BCL2 Translocations Research Articles

Clinical and Molecular Features of Patients with Double/Triple Hit Large B-Cell Lymphoma

Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a recently defined category in the latest revision of the World Health Organization (WHO) classification, which comprises approximately 5-15% of newly diagnosed DLBCL. This subtype of lymphoma poses numerous challenges from diagnosis to treatment of patients. On the one hand, diagnosis of HGBL-DH/TH requires the accomplishment of fluorescent in-situ hybridization (FISH) to identify the translocations of MYC(8q24) and BCL2(18q21) or/and BCL6(3q27), with no consensus being reached regarding the most applicable population for FISH tests due to high cost and low prevalence. On the other hand, HGBL-DH/TH displays aggressive features, including a high incidence of advanced disease at diagnosis, and inferior efficacy to standard-induced chemical immunotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Despite several treatment strategies including intensified protocols and hematopoietic cell transplantation (HCT) have been explored, there is no standard regimen to improve outcomes for HGBL patients, and participation in clinical trials is encouraged. Aims: This study aims to investigate the clinical characteristics and prognostic outcomes of HGBL-DH/TH patients, and performed genomic and transcriptomic analyses to link the oncogenic mutations and tumor microenvironment alterations to different DLBCL subgroups. Method:In this study, 107 patients with newly diagnosed HGBL-DH/TH were analyzed. Histological diagnoses were established according to the WHO classification. These HGBL-DH/THs were termed DHL-BCL2 (MYC and BCL2 translocation), DHL-BCL6 (MYC and BCL6 translocation), and THL in our study. DNA sequencing was carried out on 95 patients, for detection of genetic aberrations. Results: Patients with DHL-BCL2 (n=44) and THL (n=15) have similar clinical features, including elevated serum lactate dehydrogenase (LDH), increased proportion of double expression (DE) as well as increased prevalence of non-GCB subtype compared to those with DHL-BCL6 (n=48). With the median follow-up time of 19.3 months (2.5-114.2 months), patients in the DHL-BCL2 and THL group had dismal survival (2-year PFS: 49.3% and 34.2%; 2-year OS: 64.0% and 55.6%) when compared with those in the DHL-BCL6 group (2-year PFS: 63.5%; 2-year OS: 89.3%) (Figure A). Moreover, intensive chemoimmunotherapy or combination with novel targeted agents seemed to improve outcomes in patients with DHL/THL compared with those who received a standard R-CHOP regime. The use of autologous stem cell transplant (ASCT) did not yield a significant improvement in survival for patients who achieved remission after receiving first-line R-DA-EDOCH therapy. However, it is worth noting that there was a trend towards prolonging progression-free survival (PFS) and overall survival (OS) in patients who achieve complete remission (CR) through RCHOP-based treatment and undergo ASCT (Figure B). Compared to DHL-BCL6, the primary genetic lesions in DHL-BCL2 and THL were alterations associating epigenetic regulators like EZH2 and CREBBP, while the primary genetic lesions in DHL-BCL6 were alterations associating cellular differentiation and transcription factors like BTG2, PTPN6, CD70, and BCL6. Correlatively, the EZB genotype was more frequently observed in patients with DHL-BCL2 and THL, while the BN2 genotype was more frequently observed in patients with DHL-BCL6. Conclusion: DHL-BCL2/THL exhibits similar clinical characteristics, prognostic outcomes, and molecular features that set it apart from DHL-BCL6. This implies the need for testing novel agents or therapeutic strategies to expedite treatment development for patients with DHL-BCL2/THL.

Hnrnpu mutations Are Haploinsufficient and Alter the Transcriptome of MYC-Driven Lymphomas

Introduction: Burkitt lymphoma (BL) is an aggressive B-cell cancer, characterized by translocations that juxtapose a potent immunoglobulin enhancer with the MYC oncogene. MYC translocations are also found in 10% of tumors with diffuse large B-cell lymphoma morphology. When a BCL2 translocation is also present, these high-grade B-cell lymphomas (HGBCL-DH- BCL2) have poor prognosis. While sustained MYC expression can promote cell activation and proliferation, it also has potent and acute effects on programmed cell death, which cancer cells must overcome in order to survive. Identification and functional characterization of common mutations that cooperate with MYC are thus important for understanding its role in pathobiology. Methods: Patient samples were obtained from an ongoing meta-analysis of mature B-cell neoplasms including unpublished data from the Lymphoma/Leukemia Molecular Profiling Project. Simple somatic mutations were identified from whole genome or exome sequencing data using an ensemble of variant callers. MYC rearrangements breakpoints were identified using GRIDSS and Manta. All functional studies were performed in an EBV+ BL cell line (Raji). HNRNPU mutations were introduced into cells using IDTs ALT-R CRISPR-Cas9 system, clonally isolated, and verified by sequencing. Subsequent RNA-sequencing was performed. Knockdown and overexpression experiments were performed using an HNRNPU expression vector or siRNA targeting endogenous HNRNPU. HNRNPU eCLIP in HepG2 and K562 cells were obtained from the ENCODE project consortium. Differential gene expression was analysed with DESeq2. Peak-calling and individual crosslink sites were detected using PureCLIP. Results: Within the sequencing data, we noted that several RNA binding proteins were commonly mutated in BL and HGBCL-DH- BCL2. Mutations affecting HNRNPU were identified in tumors from 11.6% of HGBL-DH- BCL2 and 5.6% of BL patients. These mutations were predicted to be inactivating but, unlike conventional tumor suppressor genes, an inactivating mutation on the other allele was never observed. HNRNPU mutations were significantly enriched in EBV+ BLs. The gene product (hnRNP U) is an RNA- and DNA-binding protein that plays a central role in gene expression regulation. To understand their biological effect in aggressive lymphomas, we introduced heterozygous inactivating mutations in the most affected region of HNRNPU. Clones with confirmed knockout exhibited reduced expression at the mRNA and protein level. Through gene expression analysis, comparing the parental to two mutant lines, we identified 615 differentially expressed genes (358 downregulated, 219 upregulated). Pathway enrichment analysis revealed reduced expression of genes in several relevant pathways such as MYC, TP53 and DNA damage response. This was consistent with our observation of reduced MYC protein in the mutant lines. A role of hnRNPU-in MYC modulation was further in experiments when HNRNPU knockdown reduced and overexpression increased MYC expression. Overexpression of hnRNP U also resulted in cellular stress and a decrease in cell proliferation. Using actinomycin D chase experiments, we found that hnRNPU loss leads to reduced MYC transcript stability. Crosslinking immunoprecipitation suggests that hnRNP U binds directly to the MYC transcript in poly G tracts in intron 1. This region is predicted to fold into G-quadruplexes, a secondary structure that can be bound by hnRNP U. HNRNPU mutations are only observed in tumors with an intact MYC locus rather than those with a rearrangement in intron 1. Taken together, these findings suggest that the region of MYC upstream of intron 1 may be relevant for HNRNPU-mediated modulation of MYC. Conclusion: HNRNPU mutations are novel recurrent driver mutations specifically within MYC translocated B-cell lymphomas. HNRNPU acts as a modulator of MYC expression, and the most common mutations are predicted to negatively impact this role. We propose a model where HNRNPU mutations moderate MYC expression, thus buffering MYC-induced apoptosis and proteotoxic stress. Further experiments to explore the role of HNRNPU binding on MYC expression and the potential role in buffering the proteotoxic stress associated with MYC translocations are ongoing. Insights into the mechanisms contributing to disease will support ongoing work to establish in vitro and in vivo models for future therapeutic investigations.

Prognostic Utility of Minimal Residual Disease (MRD) after Curative Intent Induction Therapy for DLBCL: A Prospective Real-World Ctdna Study

Background: The prognostic value of MRD using ultra-sensitive ctDNA assays after 1L DLBCL immunochemotherapy was recently reported (Roschewski, ASH 2022). However, real-world evidence (RWE) for the utility of ctDNA MRD assays to predict survival outcomes after standard-of-care (SOC) 1L treatment for DLBCL remains less clear. Here, we report longitudinally measured ctDNA levels, without use of tumor biopsies, in a real-world population of DLBCL patients treated with curative intent using current anthracycline-based induction regimens. Methods: We prospectively enrolled patients with DLBCLs from Samsung Medical Center undergoing 1L therapy for ctDNA profiling at 3 pre-defined milestones (Baseline, Interim after 3 cycles (C4D1), and end of treatment [EOT]). A total of 99 patients had baseline pretreatment plasma available with sufficient tumor burden for inclusion in this study. 364 samples were profiled in a blinded manner by Phased Variant Enrichment & Detection Sequencing (PhasED-Seq, Foresight Diagnostics). Tumor-derived Phased Variants (PVs) were identified directly from baseline plasma (median 4 mL) without the use of tumor tissue, with matched constitutional DNA from paired PBMCs used to censor germline variants and CHIP. PVs were used to longitudinally assess ctDNA-MRD after therapy, including at the C4D1 and EOT timepoints. MRD levels were compared to standardized responses by PET/CT (Lugano 2014), PFS, and OS. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1:10 6 cfDNA molecules) corresponding to 98% specificity. Results: Among the 99 evaluable patients, the median age was 58 years and 38% were women. 67% had stage III-IV disease, 53% had IPI scores ≥3, and 6% had MYC and BCL2 translocations. All patients were treated with anthracycline based therapy, with 94% receiving RCHOP and 6% receiving DA-EPOCH-R. The median follow-up was 44 months. We tested ctDNA performance at each landmark for predicting outcomes. ctDNA-MRD levels by PhasED-Seq at each landmark were prognostic for both PFS and OS. Specifically, when dividing pretreatment ctDNA levels at the median, ctDNA significantly predicted outcomes (PFS: P=0.004, HR=2.7; OS: P=0.0004, HR=5.1). At the C4D1 and EOT landmarks, we divided patients into those with detectable versus undetectable ctDNA-MRD. Both timepoints significantly stratified patients for outcomes including at C4D1 (PFS: P=0.0002, HR=5.0; OS: P=0.0046, HR=4.1) and at EOT (PFS: P<0.0001, HR=7.8; OS: P<0.0001, HR=10.8). As the current standard for assessing response in DLBCL is imaging, we also examined the prognostic value of PET/CT scans at the same time-point as ctDNA. While interim PET/CT scans in our cohort were prognostic for PFS, they were not significantly prognostic for OS (PFS: P=0.02, HR=2.5; OS: P=0.06, HR=2.4). At EOT, PET/CT scans were prognostic for both PFS and OS (PFS: P=0.011, HR=3.4; OS: P=0.011, HR=4.0). At both milestones, PET/CT appeared less discriminatory than ctDNA-MRD. Exemplifying this, the overall survival of patients at 24 months was better separated at EOT by ctDNA-MRD than it was by PET/CT scan (OS24 ctDNA-MRD+: 37%, ctDNA-MRD-: 94%, compared with PET/CT PR/SD/PD: 50%, PET/CT CR: 86%). Finally, to further compare the prognostic value of ctDNA-MRD in the context of the current standard PET/CT scan, we performed a Cox proportional hazards model to predict PFS and OS, using both PET/CT and ctDNA-MRD as univariate predictors, as well as a multivariate model considering both PET/CT and ctDNA. As expected, interim and EOT ctDNA-MRD and PET/CT scans were predictive of outcomes when considered in univariate analysis. However, when considered in combination, while the prognostic value of ctDNA-MRD for OS remained at both interim and EOT timepoints, PET/CT scan was no longer significantly prognostic of outcomes (Fig 1). Conclusions: These data both demonstrate the feasibility and the prognostic utility of ctDNA-MRD during and after SOC induction therapy for DLBCL in a real-world population using an ultrasensitive ctDNA-MRD assay. The higher predictive value and accuracy of detectable ctDNA-MRD as compared with PET/CT suggest opportunities for integration of such assays in lymphoma response criteria, to potentially inform future clinical decision making.

Prognostic Understanding in Newly Diagnosed Older Adults with Diffuse Large B Cell Lymphoma (DLBCL) or DLBCL/High Grade B-Cell Lymphoma with MYC and BCL2 rearrangements

Background: Diffuse large B cell lymphoma (DLBCL) is an aggressive lymphoid malignancy that often affects older adults. Therapy is potentially curative, with up to two-thirds of patients cured with chemoimmunotherapy but carries substantial risk of toxicity. Treatment of older adults must balance the potential for cure with the risk of therapy toxicity and intensive healthcare utilization. Therefore, prognostic understanding is a crucial component of informed decision-making for this patient population. Yet, data regarding prognostic understanding in older adults with DLBCL are lacking. Methods: We conducted a prospective study of older adults with aggressive non-Hodgkin lymphomas at two tertiary care hospitals between 9/2020 and 01/2023. Eligible patients were age >= 65 years at time of treatment initiation receiving a combination chemoimmunotherapy. Patients could enroll up until cycle 1 day 8 of therapy. Patients completed questionnaires at baseline that assessed demographics as well as information preferences and prognostic understanding (Prognosis and Treatment Perception Questionnaire) and underwent a comprehensive geriatric assessment. We used descriptive statistics to examine patient beliefs regarding likelihood of cure, the importance of knowing about prognosis, and preferences for information about treatment at baseline. We focused on the subset of patients (N=92) with DLBCL or DLBCL/high grade B-cell lymphoma with MYC and BCL2 translocations (double hit lymphoma) in order to have a relatively homogenous population for prognosis and likelihood of cure. Results: The parent study enrolled 106/163 (65.0%) patients, with 92 patients having DLBCL or double hit lymphoma. 88/92 enrolled patients with DLBCL (95.7%) submitted the prognostic understanding questionnaire. Among DLBCL/double hit lymphoma patients (N=92), the median age was 74 years (range: 66-99), and most were male gender (63%), White race (96.7%), and married/with a life partner (73.9%). 48/91 (52.7%) had an age-adjusted International Prognostic Index of 2 or 3, 30/92 (32.6%) had a Charlson comorbidity index of 2+ (not counting the patients' lymphoma), 14/92 (15.2%) had an Eastern Cooperative Group performance status of 2-4, and 50% (46/92) were frail based on comprehensive geriatric assessment. When asked about their likelihood of cure, 37.5% (33/88) selected >90% chance, 37.5% (33/88) selected 75-90% chance, 20.5% (18/88) selected 50-74% chance, 3.4% (3/88) selected 25-49% chance, and 1.1% (1/88) selected 10-24% chance of cure. Most (88.6%;78/88) patients believed that the primary goal of their current treatment was to cure their cancer, and most (93%; 80/86) believed that was their oncologist's primary goal. When asked if they would prefer a course of treatment focused on extending life as much as possible even if it meant more pain and discomfort versus a plan of care focused on relieving pain and discomfort as much as possible, even if it meant not living as long, 30.0% (26/88) chose extending life as much as possible, 40.9% (36/88) chose relieving pain as much as possible, and 30.0% (26/88) did not know. 94.0% (78/83) reported that they had a conversation about prognosis with their oncologist. The majority (91%; 78/86) felt it was extremely important or very important to know about their prognosis over time, and felt (81.4%; 70/86) they had just the right amount of information about their prognosis. Patients stated that knowing about their prognosis was extremely helpful in making decisions about treatment (75.3%; 61/81), preparing for the future (68.3%; 56/82), maintaining hope (79.3%; 65/82), and coping with the disease (72.5%; 58/80). 74.4% (61/82) said that overall knowing about prognosis was extremely helpful. Conclusion: In this cohort of older adults receiving treatment for DLBCL or double hit lymphoma, the vast majority felt it was important to know prognostic information over time and found that this information was helpful for decision making. Our finding that the majority estimated their likelihood of cure to exceed 75% despite most having aa-IPI scores indicating average cure rates < 60 suggests that older adults with newly diagnosed DLBCL hold overly optimistic views of their prognosis.

Longitudinal Patient-Reported Outcomes and Clinical Outcomes By VES-13 and Comprehensive Geriatric Assessment in Older Adults with Aggressive Non-Hodgkin Lymphomas

Background: Older adults with aggressive non-Hodgkin lymphoma (NHL) can experience durable remissions with therapy but also substantial toxicities and healthcare utilization. The Vulnerable Elders Survey (VES-13) is a 13-item tool assessing age, self-rated health, and ability to perform physical and functional activities (score of 3+ indicates vulnerability). The Fondazione Italiana Linfomi comprehensive geriatric assessment (CGA) assesses age, comorbidities, and ability to perform physical and functional activities and classifies patients as fit, unfit, or frail. Both the VES-13 and CGA hold promise to risk stratify older adults for poor outcomes. Few data are available comparing these tools in assessing risk for poor survival and healthcare utilization, and prior studies have not examined the impact of therapy on longitudinal patient-reported outcomes (PROs). Methods: We conducted a longitudinal prospective study of 105 older adults with aggressive NHL at Massachusetts General Hospital or the Dana-Farber Cancer Institute between 9/2020 and 1/2023. Eligible patients were age >= 65 years at time of treatment initiation receiving combination chemoimmunotherapy. Patients could enroll up until cycle 1 day 8 of therapy. Patients completed questionnaires at baseline and 6 weeks, 12 weeks, 18 weeks, and 6 months post therapy initiation assessing demographics, quality of life (QOL-Functional Assessment of Cancer Therapy-Lymphoma [FACT-Lym]), depression and anxiety (Hospital Depression and Anxiety Scale [HADS]), and physical symptoms (Edmonton Symptom Assessment Scale-revised [ESAS-r]). At baseline patients also completed the VES-13 and underwent CGA evaluated by an oncologist. We utilized linear mixed models to examine longitudinal QOL, depression, anxiety, and physical symptoms. We assessed univariate factors associated with longitudinal QOL. Using Cox regression models controlling for gender, age-adjusted International Prognostic Index (IPI), hypoalbuminemia, Charlson comorbidity index (CCI), and lymphoma subtype, we assessed the relationship of VES-13 and CGA in separate models with overall survival (OS). We also examined factors associated with unplanned hospitalization within 6 months of therapy start using logistic regression. Results: We enrolled 106/163 (65.0%) patients. 1 patient was excluded due to treatment with an ineligible therapy. Patients had a median age of 73 years (range: 65-99, 38.1% >75, 21.9% >=80); most were male gender (64.8%), White race (96.2%), and married/with a life partner (72.4%). The most common lymphoma subtypes were DLBCL (73.0%), DLBCL/high grade B-cell lymphoma with MYC and BCL2 translocations (12.4%), and mantle cell lymphoma (10.5%). The majority (56/104, 53.8%) had an age-adjusted IPI of 2 or 3, over a third (34.3%) had a CCI score of 2+, and 82.9% had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-1. 45.7% (48/105) had a VES-13 score of 3+. 40.0%, 9.5%, and 50.5% were fit, unfit, and frail, respectively, on CGA. QOL, depression, anxiety, and physical symptoms all improved over time (Figure 1). QOL improved regardless of age group, VES-13 category (<3 vs 3+), or CGA result (Figure 1). Compared to other patients, those 80+ years with either VES-13 score 3+ (β= -2.90, p=0.045) or frail on CGA (β= -3.68, p=0.007) had a worse QOL trajectory. Income > $50,000 annually (β=2.41; p=0.041) was associated with better QOL trajectory. VES-13 score of 3+ (HR=6.08; p=0.014) and frail on CGA (HR=5.20; p=0.028) were both associated with worse survival. Of those with follow up (N=66), 8 died within 1 year (1-year OS=87.9%), all of whom were both frail on CGA and had VES-13 score 3+. 25.8% (8/31) of the CGA frail patients and 28.6% (8/28) of those with VES-13 3+ died within 1 year. VES-13 score (OR=1.16, p=0.028) was associated with greater likelihood of unplanned hospitalization. Conclusion: Older adults with aggressive NHL often experience improvement in QOL and other PROs with treatment, even across age and frailty categories. VES-13 score and CGA identify patient groups at high risk for early death and poor QOL trajectory, for whom novel therapies and supportive care interventions are warranted. Higher VES-13 score is also associated with higher risk of unplanned hospitalization. Routine assessment of VES-13 and CGA has high potential for early identification of high risk patients and implementation of appropriate interventions.

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 < 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<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<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 >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<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 Impact of Induction Regimens on Clinical Outcomes in Double and Triple-Hit Lymphoma: A Meta-Analysis of Comparative Studies

Background: Double and triple hit lymphomas (DTHL) are aggressive high-grade lymphomas that harbor translocations of MYC and BCL2 and/or BCL6. They constitute 6-14% of all aggressive B-cell lymphoma and have been associated with poor clinical outcomes. Though there is no established standard therapy for DTHL, induction chemotherapy vacillates between R-CHOP-like regimens, R-EPOCH, or more dose-intense regimens (DI) such as R-hyperCVAD/MA and CODOX-M/IVAC with or without consolidation with stem cell transplant. Currently, there are no randomized trials establishing a standard induction regimen. Furthermore, several retrospective studies reported conflicting results regarding the comparative benefits of these various induction approaches. Therefore, we conducted this meta-analysis to evaluate the impact of induction chemotherapy regimens on the clinical outcomes of DTHL. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language, diagnosis of DTHL, comparative studies of induction chemotherapy regimens, and studies that reported overall (OS) and progression-free (PFS) survival. Studies that reported the aggregate outcome of mixed samples of DTHL and other lymphomas were excluded. A meta-analysis using the fixed effects and random effects models was conducted. Results: Seven retrospective comparative studies with a total of 1433 patients were included. The median age was 62 years, and the median follow-up was 27.5 months. DI regimens were associated with better PFS than R-CHOP-like regimens (HR = 0.81, 95%CI 0.66-0.99, p = 0.039) but without improved OS. R-EPOCH, including R-DA-EPOCH, regimens were associated with better PFS than R-CHOP-like regimens (HR = 0.62, 95%CI 0.43-0.91, p = 0.013) but without improved OS. R-HyperCVAD/MA regimen was associated with better PFS compared to R-CHOP-like regimens (HR = 0.64, 95%CI 0.47-0.87, p = 0.005) but without improved OS. Conclusions: This is the first and largest meta-analysis to show that induction DI regimens, in general, and R-EPOCH and R-HyperCVAD/MA regimens, in particular, are associated with better PFS than R-CHOP-like regimens in patients with DTHL. However, this advantage did not translate into a survival benefit.

Primary bone diffuse large B-cell lymphoma (PB-DLBCL): a distinct extranodal lymphoma of germinal centre origin, with a common EZB-like mutational profile and good prognosis.

Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is not recognized as a separate entity by the current classification systems. Here we define and highlight its distinctive clinical presentation, morphology, phenotype, gene expression profile (GEP), and molecular genetics. We collected 27 respective cases and investigated their phenotype, performed gDNA panel sequencing covering 172 genes, and carried out fluorescence in situ hybridization to evaluate MYC, BCL2, and BCL6 translocations. We attempted to genetically subclassify cases using the Two-step classifier and performed GEP for cell-of-origin subtyping and in silico comparison to uncover up- and downregulated genes as opposed to other DLBCL. Most cases (n = 22) were germinal centre B-cell-like (GCB) by immunohistochemistry and all by GEP. Additionally, PB-DLBCL had a mutational profile similar to follicular lymphoma and nodal GCB-DLBCL, with the exception of more frequent TP53 and B2M mutations. The GEP of PB-DLBCL was unique, and the frequency of BCL2 rearrangements was lower compared to nodal GCB-DLBCL. The Two-step classifier categorized eight of the cases as EZB, three as ST2, and one as MCD. This study comprehensively characterizes PB-DLBCL as a separate entity with distinct clinical and morpho-molecular features. These insights may aid in developing tailored therapeutic strategies and shed light on its pathogenesis.

Deciphering the Clinical Benefit of Pola-R-CHP versus R-CHOP in Different Genetic Subtypes Beyond Cell of Origin in the POLARIX Study

Introduction: In the POLARIX study, polatuzumab vedotin in combination with rituximab plus cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) demonstrated prolonged progression-free survival (PFS) vs rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients (pts) with previously untreated diffuse large B-cell lymphoma (DLBCL; NCT03274492; Tilly et al. N Engl J Med 2022); the PFS benefit was sustained with longer follow-up (Herrera et al. Blood 2022). Exploratory analyses demonstrated a trend towards greater PFS improvement in activated B cell-like (ABC) DLBCL with Pola-R-CHP vs R-CHOP (Tilly et al. N Engl J Med 2022). Although not yet available for daily practice, more granular molecular subtypes of DLBCL beyond cell of origin (COO) classification have demonstrated poor prognosis with R-CHOP, including EZH2 mutations/ BCL2 translocations (EZB), MYD88/ CD79B-mutated (MCD; as defined by LymphGen; Wright et al. Cancer Cell 2020), and dark zone gene expression signature (DZsig; Alduaij et al. Blood 2023). Here, in a post hoc exploratory analysis, we investigate the prevalence and clinical outcomes of molecularly defined subtypes of DLBCL in pts treated with Pola-R-CHP vs R-CHOP in POLARIX. Methods: All genetic analyses were performed on baseline tumor biopsies. Using mutations derived from whole exome sequencing (WES), pts were allocated to different genetic subtypes, as defined by the LymphGen classifier. DZsig status was determined from global gene expression patterns (GEP) as measured by RNAseq, as previously described (Ennishi et al. J Clin Oncol 2019). COO was determined by NanoString. Hazard ratios (HR) were adjusted for International Prognostic Index score (2 vs 3-5) and age (≤60 vs >60 years). Due to the exploratory nature of the subtype analyses, all statistics are descriptive. Results: WES data were available for 594 pts (Pola-R-CHP, n=292; R-CHOP, n=302), and baseline clinical characteristics were balanced between treatment arms. The observed 2-year PFS rates with EZB and MCD subtypes were numerically higher but not statistically significant in the Pola-R-CHP vs R-CHOP arm ( Table): EZB, 83% (95% confidence interval [CI]: 76-92) vs 75% (95% CI: 65-86), HR 0.61, 95% CI: 0.33-1.13; MCD, 85% (95% CI: 73-98) vs 73% (95% CI: 63-90), HR 0.64, 95% CI: 0.24-1.70. Lower 2-year PFS rates were observed in pts with the BCL6 fusions and NOTCH2 mutations (BN2) subtype treated with Pola-R-CHP vs R-CHOP (HR 1.82, 95% CI: 0.46-7.18; Table). In the subgroup of pts with an undetermined genetic subtype, 2-year PFS estimates were numerically higher with Pola-R-CHP vs R-CHOP (74% vs 62%, respectively; HR 0.70, 95% CI: 0.46-1.07). GEP data were available for 665 pts (Pola-R-CHP, n=331; R-CHOP, n=334); 108 (16.2%) were DZsig+ (Pola-R-CHP, n=52; R-CHOP, n=56). Of those who were DZsig+, 103 (95.4%) had germinal center B cell-like (GCB) DLBCL, 3 (2.8%) had ABC DLBCL, and 2 (1.9%) had an unknown COO. In pts who were DZsig+ and had FISH results, 54/86 (62.8%) had BCL2 translocations, 41/92 (44.6%) had MYC translocations, and 6/40 (15%) had BCL6 translocations (BCL6 FISH was only done in pts with a MYC translocation), resulting in 23/90 (25.6%) pts with double/triple-hit DLBCL. Pts who were DZsig+ were predominantly within the EZB genetic subtype (60.3%). In the R-CHOP arm, pts who were DZsig+ had shorter PFS vs pts who were DZsig− (2-yr PFS: 62% [95% CI: 51-77] vs 73% [95% CI: 68-79], respectively; HR 1.61, 95% CI: 1.02-2.56; Figure). In the Pola-R-CHP arm, no significant difference in PFS was observed between pts who were DZsig+ vs DZsig− (2-yr PFS: 77% [95% CI: 66-89] vs 79% [95% CI: 74-84]; HR 0.97, 95% CI: 0.54-1.75). Improved PFS was demonstrated in pts who were DZsig+ and treated with Pola-R-CHP vs R-CHOP (HR 0.47, 95% CI: 0.24-0.95). Conclusions: In this exploratory biomarker analysis, we recapitulated that pts with molecularly defined DLBCL subtypes, including EZB and MCD by LymphGen and DZsig+ by RNAseq, have poor outcomes with R-CHOP therapy. In pts with the EZB and MCD subtypes, Pola-R-CHP appeared to improve 2-year PFS compared with R-CHOP. Pts with GCB DLBCL who were DZsig+ significantly benefited from Pola-R-CHP vs R-CHOP. In summary, our data indicate that molecular analysis beyond COO leads to identification of distinct molecular subsets of DLBCL that may respond favorably to Pola-R-CHP; future prospective validation is required.

Safety Results of a Phase I Study of Zandelisib + R-CHOP in Newly Diagnosed Diffuse Large B Cell Lymphoma (DLBCL)

Introduction: Emerging data have identified distinct genetic subtypes of DLBCL based on the diverse oncogenic molecular drivers with varying clinical outcomes when treated with standard R-CHOP. Relapse post frontline treatment could be curbed by integrating novel agents upfront. The phosphatidylinositol 3 kinase (PI3K) signalling pathway contributes to the oncogenesesis of both germinal center derived and activated B-cell (ABC) DLBCL subtypes (Wright, et al Cancer Cell, 2020). Zandelisib, a highly selective oral PI3Kδ inhibitor has proven efficacy in B cell lymphoma with a favorable toxicity profile when administered in intermittent dosing schedule (Pagel, et al Lancet Oncol 2022). We conducted a phase I/2 trial to evaluate the safety and efficacy of zandelisib in combination with R-CHOP chemotherapy in newly diagnosed DLBCL. The study enrollment was closed in 12/2022 after the development of zandelisib was discontinued. We present the results of the phase I (P1) trial. Methods: In this investigator-initiated trial, patients (pts) with untreated DLBCL with ECOG performance status (PS) ≤ 2, adequate bone marrow (BM) and organ function were enrolled from 08/2021-12/2022 at Cleveland Clinic (NCT04517435). ECOG PS 3 was allowed if it was attributed to the lymphoma diagnosis. The study was supported by the MEI Pharma. The P1 study had 2 dose escalation cohorts in combination with standard doses of R-CHOP every 21 days for 6 cycles; zandelisib was dosed 60 mg daily on days 1-4 in cohort 1 and daily on days 1-7 in cohort 2. If the 3 subjects in dose level 1 (D1) did not experience any dose limiting toxicity (DLT) during cycle 1 (C1), pts were enrolled into D2. In phase 2 (P2), a cycle of R-CHOP prior to the study treatment was allowed, if clinically deemed. All pts received pneumocystis jirovecii (PJP) and viral prophylaxis. Disease response was assessed after C3 and C6. Primary end point for P1 was to assess safety and tolerability of the combination and to determine recommended P2 dose (RP2D) of zandelisib. Pts who received at least 1 cycle of study treatment were included in the safety analysis. Primary objective of P2 is progression free survival (PFS) at 1 year. Pt and disease characteristics were analyzed using descriptive statistics. Results: We enrolled a total of 13 pts (P1: 9 and P2: 4) in the study prior to termination. The median age was 67 (range 46-79) years, with majority being female (53.8%), with an ECOG PS of 0-2 (100%). Most pts had stage III-IV disease 10/13 (77%), IPI ≥3 in 8/15 (62%), elevated LDH 9/13 pts (69%), and extranodal involvement 7/13 (54%). 3 pts had BM involvement. DLBCL was germinal center (GC) subtype in 77% of the pts. FISH studies revealed translocations in BCL2 (46%), BCL6 (8%) and c-myc (0%). There was no DLT observed in cohort 1 and zandelisib 60 mg daily on D1-7 was the RP2D. 12 of 13 enrolled pts completed 6 cycles of the study regimen prior to study closure. Of the 12 evaluable pts, the ORR was 100% with 9 pts (75%) achieving CR and 3 PR (25%). No pt experienced disease progression prior to study closing. Median time from treatment start to response was 165 (range 48-189) days. The most common adverse events were diarrhea (38%), neutropenia and anemia (31% each), fatigue, constipation, leukopenia and thrombocytopenia (23% each). Other AEs include colitis, nausea, hypoalbuminemia and hypokalemia (15% each). Of the 2 pts who developed colitis, 1 pt had G1 colitis which resolved, while the other pt discontinued the study after 2 cycles due to G3 colitis and bowel obstruction. Another pt experienced G3 jejunal perforation after completing cycle 6. Other G≥3 AE observed were neutropenia and leukopenia each (23% each), lung infection and lymphopenia (8% each). All pts were alive at the time of study closure. Conclusion: In this phase 1/II study, encouraging efficacy was observed with zandelisib + R-CHOP, but this should be interpreted with caution as the study was incomplete. There is a potential signal for increased gastrointestinal toxicity (one G3 colitis, one G3 bowel perforation) in this small sample size and larger prospective trials are needed to further assess the toxicity profile of the combination of oral PI3K inhibitors with R-CHOP.

Plasma Circulating Tumor DNA (ctDNA) as an Alternative to Tissue DNA for Genotyping of DLBCL: Results from the POLARIX Study

Introduction: In the POLARIX study (NCT03274492), polatuzumab vedotin in combination with rituximab plus cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) demonstrated prolonged progression-free survival (PFS) versus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with previously untreated diffuse large B-cell lymphoma (DLBCL; Tilly et al. N Eng J Med 2022); the PFS benefit was maintained with longer follow-up (Herrera et al. Blood 2022). We previously validated the prognostic value of ctDNA at baseline, on treatment, and at the end of treatment in POLARIX (Herrera et al. Blood 2022). Here, we assess and compare the mutation landscape and molecular subtyping of DLBCL by plasma ctDNA versus tissue DNA using the LymphGen classifier. Methods: Baseline plasma ctDNA single nucleotide variants (SNVs) were identified using the AVENIO ctDNA NHL assay (Research Use Only) with an allelic frequency (AF) of 0.5% (Stokowski et al. Blood 2022). Tissue SNVs at baseline were determined by whole exome sequencing (WES) with an AF cutoff of 5%. Plasma-depleted whole blood at baseline was used as a source of germline DNA to filter non-tumor-specific variants for both ctDNA and WES assays. Mutation landscapes in tissue DNA and plasma ctDNA were compared, focusing on genomic regions covered by both assays and variants within the coding regions. LymphGen uses genetic mutations, copy number data, and BCL2/BCL6 rearrangements to classify samples into at least one of seven genetic subtypes (Wright et al. Cancer Cell 2020). Genetic subtypes were defined by the LymphGen classifier using tissue and ctDNA SNVs, per the following: A53 ( TP53 and TP53BP1 mutations), BN2 ( BCL6 translocations and NOTCH2 mutations), EZB ( EZH2 mutations and BCL2 translocations), MCD ( MYD88L265P and CD79B mutations), N1 ( NOTCH1 mutations), and ST2 ( SGK1 and TET2 mutations). Hazard ratios (HR) were adjusted for International Prognostic Index score (2 vs 3-5) and age (≤60 vs >60 years). Results: At baseline, 443 patients had both WES and ctDNA data. When comparing mutation landscapes between tissue DNA and ctDNA, a median 82% (lower quartile: 0.5, upper quartile: 1.0) of tissue SNVs per patient were also found in their matched ctDNA samples. In total, tissue and ctDNA SNVs were found in 163 and 202 genes, respectively. Among these genes, 154 had SNVs in both tissue and ctDNA samples, representing 96% and 78% of mutated genes identified in tissue and ctDNA, respectively. Eleven genes showed statistically significant differences in distribution between tissue and ctDNA (Fisher's exact test, false discovery rate<0.01; Benjamini and Hochberg. J R Stat Soc 1995), including MEF2BNB-MEF2B, MEF2B, BCL10, BNC2, TNFAIP3, RIMS2, CDH19, KIF2B, BCL6, KMT2D and MYC; all but one ( MEF2B) showed higher mutation frequencies in ctDNA than in tissue. The distribution of molecular subtypes is listed in Table 1; due to a lack of information on copy number alterations, the A53 subtype was not included. Based on the tissue SNV data, LymphGen was able to determine the molecular subtypes of 442 patients. Using LymphGen results from tissue SNVs as a reference, 333 (75.3%) patients had the same subtype designation by tissue and ctDNA SNVs, and 109 (24.7%) had different designations ( Table 2). Among the 109 patients, 9 (8.2%) were assigned to a different molecular subtype by ctDNA, 50 (45.9%) patients with tissue SNV-defined molecular subtype were assigned to the undetermined subtype by ctDNA SNVs, and 50 (45.9%) patients with undetermined status by tissue SNVs were classified into distinctive genetic subtypes by ctDNA variants. In a pooled analysis, the 2-year PFS estimates of individual subtypes by tissue versus ctDNA SNVs were consistent ( Table 1). Conclusion: Our analyses demonstrated that the mutation landscape of ctDNA in DLBCL characterized by the AVENIO ctDNA NHL assay resembles that of tumor tissue determined by WES. Patients with molecular subtypes defined by WES or ctDNA had similar PFS outcomes. Overall, these findings support the use of plasma ctDNA as an alternative to tumor tissue for the genotyping of DLBCL.

EZH2 Inhibitors Enhance CART Cell Quality, Efficacy, In Vivo Homing, Tumor Cell Binding and Killing of Fully Syngeneic Primary B Cell Lymphomas, As Well As Reprogramming Lymphoma Cells to a Highly Immunogenic and T Cell Adherent Phenotype

Although having efficacy in a subset of diffuse large B cell lymphoma (DLBCL) patients, cellular immunotherapies are currently administered without guidance as to whether the underlying biology or immunology are conducive to durable response. Moreover, mechanisms explaining CART resistance in DLBCLs remain largely unknown. One critical missing link to address these unmet needs are syngeneic animal models precisely reflecting the genetics and immunology of human DLBCLs. To overcome these hurdles, we developed a suite of genetically engineered mouse models (GEMM) faithfully recapitulating different types of DLBCL. A retrospective study showed that C3/EZB DLBCLs, featuring EZH2 mutations and BCL2 translocation, experienced inferior outcomes after CART therapy. We reported that Ezh2 mutations cause aberrant repression of genes required for productive immune synapses with T cells. We hypothesized that this phenotype limits CART cell activity in vivo, and that EZH2 inhibitors could overcome CART resistance. We generated EZB GEMMs with conditional expression of Ezh2 Y641F and BCL2 in germinal center (GC) B cells. These mice developed T cell depleted lymphomas, histologically similar to human C3/EZB DLBCLs. We generated several EZB cell lines from these mice, which recapitulated the epigenetic, transcriptional, histological, and immune microenvironment of EZB DLBCLs in humans when engrafted in immunocompetent recipients. Strikingly, treating EZB cells with EZH2 inhibitor tazemetostat (taz) ex vivo reprogrammed them to re-express the full spectrum of T cell engagement genes such as ICOSL, ITGB7, 4-1BBL, and OX40L and rendered them highly immunogenic. For example, mixing endogenous T cells with taz pre-treated EZB cells yielded markedly enhanced killing (p<0.001). Taz pre-treated EZB cells failed to engraft in syngeneic mice due to immunological rejection, but caused fatal DLBCL in Rag1-/- mice. EZB cell lines did not undergo proliferation arrest after taz ex vivo, but showed a significant reduction in tumor burden (p<0.05) when treated in vivo, along with a 3-fold increase in tumor CD4 and CD8 cells (p<0.05), and 50% reduction in Tregs (p<0.01). Taz also increased the proportion of naïve/memory CD8 and reduced effector CD8 cells. EZH2 inhibitors therefore mediate their effects at least in part through immunological mechanisms. We next examined whether taz had any deleterious effect on CART cells. Pre-treating donor mice with taz prior to harvesting T cells yielded significant increase in memory CD8 CART cells after transduction (p<0.05). Taz pre-treated CART cells displayed superior killing of EZB cells (p<0.001) and proliferative capacity ex vivo (p<0.001). Infusing taz pre-treated CART cells in EZB mice resulted in significantly reduced tumor burden in vivo (p<0.001) and reduction in PD1+CD38+ exhausted CD8 CART cells (p<0.001). On the other hand, pre-treatment of EZB cells (but not CART cells) with taz ex vivo enhanced CART cell killing by 3-fold (p<0.001). Administering CART cells in vivo in EZB mice after taz treatment significantly prolonged their survival (100% vs 30%, p<0.01) and was associated with significant reduction in tumor burden assessed by IVIS and post-mortem examination. Therefore, EZH2 inhibition enhances CART cell activity through direct effects on CART cells, in addition to making EZB immunogenic. Finally, to directly demonstrate the functional consequence of rendering EZB lymphomas immunogenic on CART performance in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CART cells and GFP+ EZB lymphomas. Remarkably, pre-treatment of EZB cells with taz, doubled the recruitment of CART cells into lymphomas (p<0.001) and significantly enhanced the CART to EZB lymphoma cell surface engagement index (strength and duration of binding, p<0.001). This was associated with increased killing, and CART-engaged lymphoma cells were observed to be ingested/engulfed or phagocyted by tumor resident macrophages. Collectively, EZH2 inhibitors yield a potent boost to CART mediated anti-lymphoma effects by enhancing CART cell functions and B cell immunogenicity, which would likely yield a significant clinical benefit for these patients where CART cells are less active. These results prompted us to initiate a clinical trial evaluating the safety and efficacy of this combination in B cell lymphomas (NCT05934838).

Oral follicular lymphoma: a clinicopathologic and molecular study.

Follicular lymphoma is a hematolymphoid neoplasm that originates from germinal center B cells. It is made up of a combination of small cleaved centrocytes and a varying quantity of larger non-cleaved centroblasts to describe the clinical, microscopic, immunohistochemical, and molecular features of oral follicular lymphomas. Follicular lymphomas affecting the oral cavity were retrieved from pathology files. Immunohistochemistry was performed to confirm the diagnosis, and fluorescence in situ hybridization (FISH) was employed to detect rearrangements in BCL2, BCL6, and MYC genes. Clinical and follow-up data were obtained from the patient's medical and pathology files. Twenty cases were obtained. There was an equal sex distribution (10 males: 10 females) and a mean age of 60.9 years (range: 10-83 years-old). Lesions presented as asymptomatic swellings, usually in the palate (10 cases) and the buccal mucosa (7 cases). Five patients presented with concomitant nodal involvement. Microscopic evaluation depicted the follicular growth pattern with diffuse areas in six cases. Grades 1 and 2 follicular lymphomas represented 12 cases, while grade 3A neoplasms accounted for other 8 cases. Two cases showed rearrangements in MYC, BCL2, and BCL6 genes, while single BCL2 translocation was found in eight cases. Two cases had no translocation. Three patients deceased and the 2-year overall survival achieved 88%. Follicular lymphoma affecting the oral cavity is uncommon, usually affects the palate as a non-ulcerated swelling and the presence of a systemic disease most always be ruled out.

Genomic Profiling of Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System Suggests Novel Potential Therapeutic Targets

Primary diffuse large B-cell lymphoma of the primary central nervous system (CNS-DLBCL) is an aggressive disease, with dismal prognosis despite the use of high-dose methotrexate-based polychemotherapy. Our study aimed to expand the biologic profiles of CNS-DLBCL and to correlate them with clinical/imaging findings to gain diagnostic insight and possibly identify new therapeutic targets. We selected 61 CNS-DLBCL whose formalin-fixed paraffin-embedded samples were available at first diagnosis. These were investigated by immunohistochemistry, cMYC rearrangements were explored by fluorescence in situ hybridization, and CNS-DLBCL mutated genes were evaluated by next-generation sequencing. CD10, BCL6, and IRF4 were observed in 16%, 83.6%, and 93% of cases, respectively. As typical of CNS lymphoma, 10 (16.4%) of 61 cases were classified as germinal center (GCB) type and 51 (83.6%) of 61 as non-germinal center (non-GCB) type according to the Hans algorithm. Double-expression status for BCL2 and cMYC was detected in 36 (59%) of 61 cases whereas 25 (41%) of 61 were non-DE. Rearrangement of the cMYC gene was detected in 2 cases, associated with BCL6 translocation only in 1 case MYD88, PIM1, CD79B, and TP53 were mutated in 54.5%, 53.5%, 30.2%, and 18.4% cases, respectively. Novel mutations not previously reported in CNS-DLBCL were found: AIP in 23.1%, PI3KCA in 15%, NOTCH1 in 11.4%, GNAS in 8.1%, CASP8 in 7.9%, EGFR in 6.4%, PTEN in 5.1, and KRAS in 2.6% of cases. Survival was significantly longer for patients with mutated MYD88 (8.7 months vs 1.7 months; log-rank test = 5.43; P = .020) and for patients with mutated CD79B (10.8 months vs 2.5 months; log-rank test = 4.64; P = .031). MYD88 and CD79B predicted a longer survival in patients affected by CNS-DLBCL. Notably, we identified novel mutations that enrich the mutational landscape of CNS-DLBCL, suggest a role of PTEN-PI3K-AKT and receptor tyrosine kinase-RAS-mitogen-activated protein kinase signaling in a subset of CNS-DLBCL, and provide new potential therapeutic targets.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type associated with diffuse large B-cell lymphoma: a clinicopathological study

Objective: To investigate the clinicopathological features and molecular genetics of diffuse large B-cell lymphomas (DLBCL) with concurrent or secondary to nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (nTFHL-AI). Methods: The clinicopathological features and molecular genetics of DLBCL associated with nTFHL-AI diagnosed between January 2015 and October 2022 at the First Affiliated Hospital of Zhengzhou University were analyzed using histology, immunohistochemistry, PCR, EBV-encoded RNA in situ hybridization and fluorescence in situ hybridization (FISH). Clinical information was collected and analyzed. Results: A total of 6 cases including 3 nTFHL-AI with secondary DLBCL and 3 composite lymphomas were reviewed. There were 4 male and 2 female patients, whose ages ranged from 40 to 74 years (median 57 years). All patients presented with nodal lesions at an advanced Ann Arbor stage Ⅲ/Ⅳ (6/6). Bone marrow involvement was detected in 4 patients. All cases showed typical histologic and immunophenotypic characteristics of nTFHL-AI. Among them, 5 cases of DLBCL with concurrent nTFHL-AI exhibited numerous large atypical lymphoid cells and the tumor cells were CD20 and CD79α positive. The only case of DLBCL secondary to nTFHL-AI showed plasma cell differentiation and reduced expression of CD20. All of cases were activated B-cell (ABC)/non-germinal center B-cell (non-GCB) subtype. Three of the 6 cases were EBV positive with>100 positive cells/high power field, meeting the diagnostic criteria of EBV+DLBCL. The expression of MYC and CD30 protein in the DLBCL region was higher than that in the nTFHL-AI region (n=5). C-MYC, bcl-6 and bcl-2 translocations were not detected in the 4 cases that were subject to FISH. Four of the 6 patients received chemotherapy after diagnosis. For the DLBCL cases of nTFHL-AI with secondary DLBCL, the interval was between 2-20 months. During the follow-up period ranging from 3-29 months, 3 of the 6 patients died of the disease. Conclusions: DLBCL associated with nTFHL-AI is very rare. The expansion of EBV-infected B cells in nTFHL-AI may progress to secondary EBV+DLBCL. However, EBV-negative cases have also been reported, suggesting possible other mechanisms. The up-regulation of MYC expression in these cases suggests a possible role in B-cell lymphomagenesis. Clinicians should be aware that another biopsy is still necessary to rule out concurrent or secondary DLBCL when nodal and extranodal lesions are noted after nTFHL-AI treatment.

A validated composite comorbidity index predicts outcomes of CAR T-cell therapy in patients with diffuse large B-cell lymphoma.

Chimeric antigen receptor T-cell therapy (CART) has extended survival of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). However, limited durability of response and prevalent toxicities remain problematic. Identifying patients who are at high risk of disease progression, toxicity, and death would inform treatment decisions. Although the cumulative illness rating scale (CIRS) has been shown to correlate with survival in B-cell malignancies, no prognostic score has been independently validated in CART recipients. We retrospectively identified 577 patients with relapsed/refractory DLBCL indicated for CART at 9 academic centers to form a learning cohort (LC). Random survival forest modeling of overall survival (OS) and progression-free survival (PFS) was performed to determine the most influential CIRS organ systems and severity grades. The presence of a severe comorbidity (CIRS score ≥ 3) in the respiratory, upper gastrointestinal, hepatic, or renal system, herein termed "Severe4," had the greatest impact on post-CART survival. Controlling for other prognostic factors (number of prior therapies, Eastern Cooperative Oncology Group performance status, BCL6 translocation, and molecular subtype), Severe4 was strongly associated with shorter PFS and OS in the LC and in an independent single-center validation cohort (VC). Severe4 was also a significant predictor of grade ≥3 cytokine release syndrome in the LC, while maintaining this trend in the VC. Thus, our results indicate that adverse outcomes for patients with DLBCL meant to receive CART can be predicted using a simplified CIRS-derived comorbidity index.

PROGNOSTIC SIGNIFICANCE OF CD30 EXPRESSION IN DIFFUSE LARGE B-CELL LYMPHOMA: A SYSTEMATIC REVIEW WITH META-ANALYSIS

Objective To determine the frequency of CD30 expression in DLBCL and its potential for prognostic determination. Study Design A systematic review was performed, followed by a quantitative meta-analysis to assess the frequency of CD30 expression with positivity cut-off values of >0% and >20%, and determine its association with clinicopathological features and patient survival. Results Using a cut-off value >0%, we observed that 3.5%-59.1% of the cases were considered positive for CD30. There was a significant association of the protein expression with a lower number of extra-nodal sites affected by the neoplasm, with Ann Arbor advanced stage, the absence of B-symptoms, the lack of MYC and BCL2 translocations, and a lower ECOG performance. Using a cut-off value >20%, we observed that 2.5%-36.7% of the cases were considered positive for CD30, being significantly associated with a lower number of extra-nodal sites affected by the neoplasm, Ann Arbor stages III/IV, non-GCB tumours, the lack of MYC and BCL2 translocations, and a lower ECOG value. Conclusion Despite variations in the cut-off values used to determine CD30 positivity in DLBCL, the expression of this protein seems to be associated with a higher survival rate and better prognosis. To determine the frequency of CD30 expression in DLBCL and its potential for prognostic determination. A systematic review was performed, followed by a quantitative meta-analysis to assess the frequency of CD30 expression with positivity cut-off values of >0% and >20%, and determine its association with clinicopathological features and patient survival. Using a cut-off value >0%, we observed that 3.5%-59.1% of the cases were considered positive for CD30. There was a significant association of the protein expression with a lower number of extra-nodal sites affected by the neoplasm, with Ann Arbor advanced stage, the absence of B-symptoms, the lack of MYC and BCL2 translocations, and a lower ECOG performance. Using a cut-off value >20%, we observed that 2.5%-36.7% of the cases were considered positive for CD30, being significantly associated with a lower number of extra-nodal sites affected by the neoplasm, Ann Arbor stages III/IV, non-GCB tumours, the lack of MYC and BCL2 translocations, and a lower ECOG value. Despite variations in the cut-off values used to determine CD30 positivity in DLBCL, the expression of this protein seems to be associated with a higher survival rate and better prognosis.

MYC ALTERATIONS IN HIV‐ASSOCIATED B CELL LYMPHOMAS: RESULTS OF “EUROMYC” STUDY (A EUROPEAN RETROSPECTIVE STUDY)

Introduction: HIV negative patients (pts) with B cell lymphoma (Ly) carrying MYC rearrangements and BCL2+/-BCL6 translocations [double hit (DHL) or triple hit Ly (THL)] have shown a dismal prognosis when treated with standard chemotherapy. Data about the prognosis of “single hit” Ly, (SHL) are more controverse. In HIV-associated B cell lymphomas (HIV+Ly), scanty data are available on the prevalence and prognostic impact of MYC rearrangements. Methods: This is a retrospective study conducted in 11 European centers with the aim to evaluate the clinical and prognostic impact of MYC rearrangement, evaluated by FISH analysis, in HIV+Ly. We compared HIV+ Ly [diffuse large B cell Ly (DLBCL), B cell Ly unclassifiable, with features intermediate between DLBCL and Burkitt (BCLU), and High grade B cell Ly (HGBL)] with (MYC+) and without (MYC-) MYC rearrangements, treated with standard (R-CHOP or CHOP like) or intensive therapy (iCT) that is, CODOX-IVAC, Carmen trial, GMALL. Results: A total of 161 consecutive pts were enrolled: 49 (30%) were MYC+ and 112 (70%) MYC-. MYC+ pts had higher involvement of central nervous system (CNS) at presentation, higher ki67%, more frequent histology other than DLBCL, translocation of BCL2 and germinal center B phenotype. MYC+ pts received more frequently iCT (45% vs. 20%, p = 0.002). With a median follow-up of 57 months, there were no significant differences in overall survival (OS) and progression-free survival (PFS) between MYC+ and MYC- pts (5 years (y) OS and PFS 55% and 47% in MYC+ and 59% and 53% in MYC- pts). DHL/THL were 10 (13 MYC+ pts did not have data on BCL2/BCL6): compared to 26 SHL they had similar clinical characteristics, but worse outcome with 5y PFS (30% vs. 60%, p = 0.02) and 5y OS (50% vs. 67%, p = 0.07). In univariate analysis IPI≥3, ECOG≥ 2, increased LDH and ki67 < 90% were related to worse OS and PFS while BCL2 translocation with shorter PFS. In multivariate analysis ECOG≥2, elevated LDH and ki67 < 90% maintained their negative impact. In MYC+ pts, iCT was related to better PFS compared to standard therapy (5 y PFS 62% and 35%, p = 0.04) and to a trend of better OS (5 y OS 61% and 52%, p = 0.25) (Figure 1). No pts treated with iCT died from toxicity. Keyword: aggressive B-cell non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract M. Spina Honoraria: GILEAD, Servier, Novartis, Incyte, BeiGene, Istituto Gentili Research funding: MENARINI L. Arcaini Consultant or advisory role Roche, Janssen-Cilag, Verastem, Incyte, EUSA Pharma, Celgene/Bristol Myers Squibb, Kite/Gilead, ADC Therapeutics Research funding: Gilead Sciences Other remuneration: Speakers’ Bureau: EUSA Pharma, Novartis D. Dalu Educational grants: Roche, Gentili, Lilly and Eisai Other remuneration: Gentili, Daikii Sanchio A. Tucci Consultant or advisory role Gentili, Sanofi, Jannsen Honoraria: Kiowa Kyrin, Takeda

Progression of follicular lymphoma and related entities: Report from the 2021 SH/EAHP Workshop.

The 2021 Society for Hematopathology and European Association for Haematopathology Workshop addressed the molecular and cytogenetic underpinnings of transformation and transdifferentiation in lymphoid neoplasms. Session 4, "Transformations of Follicular Lymphoma," and session 5, "Transformations of Other B-Cell Lymphomas," included 45 cases. Gene alteration analysis and expression profiling were performed on cases with submitted formalin-fixed, paraffin embedded tissue. The findings from session 4 suggest that "diffuse large B-cell lymphoma/high-grade B-cell lymphoma with rearrangements of MYC and BCL2" is a distinct category arising from the constraints of a preexisting BCL2 translocation. TdT expression in aggressive B-cell lymphomas is associated with MYC rearrangements, immunophenotypic immaturity, and a dismal prognosis but must be differentiated from lymphoblastic -lymphoma. Cases in session 5 illustrated unusual morphologic and immunophenotypic patterns of transformation. Additionally, the findings support the role of cytogenetic abnormalities-specifically, MYC and NOTCH1 rearrangements-as well as single gene alterations, including TP53, in transformation. Together, these unique cases and their accompanying molecular and cytogenetic data suggest potential mechanisms for and unusual patterns of transformation in B-cell lymphomas and indicate numerous opportunities for further study.