA3+ Th2 cells, FOXP3+ Tregs and CD33+ myeloid-derived suppressor cells (MDSCs) were significantly higher in A-DLBCL than in ordinary DLBCL. The associations between clinicopathological features and tumor immune microenvironment cell frequency were analyzed in A-DLBCL patients. Briefly, the number of PD-1+ TILs was lower and the number of CD33+ MDSCs was higher in patients with mutated TP53 compared to those with wild-type TP53. The number of FOXP3+ Tregs was much lower in patients with a noncomplete response (CR) to chemotherapy than in those with a complete response. The number of CD8+ T cells showed a decreasing trend in patients with high International Prognostic Index (IPI) scores and in those with concurrent MYC and BCL2 and/or BCL6 abnormalities. Univariate survival analysis showed that patients with PD-L1+, mPD-L1+(PD-L1+ nonmalignant stromal cells) or mPD-L1+ status had a significantly poorer overall survival (OS) than those with PD-L1- status. An increase in the number of CD3+ T cells, FOXP3+ Treg cells and T-bet+ Th1 cells was significantly associated with prolonged OS in patients with A-DLBCL.ConclusionOur study suggests that A-DLBCL displays a distinct pattern of tumor immune microenvironment components and checkpoint molecules that distinguish it from ordinary DLBCL. The analysis of tumor immune microenvironment components and checkpoint molecules could help in predicting the prognosis of A-DLBCL patients and determining therapeutic strategies targeting the tumor immune microenvironment.

The impact of MYC and BCL2 structural variants in tumors of DLBCL morphology and mechanisms of false-negative MYC IHC

AbstractWhen the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An “atypical double-hit” category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.

High-Grade B-Cell Lymphoma (HGBL) with MYC and BCL2 and/or BCL6 Rearrangements Is Predominantly BCL6-Rearranged and BCL6-Expressing in Taiwan

Simple SummaryThis study highlights the epidemiological, cytogenetic and clinical difference between patients with multiple hit diffuse large B-cell lymphoma in Taiwan and those from western countries. Unlike in the West, the majority of patients with multiple hit lymphoma in Taiwan harbor a BCL6 rearrangement. Almost three in every five BCL6-rearranged double hit lymphoma cases in Taiwan are non-GCB phenotype, indicating, at least in part, that the preferential screening for double hit with BCL6 rearrangement may be a clinically-informative modality for patients with non-GCB phenotype DLBCL in Taiwan. This also suggests the need for a different treatment approach than is obtained in the West where BCL6 double hit lymphomas are seemingly GCB. Consistent with our present findings, mandatory screening for BCL6-rearrangement in suspected DLBCL cases in Taiwan may aid early diagnosis, therapy decision, and clinical outcome forecast.This study investigated the epidemiological and clinical peculiarities of BCL2 and BCL6 rearrangement in patients with high grade B-cell lymphoma (HGBL) from Taiwan, compared with data from Western countries. Two hundred and eighty-two DLBCL cases from Taipei Medical University-affiliated hospitals (n = 179) and Tri-Service General Hospital (n = 103) were enrolled for this study. From the 282, 47 (16.7%) had MYC translocation; 24 of these harbored concurrent BCL2 and/or BCL6 translocation (double-hit, DH or triple-hit, TH). Twelve DH-HGBL cases had simultaneous MYC and BCL6 translocations, 8 harbored MYC and BCL2 rearrangement, while the remaining 4 patients exhibited TH. Together, 66.7% of DH/TH-HGBL patients were BCL6 rearrangement positive. Among these BCL6-rearranged DH/TH-HGBL patients, only 6 (37.5%) overexpressed MYC and BCL6 proteins simultaneously, indicating that MYC-BCL6 co-overexpression may not be plausible surrogate biomarker for screening BCL6-rearranged DH-HGBL. By the end of year 5, all patients with TH-HGBL, BCL2 DH-HGBL and all but one BCL6 DH-HGBL cases had expired or were lost to follow-up. Progression-free survival (PFS) was longer for the non-DH/TH-HGBL group compared with the DH/TH-HGBL group. While the patients with BCL2 DH-HGBL were lost to follow-up by day 800, their remaining TH-HGBL and BCL6 DH-HGBL peers exhibited very poor PFS, regardless of age strata. More so, patients with BCL6 rearrangement were 5.5-fold more likely associated with extranodal involvement compared with their BCL2-rearranged peers. Moreover, ~60.0% of the BCL6-rearranged DH-HGBL cases were non-GCB, suggesting that including screening for BCL6 rearrangement in patients with the non-GCB phenotype may aid medical decision-making and therapeutic strategy. Contrary to contemporary data from western countries, 2 in every 3 patients with DH/TH-HGBL in Taiwan harbor BCL6 rearrangement. Consistent with present findings, we recommend mandatory screening for BCL6 rearrangement in patients with aggressive HGBL in Taiwan.

49. Cryptic Triple Hit Lymphoma: What's hiding in those chromosomes? A chromosome, FISH and microarray story

High-grade B-cell lymphoma with concurrent MYC, BCL2, and BCL6 rearrangements (i.e. Triple Hit Lymphoma (TH)) is an aggressive lymphoma generally detected by conventional cytogenetics and/or FISH. We present a patient referred for high grade diffuse large B-cell lymphoma with a complex karyotype. The diagnosis was consistent with follicular lymphoma and clonal evolution, based only on the karyotype analysis. Dual fusion and breakapart (BAP) FISH strategies and microarray testing were required to diagnosis TH lymphoma. The karyotype included a t(14;18) with an extra derivative 14 (no normal 14), a t(3;22)(q27;q11.2), a t(2;6) and trisomy 8. IGH/BCL2 FISH identified three fusions, consistent with the novel double derivative (14)t(14;18) and derivative 18. BCL2 -BAP FISH showed one germline fusion, a 3’ signal and two 5’ signals rather than the typical evolution related extra 3’ signal, but consistent with the double derivative 14. MYC-BAP FISH showed three un-rearranged fusions discordant with the double fusion IGH/MYC result. This apparent incongruence could be explained by an IGH insertion into MYC which was confirmed by metaphase FISH. The BCL6 FISH was also atypical with 2 germ line fusions and a small extra 5’ BCL6 signal. Since the microarray showed no 3q27 gain, a balanced rearrangement between the 5’BCL6 promotor and the lambda-light gene at 22q11.2 was concluded. Copy neutral LOH (CN-LOH) of 14q32.13->qter in the array analysis (spanning IGH) supports mitotic recombination based clonal evolution for the novel double derivative 14, consistent with a reversal of the common oncogenic fusion derivative. A second CN-LOH region was observed spanning distal 6p, although the driver gene for that evolution is not clear. In summary, the TH would have been missed without multiple disciplinary ancillary testing, highlighting their importance in accurately evaluating complex clones in facilitating optimal patient care and management.