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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

Navigating Lymphomas through BCR Signaling and Double-Hit Insights: Overview.

Non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of lymphoproliferative disorders originating from B, T, or NK lymphocytes. They represent approximately 4-5% of new cancer cases and are classified according to the revised WHO system based on cell lineage, morphology, immunophenotype, and genetics. Diagnosis requires adequate biopsy material, though integrated approaches are used for leukemic presentations. Molecular profiling is improving classification and identifying prognostic markers. Indolent NHLs, such as follicular lymphoma and marginal zone lymphoma, typically pursue a non-aggressive clinical course with long survival. Aggressive diffuse large B-cell lymphoma (DLBCL) is the most common subtype. Recent studies have elucidated pathogenic mechanisms like MYC translocations and BCR pathway mutations. "Double hit" lymphomas with MYC and BCL2/BCL6 alterations confer a poor prognosis. Treatment approaches are evolving, with chemoimmunotherapy remaining standard for many indolent cases while intensified regimens and targeted agents show promise for refractory or high-risk aggressive disease. Continued elucidation of the genetic and microenvironmental underpinnings of lymphomagenesis is critical for developing personalized therapeutic strategies.

Do Double-Expressor High-Grade B-Cell Lymphomas Really Need Intensified Treatment? A Report from the Real-Life Series of High-Grade B-Cell Lymphomas Treated with Different Therapeutic Protocols at the Institute of Oncology Ljubljana.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements are known for their aggressive clinical course and so are the ones with MYC and BCL2 protein overexpression. The optimal therapy for these lymphomas remains to be elucidated. A retrospective analysis of all diffuse large B-cell lymphomas and high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements diagnosed between 2017 and 2021 at the Institute of Oncology Ljubljana, Slovenia, has been performed. Only patients with double-expressor lymphoma (DEL), double-hit lymphoma (DHL), or triple-hit lymphoma (THL) were included. Demographic and clinical parameters were assessed, as well as progression-free survival (PFS) and overall survival (OS). In total, 161 cases out of 309 (161/309; 52,1%) were classified as DEL. Sixteen patients had DHL, MYC/BCL2 rearrangement was observed in eleven patients, and MYC/BCL6 rearrangement was observed in five patients. Five patients were diagnosed with THL. Out of 154 patients (according to inclusion/exclusion criteria) included in further evaluation, one-hundred and thirty-five patients had double-expressor lymphoma (DEL), sixteen patients had DHL, and three patients had THL. In total, 169 patients were treated with R-CHOP, 10 with R-CHOP and intermediate-dose methotrexate, 19 with R-DA-EPOCH, and 16 with other regimens. The median follow-up was 22 months. The 5-year OS for the whole DEL group was 57.1% (95% CI 45.9-68.3%) and the 5-year PFS was 76.5% (95% CI 72.6-80.4%). The log-rank test disclosed no differences in survival between treatment groups (p = 0.712) while the high-risk international prognostic index (IPI) carried a significantly higher risk of death (HR 7.68, 95% CI 2.32-25.49, p = 0.001). The 5-year OS for DHL patients was 32.4% (95% CI 16.6-48.2%) while all three TH patients were deceased or lost to follow-up. Our analyses of real-life data disclose that the R-CHOP protocol with CNS prophylaxis is a successful and curative treatment for a substantial proportion of DEL patients.

Anti-CD19 Chimeric Antigen Receptor T Cell Therapy With Tisagenlecleucel for Secondary Central Nervous System Lymphoma: A Case Series.

Relapsed or refractory (R/R) large B cell lymphoma (LBCL) presenting as secondary central nervous system lymphoma (SCNSL) carries a poor prognosis, with a median survival time of two to five months. Chimeric antigen receptor (CAR)-T cell therapy has been approved in R/R LBCL, but studies are ongoing to understand its efficacy and safety for SCNSL. Axicabtagene ciloleucel or tisagenlecleucel have been shown to attain high response rates in some retrospective studies; however, response durability continues to be unclear. Our study is a case series of three patients with R/R SCNSL who were treated with tisagenlecleucel. One patient achieved a complete response 30 days after CAR-T therapy but developed disease progression on day +100 imaging. The second patient had a partial response and eventual disease progression with ultimately death. The third patient died from central nervous system complications of CAR-T therapy. Two of the three patients developed immune effector cell-associated neurotoxicity syndrome grade 4 and cytokine release syndrome grade 1 toxicities. Our series of three patients demonstrates that R/R SCNSL can elicit a response with CAR-T therapy, although with a limited duration response.

Induction treatment in high-grade B-cell lymphoma with a concurrent MYC and BCL2 and/or BCL6 rearrangement: a systematic review and meta-analysis.

Background and aim: High-grade B cell lymphomas with concomitant MYC and BCL2 and/or BCL6 rearrangements (HGBCL-DH/TH) have a poor prognosis when treated with the standard R-CHOP-like chemoimmunotherapy protocol. Whether this can be improved using intensified regimens is still under debate. However, due to the rarity of HGBCL-DH/TH there are no prospective, randomized controlled trials (RCT) available. Thus, with this systematic review and meta-analysis we attempted to compare survival in HGBCL-DH/TH patients receiving intensified vs. R-CHOP(-like) regimens. Methods: The PubMed and Web of Science databases were searched for original studies reporting on first-line treatment in HGBCL-DH/TH patients from 08/2014 until 04/2022. Studies with only localized stage disease, ≤10 patients, single-arm, non-full peer-reviewed publications, and preclinical studies were excluded. The quality of literature and the risk of bias was assessed using the Methodological Index for Non-Randomized Studies (MINORS) and National Heart, Lung, and Blood Institute (NHLBI) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. Random-effect models were used to compare R-CHOP-(like) and intensified regimens regarding 2-year overall survival (2y-OS) and 2-year progression-free survival (2y-PFS). Results: Altogether, 11 retrospective studies, but no RCT, with 891 patients were included. Only four studies were of good quality based on aforementioned criteria. Intensified treatment could improve 2y-OS (hazard ratio [HR]=0.78 [95% confidence interval [CI] 0.63-0.96]; p=0.02) as well as 2y-PFS (HR=0.66 [95% CI 0.44-0.99]; p=0.045). Conclusions: This meta-analysis indicates that intensified regimens could possibly improve 2y-OS and 2y-PFS in HGBCL-DH/TH patients. However, the significance of these results is mainly limited by data quality, data robustness, and its retrospective nature. There is still a need for innovative controlled clinical trials in this difficult to treat patient population. Systematic review registration: https://www.crd.york.ac.uk/prospero, identifier CRD42022313234.

Double-hit and double-expressor primary central nervous system lymphoma: Experience from North India of an infrequent but aggressive variant.

Background: High-grade non-Hodgkin B-cell lymphoma is an aggressive mature B-cell lymphoma that depicts poor treatment response and worse prognosis. The presence of MYC and B-cell lymphoma 2 (BCL2) and/or B-cell lymphoma 6 (BCL6) rearrangements qualifies for triple-hit and double-hit lymphomas (THL/DHL), respectively. We attempted to explore the incidence, distribution, and clinical characteristics of the primary high-grade B-cell lymphoma of the central nervous system (CNS) in our cohort from North India. Methods: All the histologically confirmed cases of primary CNS diffuse large B-cell lymphoma (PCNS-DLBCL) over a period of 8 years were included. Cases showing MYC and BCL2 and/or BCL6 expression on immunohistochemistry (IHC) (double- or triple-expressor) were further analyzed by fluorescence in situ hybridization for MYC, BCL2 and /or BCL6 rearrangements. The results were correlated with other clinical and pathological parameters, and outcome. Results: Of total 117 cases of PCNS-DLBCL, there were seven (5.9%) cases of double/triple-expressor lymphomas (DEL/TEL) (six double- and one triple-expressor) with median age of 51 years (age range: 31-77 years) and slight female predilection. All were located supratentorially and were of non-geminal center B-cell phenotype. Only triple-expressor case (MYC+/BCL2+/BCL6+) demonstrated concurrent rearrangements for MYC and BCL6 genes indicating DHL (n = 1, 0.85%), while none of the double-expressors (n = 6) showed MYC, BCL2, or BCL6 rearrangements. The mean overall survival of the DEL/TEL was 48.2 days. Conclusion: DEL/TEL and DHL are uncommon in CNS; mostly located supratentorially and are associated with poor outcome. MYC, BCL2, and BCL6 IHC can be used as an effective screening strategy for ruling out double/ triple-expressor PCNS-DLBCLs.

A coordinated strategy for a simple, pragmatic approach to the early identification of the ultra-high-risk patient with diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent aggressive lymphoma seen in clinical practice. Despite huge strides in understanding its biology, front-line therapy has remained unchanged for decades. Roughly one-third of patients have primary refractory or relapse following the end of conventional first-line therapy. The outcome of patients with primary refractory disease and those with early relapse (defined as relapse less than 1 year from the end of therapy) is markedly inferior to those with later relapse and is exemplified by dismal overall survival. In this article, the authors term patients with features that identify them as being at particularly high-risk for either primary refractory disease or early relapse, as 'ultra-high-risk'. As new treatment options become established (e.g. bispecific T-cell engagers, chimeric antigen receptor 'CAR' T-cells and antibody-drug conjugates), it is likely that there will be a push to incorporate some of these agents into the first-line setting for patients identified as ultra-high-risk. In this review, the authors outline advances in positron emission tomography, widely available laboratory assays and clinical prognosticators, which can detect a high proportion of patients with ultra-high-risk disease. Since these approaches are pragmatic and able to be adopted widely, they could be incorporated into routine clinical practice.

AMG176, an MCL-1 inhibitor, is active in pre-clinical models of aggressive B-cell lymphomas.

Upregulation of the anti-apoptotic protein MCL-1 has been implicated in chemotherapy resistance and poor clinical outcomes in B-cell lymphoma (BCL). We report the activity of AMG176, a direct, selective MCL-1 inhibitor, in preclinical models of BCL. A panel of cell lines representing diffuse large B-cell lymphoma (DLBCL), double-hit lymphoma (DHL) and Burkitt's lymphoma (BL) was selected. AMG176 induced apoptotic cell death in a dose- and time-dependent manner in all BCL cell lines. Baseline MCL-1 expression was not predictive of response. AMG176 exhibited impressive synergy with venetoclax and chemotherapeutic agents, less so with proteasomal inhibitors, and antagonism with anti-CD20 monoclonal antibodies. The activity of AMG176 could not be confirmed in murine models of BCL. Combination therapy targeting MCL-1 and BCL-2 may provide an alternative therapeutic approach in BCL, however optimal patient selection will remain the key to obtaining high response rates and tolerability.

Integrated Genomic DNA/RNA Profiling vs Fluorescence in Situ Hybridization in the Detection of MYC and BCL2 (and BCL6) Rearrangements in Large B-Cell Lymphomas: Updates Amid the New WHO Classification of Lymphoid Neoplasms.

OBJECTIVES: Large B-cell lymphomas (LBCLs) are a heterogeneous group of lymphoid neoplasms whose molecular and cytogenetic profile has predictive and prognostic implications. The concept of double-hit lymphomas (DHLs) was recently updated in the fifth edition of the World Health Organization classification, with the exclusion of MYC and BCL6 rearranged tumors from the group. Now, DHLs are referred to as diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Fluorescence in situ hybridization (FISH) is the current gold standard for detecting rearrangements in LBCLs, but comprehensive genomic profiling (CGP) has recently been suggested to be at least as accurate as FISH in classifying these neoplasms and providing additional genetic information. METHODS: We analyzed a cohort of 131 patients in whom FISH and CGP studies were performed as part of our normal clinical workflow and compared the effectiveness of FISH and CGP in detecting these clinically relevant rearrangements. RESULTS: Our findings are in agreement with our previously published study, which analyzed a cohort of 69 patients, supporting our hypothesis that the best approach to maximize detection of DHLs while limiting waste seems to be a combination of CGP and MYC break-apart FISH testing, the latter to capture the presence of non-IGH::MYC events. CONCLUSIONS: Our study supports the combined use of FISH and GCP rather than either method alone to better detect MYC and BCL2 (and BCL6) gene rearrangements.

Transformed Plasmablastic Lymphoma Presenting With Marked Lymphocytosis and Spontaneous Tumor Lysis Syndrome.

The clinicopathology entity of plasmablastic lymphoma (PBL), despite broad recognition by the World Health Organization (WHO), represents a diagnostic challenge due to its overlapping features and scarce occurrence. Often, PBL arises in immunodeficient, elderly male patients, most notably those who are human immunodeficiency virus (HIV)-positive. More infrequent, cases of transformed PBL (tPBL) evolved from another hematologic disease have been identified. Herein, we describe a case of a 65-year-old male transferred from a neighboring hospital with pronounced lymphocytosis and spontaneous tumor lysis syndrome (sTLS) presumed to be chronic lymphocytic leukemia (CLL). Utilizing a complete clinical, morphologic, immunophenotypic, and molecular evaluation, we arrived at a final diagnosis of tPBL with sTLS, suspected to have evolved from the NF-κB/NOTCH/KLF2 (NNK) genetic cluster of splenic marginal zone lymphoma (SMZL) (NNK-SMZL), a potential transformation and presentation, to our knowledge, not previously reported. However, definitive clonality testing was not performed. In this report, we also outline the diagnostic and educational considerations we faced in discerning tPBL from other more common B-cell malignancies which can present similarly, such as CLL, mantle cell lymphoma, or plasmablastic myeloma. We summarize recently reported molecular, prognostic, and therapeutic considerations for the treatment and recognition of PBL, including the successful implementation, in our patient, of bortezomib to an EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) regimen with prophylactic intrathecal methotrexate, who has since achieved complete remission (CR) and entered clinical surveillance. Lastly, this report briefly highlights the challenge we faced in this area of hematologic typification that necessitates additional review and discussion by the WHO: tPBL with potential double-hit cytogenetic versus double-hit lymphoma with a plasmablastic phenotype.

A Rare Case of Ovarian Double-Hit/Diffuse Large B-Cell Lymphoma: A Case Report and Review of Literature.

Primary ovarian non-Hodgkin lymphoma is a rare lymphoma that is often associated with diagnostic delays, initial misdiagnosis, and inappropriate management. We report a case of ovarian diffuse large B-cell lymphoma (DLBCL) in a young female who initially presented with generalized fatigue, lower abdominal discomfort, and 40 pounds of unintentional weight loss. She subsequently had a computed tomography of abdomen done that showed fatty liver, hepatomegaly, and a left heterogeneous ovarian mass measuring about 4 × 4.2 cm. Transvaginal ultrasound was also done that showed a heterogeneous solid left adnexal mass measuring 7.4 × 5.6 × 6.6 cm. She subsequently had a total abdominal hysterectomy with bilateral salpingo-oophorectomy. Immunohistochemistry (IHC) showed the malignant cells expressing PAX5, CD20, and BCL2 with a Ki-67 proliferation index greater than 90%. The cells were negative for AE1/AE3, S100, CD30, and cyclin D1. Aggressive B-cell lymphoma fluorescence in situ hybridisation (FISH) panel was positive for rearrangement of BCL6 and MYC, with no evidence of BCL2 rearrangement, consistent with a double-hit high-grade B-cell lymphoma. Immunohistochemistry for BCL6 and MU M1 showed positive staining in the malignant cells. CD10 was negative. The staining profile was consistent with nongerminal center B-cell-like type of DLBCL. Ovarian lymphoma is a very rare entity; the presence of an enlarged ovarian tumor should raise the suspicion of ovarian lymphoma, and our case also emphasizes on the use of IHC markers in diagnosing the ovarian DLBCL.

Peripheral immune cell profiling of double-hit lymphoma by mass cytometry.

BACKGROUND: Double-hit or Triple-hit lymphoma (DHL/THL) is a subset of high-grade B cell lymphoma harboring rearrangements of MYC and BCL2 and/or BCL6, and usually associate with aggressive profile, while current therapies tend to provide poor clinical outcomes and eventually relapsed. Further explorations of DHL at cellular and molecular levels are in demand to offer guidance for clinical activity. METHODS: We collected the peripheral blood of DHL patients and diffused large B cell lymphoma (DLBCL) patients from single institute and converted them into PBMC samples. Mass cytometry was then performed to characterize these samples by 42 antibody markers with samples of healthy people as control. We divided the immune cell subtypes based on the expression profile of surface antigens, and the proportion of each cell subtype was also analyzed. By comparing the data of the DLBCL group and the healthy group, we figured out the distinguished immune cell subtypes of DHL patients according to their abundance and marker expression level. We further analyzed the heterogeneity of DHL samples by pairwise comparison based on clinical characteristics. RESULTS: We found double-positive T cells (DPT) cells were in a significantly high percentage in DHL patients, whereas the ratio of double-negative T cells (DNT) was largely reduced in patients. Besides, CD38 was uniquely expressed at a high level on some naïve B cells of DHL patients, which could be a marker for the diagnosis of DHL (distinguishing from DLBCL), or even be a drug target for the treatment of DHL. In addition, we illustrated the heterogeneity of DHL patients in terms of immune cell landscape, and highlighted TP53 as a major factor that contributes to the heterogeneity of the T cells profile. CONCLUSION: Our study demonstrated the distinct peripheral immune cell profile of DHL patients by contrast to DLBCL patients and healthy people, as well as the heterogeneity within the DHL group, which could provide valuable guidance for the diagnosis and treatment of DHL.

Long-Term Complete Remission of a Patient With Double-Hit Diffuse Large B-Cell Lymphoma Treated by Chemoimmunotherapy and Chinese Herbal Medicine.

Double-hit diffuse large B-cell lymphoma (DHL) is an uncommon subtype of lymphoma which poorly responds to current drug therapies and has low rates of long-term survival in the patients. Herein, we report a case of a 73-year-old Caucasian male who was diagnosed with DHL with double-hit mutations of rearrangement of both c-MYC and BCL2 in November 2013. He commenced the standard R-CHOP-14 chemotherapy (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone) but changed to dose-adjusted DA-EPOCH-R protocol (etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone, and rituximab) in the second and third cycles due to double-hit mutation. Because of intolerance to the intensive therapy, the patient decided to switch to Chinese medicine intervention. From March 2014 to December 2019, he was prescribed with a classical Chinese herbal formula-Sijunzi Decoction plus Prunella vulgaris based prescriptions. After 2 months of the Chinese herbal medicine intervention, the patient felt his right groin mass disappeared. Imaging follow-up showed no residual masses, and no lymphadenopathy was seen. During the period of Chinese herbal medicine treatment, his adherence and tolerability were well maintained with no adverse events. Imaging surveillances afterward found no evidence of lymphoma recurrence. His regular blood tests indicated that the patient's blood counts were normal and stable; no hematologic toxicity, hepatoxicity, or nephrotoxicity associated with Chinese herbal medicine were found. Follow-up visits until 2020 found that he had been living and enjoying a good quality of life for over 8 years post-diagnosis. This case study illustrates the potential values of Chinese herbal medicine in DHL treatment, alongside chemo-immunotherapy, and in maintaining long-term survival and satisfactory quality of life for DHL patients. The case report provides clinicians with preliminary evidence of the use of Chinese herbal medicine as a therapeutic strategy in the management of DHL.

No difference in treatment outcome between patients with nodal versus extranodal diffuse large B-cell lymphoma.

Background and Aim: Diffuse large B-cell lymphoma (DLBCL) has been classified using various parameters, including the site of origin. Studies have reported conflicting outcomes when DLBLC patients were stratified according to the site of origin. This study aimed to investigate the response rate and survival outcomes in nodal versus extranodal DLBCL and compare the results to a region-matched study covering the 1988 - 2005 period. Methods: A single-center retrospective cohort study was conducted on all patients diagnosed with DLBCL and treated in a tertiary care hospital in Pakistan during 2014 - 2019. We calculated the mean and median for continuous variables and frequency and percentages for all categorical variables. Progression-free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier survival curves. A Cox proportional hazards model was used to determine the hazard ratio (HR) for OS. Results: Of the 118 patients, 49 patients (41.5%) had nodal disease and 69 patients (58.5%) were diagnosed with extranodal DLBCL. The majority of patients in the nodal and extranodal cohorts presented with Stages III and IV disease (73.4% and 62.3%, respectively). A complete response to (immuno) chemotherapy was achieved in 71.4% of nodal DLBCL patients and 65.2% of extranodal DLBCL patients. The 5-year PFS and median PFS in the entire cohort were 0.8% and 17 m, respectively. The PFS and median PFS in the nodal and extranodal DLBCL cohort were 0% and 1.4%, respectively, and 15 m and 19 m, respectively. The 5-year OS and median OS in the entire cohort were 16.1% and 19 m, respectively. The OS and median OS in the nodal and extranodal DLBCL cohort were 8.2% and 21.7%, respectively, and 19 m and 21 m, respectively. Multivariable linear regression revealed that the ABC phenotype (nodal, HR = 1.37, 95% CI = 1.37 - 3.20; extranodal, HR = 1.65, 95% CI = 1.46 - 3.17; GBC as reference) and double and triple hit DLBCL (nodal, HR = 1.29, 95% CI = 1.19 - 2.81; extranodal, HR = 1.87, 95% CI = 1.28 - 2.43; and non-expressors as reference) are independent negative predictors of OS. Conclusions: DLBCL incidence in the Karachi region has remained comparable but patient composition in the extranodal DLBCL cohort has shifted to predominantly advanced stage. Nodal and extranodal DLBCL were associated with similar PFS and OS profiles and first- and second-line treatment responses. Cell of origin and antigen expression status was independent negative predictors of OS, disfavoring the ABC phenotype and lesions with c-MYC and BCL2 and/or BCL6 overexpression. Relevance for Patients: DLBCL is an aggressive type of non-Hodgkin's lymphoma, however; patients respond well to standard systemic chemotherapy. Extranodal type of DLBCL patients tend to have more residual disease after first-line systemic chemotherapy, but physicians should keep in mind that the subsequent line treatment mitigates its negative impact on survival.

Synergistic disruption of BTK and BCL-2 causes apoptosis while inducing ferroptosis in double-hit lymphoma.

Double-hit lymphoma (DHL) is an aggressive subset of Diffuse Large B-cell Lymphoma (DLBCL) with poor outcomes and without satisfying treatment options. BTK inhibitor monotherapy is ineffective to suppress aggressive lymphoma. Hence, combination with other potential agents is warranted. Here, we demonstrated the second generation of BTK inhibitor, zanubrutinib, and a BCL-2 inhibitor, navitoclax, worked in synergistic manner to suppress DHL. Comprehensive in silico approach by interrogating single-cell to bulk-level profiling was employed along with in vitro and in vivo validation in DHL cell lines. Ablation of BTK enhanced sensitivity to navitoclax and suppressed proliferation of DHL cells. Combination of second generation of BTK inhibitor with navitoclax synergistically suppressed DLBCL cells with higher synergy score in DHL subset. The drug combination triggered apoptosis and ferroptosis, with the latter being characterized by reactive oxygen species (ROS) accumulation, extensive lipid peroxidation, and depletion of reduced glutathione. Moreover, ablation of BTK sensitized DHL cells to ferroptosis. Mechanistically, disruption of BTK and BCL-2 triggered ferroptosis by downregulating NRF2 and HMOX1, while deactivating GPX4. Combination of zanubrutinib and navitoclax effectively suppressed tumor growth in vivo. Our data suggest that zanubrutinib and navitoclax synergistically suppressed DHL by inducing apoptosis and ferroptosis.

Clinical features, gene mutation profile and prognosis analysis of diffuse large B-cell lymphoma complicated with follicular lymphoma / 白血病·淋巴瘤

Objective:To investigate the clinicopathologic characteristics, gene mutation profile and prognostic influencing factors of diffuse large B-cell lymphoma (DLBCL) complicated with follicular lymphoma (FL) (DLBCL/FL).Methods:The clinicopathological data of 50 DLBCL/FL patients admitted to Rui Jin Hospital Affiliated of Shanghai Jiao Tong University School of Medicine from February 2018 to November 2021 were retrospectively analyzed. Targeted sequencing was performed to assess the mutation profile of 55 lymphoma-related genes. The clinicopathological characteristics were summarized to evaluate the short-term therapeutic efficacy of all patients. Kaplan-Meier method was used to analyze the overall survival (OS) and progression-free survival (PFS) of patients. Cox regression risk models were used to assess the factors affecting the OS and PFS.Results:Among 50 DLBCL/FL patients, 23 cases (46%) were male, 22 cases (44%) had an international prognosis index (IPI) score ≥ 2 points, 16 cases (32%) were double-expression lymphoma (DEL) and 4 cases (8%) were double-hit lymphoma (DHL). The complete response (CR) and overall response rates were 68% (34/50) and 78% (39/50), respectively after the first-line therapy. The median follow-up time was 23.3 months (5.1-50.9 months). The 2-year OS rate was 82.1% and 2-year PFS rate was 67.1%; and the median OS and PFS were not reached. Targeted sequencing results showed that the mutation frequencies of KMT2D, MYD88, TP53, BTG2, DTX1, EZH2, CD70, CREBBP, DUSP2, HIST1H1C, HIST1H1E and PRDM1 genes in this cohort were more than 15%. Multivariate Cox regression analysis showed that male ( HR = 4.264, 95% CI 1.144-15.896, P = 0.031) and IPI score ≥ 2 points ( HR = 6.800, 95% CI 1.771-37.741, P = 0.007) were independent risk factors of PFS in newly diagnosed DLBCL/FL patients, and TP53 mutation ( HR = 4.992, 95% CI 1.027-24.258, P = 0.046) was an risk influencing factor of OS. Conclusions:The proportion of male and female DLBCL/FL patients is similar, with a small proportion of DHL. Mutations of KMT2D, MYD88 and TP53 genes are commonly found in DLBCL/FL patients. Generally, DLBCL/FL patients can have a high overall response and good prognosis. Male and IPI score ≥ 2 points are the independent risk factors of PFS, and TP53 mutation is an independent risk factor of OS in DLBCL/FL patients.

Favorable Outcomes with R-CHOP Induction and Consolidative Autologous Stem Cell Transplantation for Double-Hit Lymphoma.

Double-hit lymphoma (DHL) is an aggressive large B cell lymphoma associated with a poor prognosis with R-CHOP chemotherapy. The optimal treatment is unknown, but outcomes might be improved with intensive induction regimens or consolidative high-dose chemotherapy and autologous stem cell transplantation (HDT/ASCT). The purpose of this study was to determine the real-world outcomes of patients with DHL treated with primarily R-CHOP induction and consolidative HDT/ASCT. This retrospective, multicenter study included consecutive patients age 18 to 70 years with newly diagnosed DHL intended for consolidative HDT/ASCT in Alberta, Canada. Progression-free survival (PFS) and overall survival (OS) were determined using the Kaplan-Meier method. The cohort comprised 58 patients with a median age of 59.5 years (range, 30 to 69 years). High-risk features at diagnosis included International Prognostic Index score 3 to 5 in 45 patients (78%), transformed indolent lymphoma in 25 (43%), and central nervous system involvement in 3 (5%). Forty-six patients (79%) patients received R-CHOP induction, and 45 (78%) proceeded to consolidative HDT/ASCT. With a median follow-up of 4.6 years, the 4-year PFS and OS rates were 67% (95% confidence interval [CI], 53% to 78%) and 68% (95% CI, 54% to 79%), respectively, for all patients and 86% (95% CI, 72% to 93%) and 88% (95% CI, 73% to 95%) for those undergoing HDT/ASCT. R-CHOP induction and consolidative HDT/ASCT result in excellent outcomes for patients with chemosensitive DHL, whereas patients with primary refractory disease might benefit from alternative strategies, such as earlier use of chimeric antigen receptor T cell therapy.

Decreased CD11c-positive dendritic cells in the tumor microenvironment predict double-hit/triple-hit genotype and survival in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma and is a potentially curable disease. However, it is heterogenous, and the prognosis is poor if the tumor cells harbor fusions involving MYC and BCL2 or MYC and BCL6 (double-hit [DH] lymphoma), or fusions involving all three genes (triple-hit [TH] lymphoma). Fluorescence in situ hybridization is currently the gold standard for confirming the presence of DH/TH genotypes. However, the test is laborious and not readily available in some laboratories. Germinal center B (GCB) signatures and dual expression of MYC and BCL2 are commonly used as initial screening markers (traditional model) in clinical practice. Our study proposes immunohistochemical markers for more conveniently and accessibly screening DH/TH genotypes in DLBCL. We retrospectively reviewed the clinical and pathological parameters of patients with DLBCL. We assessed the proliferative index, apoptotic index, and tumor microenvironment (TME), with regard to T cells and CD11c(+) dendritic cells, in formalin-fixed paraffin-embedded tissue. We then generated a decision tree as a screening algorithm to predict DH/TH genotypes and employed decision curve analysis to demonstrate the superiority of this new model in prediction. We also assessed the prognostic significance of related parameters. Our study revealed that GCB subtypes, a Ki67 proliferative index higher than 70%, and BCL2 expression were significantly associated with DH/TH genotypes. Decreased CD11c(+) dendritic cells in the TME indicated additional risk. Our proposed screening algorithm outperformed a traditional model in screening for the DH/TH genotypes. In addition, decreased CD11c(+) dendritic cells in the DLBCL TME were an independent unfavorable prognosticator. In conclusion, we provide a convenient, well-performing model that predicts DH/TH genotypes in DLBCL. The prognostic significance of CD11c(+) dendritic cells in the TME might influence the classification and development of immunotherapy for DLBCL in the future.

Leukemic High Grade B Cell Lymphoma is Associated With MYC Translocation, Double Hit/Triple Hit Status, Transformation, and CNS Disease Risk: The Mayo Clinic Experience.

INTRODUCTION: Leukemic involvement in high grade B cell lymphoma (L-HGBL) is rare and has been sparsely described in the literature. We report our experience in a large single institution multicenter academic setting. MATERIALS AND METHODS: Medical records of patients with HGBL who received care at Mayo Clinic between 2003 and 2020 were reviewed. L-HGBL was confirmed by peripheral blood smear and flow cytometry with corroboration from tissue and bone marrow biopsy findings. RESULTS: Twenty patients met inclusion criteria. All patients had significant bone marrow involvement by HGBL. Leukemic involvement presented in 11 of 20 (55%) in the de novo and 9 of 20 (45%) in the relapsed setting. Seven of 20 patients had DLBCL, NOS, 6 of 20 had transformation (t-DLBCL), 3 of 20 had transformed double/triple hit lymphoma (t-DHL/THL), 2 of 20 had double hit lymphoma (DHL), and 2 of 20 had HGBL with intermediate features between DLBCL and Burkitt lymphoma. Nine of 15 patients had MYC translocation. Based on Hans criteria, 11 of 20 had germinal center B-cell (GCB) cell of origin (COO) and 9/20 had non-GCB COO. Five of 11 de novo patients experienced CNS relapse/progression. All de novo patients received anthracycline-based chemoimmunotherapy. Eighteen of 20 patients died of progressive disease. Median overall survival was significantly better in the de novo compared to relapsed group (8.9 months vs. 2.8 months, P = .01). COO, MYC status, DHL/THL status, HGBL subtype, or treatment group did not demonstrate a significant effect on overall survival. CONCLUSION: L-HGBL carries a poor prognosis and is associated with MYC translocation, DHL/THL status, transformation, and high CNS risk. Novel therapeutic approaches are needed for L-HGBL.