Double-hit Lymphoma Research Articles

The outcomes of Tiantan first-aid protocol on critically ill patients with primary central nervous system lymphoma

Objective: To examine the outcomes of Tiantan first-aid protocol on critically ill patients with primary central nervous system lymphoma (PCNSL). Methods: The clinical data of 18 patients with PCNSL who were treated according to Tiantan first-aid protocol at Department of Neurosurgery,Beijing Tiantan Hospital, Capital Medical University from November 2019 to December 2021 were retrospectively analyzed. There were 9 males and 9 females, aged (56.9±11.1)years (range: 29 to 77 years). The median Karnofsky performance status(KPS) score at admission was 40 (range: 20 to 60). Three patients were mild coma, 3 were lethargy and 12 were conscious. The mean midline shift was 0.7 cm (range: 0 to 1.8 cm). After admission, all patients were treated according to the plan of rapid biopsy, rapid routine pathology and rapid salvage chemotherapy. The treatment procedures, clinical and radiographic outcomes, KPS score and adverse reactions of patients after chemotherapy were collected. Results: All of the 18 patients completed the first-aid treatment. The median duration from admission to the biopsy was 1 day (range: 0 to 5 days), from biopsy to routine pathological diagnosis was 1 day (range: 1 to 4 days) and from routine pathology to salvage chemotherapy was 1 day (range: 0 to 4 days). All the patients were pathologically confirmed with diffuse large B cell lymphoma, 1 patient was double-hit lymphoma. Seventeen patients underwent clinical remission and 1 died of cardiac dysfunction. The successful salvage rate was 17/18. Radiologically, complete remission was observed in 1 case, partial remission in 16 cases, and stable disease in 1 case. The median KPS score at discharge was 60 (range: 30 to 80). The mild gastrointestinal, hematological and hepatic adverse effects were observed after chemotherapy. Conclusion: Tiantan first-aid protocol is effective for critically ill patients with PCNSL, which has the merit to be popularly used and improved.

ORAL AND OROPHARYNGEAL HIGH-GRADE B-CELL LYMPHOMA: A CASE SERIES

High-grade B-cell lymphomas (HGBLs) are aggressive B-cell neoplasms also known as double-hit and triple-hit lymphomas, that harbor translocations in BCL2, BCL6, and/or MYC genes. This study describes the clinicopathologic data, immunohistochemical features, EBV status, and molecular findings of 8 cases of HGBLs that were retrieved from the files of 3 oral pathology centers from 2001 to 2019. The median age at presentation was 64.4 years without sex predominance. Most cases involved the oral cavity as asymptomatic swellings. Microscopically, the tumors exhibited a diffuse growth pattern, mostly composed of large centroblasts. Three cases showed the starry sky appearance. Most cases were positive for Bcl2, MUM1, Bcl6, and c-Myc proteins. The mean proliferative index was 74.5%. Double translocations of MYC/BCL2 and MYC/BCL6 genes were observed in 3 and 2 cases, respectively. Three cases demonstrated simultaneous rearrangements of MYC/BCL2/BCL6. HGBL can be found in the oral/oropharyngeal region and is associated with aggressive clinical presentation. High-grade B-cell lymphomas (HGBLs) are aggressive B-cell neoplasms also known as double-hit and triple-hit lymphomas, that harbor translocations in BCL2, BCL6, and/or MYC genes. This study describes the clinicopathologic data, immunohistochemical features, EBV status, and molecular findings of 8 cases of HGBLs that were retrieved from the files of 3 oral pathology centers from 2001 to 2019. The median age at presentation was 64.4 years without sex predominance. Most cases involved the oral cavity as asymptomatic swellings. Microscopically, the tumors exhibited a diffuse growth pattern, mostly composed of large centroblasts. Three cases showed the starry sky appearance. Most cases were positive for Bcl2, MUM1, Bcl6, and c-Myc proteins. The mean proliferative index was 74.5%. Double translocations of MYC/BCL2 and MYC/BCL6 genes were observed in 3 and 2 cases, respectively. Three cases demonstrated simultaneous rearrangements of MYC/BCL2/BCL6. HGBL can be found in the oral/oropharyngeal region and is associated with aggressive clinical presentation.

Double Hit Lymphoma Mimicking B Cell Precursor Phenotype Burkitt Lymphoma/Leukemia in an Elderly

Background: The differential diagnosis of Burkitt lymphoma/leukemia (BL) and the double or triple hit lymphomas remains often problematic in terms of immunophenotyping in association with MYC gene analysis. In the past, BL-like B-cell malignancy with B-cell precursor phenotype (BCP-ALL) was described. Case Report: We report here an 84-year-old male with massive ascites without superficial lymphadenopathy. Abdominal paracentesis revealed chylous (non-bloody) ascites consisting of mostly abnormal BL-like blasts showing CD20-negative BCP phenotype. However, molecular study indicated no MYC/IGH, but probable involvements of MYC/IGL and BCL6/IGH translocations, which were confirmed by FISH studies. The patient was diagnosed as double hit lymphoma mimicking BCP-ALL.

Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

Classifying Germinal Center Derived Lymphomas-Navigate a Complex Transcriptional Landscape.

Simple SummaryGerminal center-derived B-cell lymphomas constitute a very heterogeneous group of neoplasms with diverse clinical presentations, prognoses, and responses to therapy. They divide into a series of subtypes, such as Diffuse Large B-cell lymphomas (DLBCL), Burkitt Lymphomas (BL), Follicular lymphomas (FL), and further, into several subclasses and rarer subtypes of finer granularity, which makes them one of the most heterogeneous cancer entities. Molecular classification schemes, first of all, derived from whole transcriptome gene expression data largely improved subtyping and functional understanding. Based on a whole transcriptome landscape of B-cell lymphoma, we show that one major caveat in the task of classification is represented by the rather fuzzy distribution of individual tumors without clear-cut borderlines between most of the subtypes, preventing their unambiguous association with clear-cut entities. This landscape is governed by the germinal center (GC) reaction and relates different subtypes to different states along the reaction path. We discuss the relatedness between the expression landscape and classifier signatures and their functional cell of origin background. This view helps to stratify lymphomas in terms of modular building blocks of signature genes and to interpret rarer subclasses in the context of larger ones.Classification of lymphoid neoplasms is based mainly on histologic, immunologic, and (rarer) genetic features. It has been supplemented by gene expression profiling (GEP) in the last decade. Despite the considerable success, particularly in associating lymphoma subtypes with specific transcriptional programs and classifier signatures of up- or downregulated genes, competing molecular classifiers were often proposed in the literature by different groups for the same classification tasks to distinguish, e.g., BL versus DLBCL or different DLBCL subtypes. Moreover, rarer sub-entities such as MYC and BCL2 “double hit lymphomas” (DHL), IRF4-rearranged large cell lymphoma (IRF4-LCL), and Burkitt-like lymphomas with 11q aberration pattern (mnBLL-11q) attracted interest while their relatedness regarding the major classes is still unclear in many respects. We explored the transcriptional landscape of 873 lymphomas referring to a wide spectrum of subtypes by applying self-organizing maps (SOM) machine learning. The landscape reveals a continuum of transcriptional states activated in the different subtypes without clear-cut borderlines between them and preventing their unambiguous classification. These states show striking parallels with single cell gene expression of the active germinal center (GC), which is characterized by the cyclic progression of B-cells. The expression patterns along the GC trajectory are discriminative for distinguishing different lymphoma subtypes. We show that the rare subtypes take intermediate positions between BL, DLBCL, and FL as considered by the 5th edition of the WHO classification of haemato-lymphoid tumors in 2022. Classifier gene signatures extracted from these states as modules of coregulated genes are competitive with literature classifiers. They provide functional-defined classifiers with the option of consenting redundant classifiers from the literature. We discuss alternative classification schemes of different granularity and functional impact as possible avenues toward personalization and improved diagnostics of GC-derived lymphomas.

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis

AbstractImmunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with “double-hit” diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.

Molecular Profiling of Large B-Cell Lymphomas

Background: Large B-cell non-Hodgkin's lymphoma (NHL) comprises of a heterogeneous group of lymphomas with a high-grade morphology and aggressive nature. The diagnosis has gradually evolved from morphological characterization to classification of this group based on ancillary techniques such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and molecular studies. Diffuse large B-cell lymphomas (DLBCL), not otherwise specified (NOS) is the most common B-cell NHL reported and a new diagnostic entity termed high-grade B-cell lymphoma harboring an MYC rearrangement with a BCL2 and/or BCL6 have been introduced by the WHO in 2017. MYC and BCL2/BCL6 proteins expression on IHC due to mutations leading to nuclear factor kappa B pathway activation is considered as double-expressor lymphoma (DEL). Materials and Methods: Sixty-two patients diagnosed with DLBCL, NOS on histopathology were subjected to IHC markers such as (CD20, CD79a, PAX5, CD10, Bcl6, Bcl2, MUM1, TDT, and Myc) and classified into activated B-cell and germinal center B-cell based on Hans' Algorithm. The samples were consequently subjected to tissue FISH for the detection of MYC, BCL2, and BCL6 gene translocations and classified as double-hit lymphoma (DHL)/triple-hit lymphoma (THL). The FISH results were subsequently compared for IHC expression of c-myc, Bcl2, and Bcl6. The staging, international prognostic index (IPI) scoring and lactate dehydrogenase levels were compared with progression-free survival (PFS) of 15 months among DHL/THL and DEL/TEL. Results: The median age of presentation among DLBCL-NOS patients is 58 years, while males (66.7%) were affected more commonly than females (33.3%). The majority of the patients presented with nodal involvement (71%) while extranodal involvement was seen in 29% cases. Hans' algorithm showed a significant P value with the IHC expression of BCL2, BCL6 and C-MYC. IHC and FISH correlation for BCL2 and BCL6 showed 100% sensitivity and 100% negative predictive value. IHC and FISH for c-MYC showed concordant results with a significant P < 0.03. The clinicopathological results of S/D/THL showed association with higher stage disease, higher IPI scoring, and high Ki-67 index with inferior PFS. Conclusions: IHC MYC is a sensitive screening modality for MYC translocation and can be used for the identification of rearrangement in lower socioeconomic areas. Based on clinicopathological studies, all patients with DLBCL must undergo MYC FISH testing as these patients behave as high-grade lymphomas. Hence, a new entity DLBCL with MYC rearrangement without BCL-2/6 rearrangements maybe considered as a novel entity and to be studied in future cohorts.

P1291: BTM-3566, A NOVEL ACTIVATOR OF THE MITOCHONDRIAL STRESS RESPONSE ERADICATES DIFFUSE LARGE B-CELL LYMPHOMA IN MICE

Background: Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or after CAR-T-cell therapy. Aims: We describe and characterize BTM-3566, a first-in-class compound that activates the OMA1-dependent mitochondrial stress pathway. BTM-3566 is active against a variety of B-cell malignancies but has the greatest effect in DLBCL, inducing robust therapeutic responses in vitro and in vivo. Methods: Chemical biology, animal models, functional genetics Results: BTM-3566 is a small molecule based on a pyrazolothiazol-backbone. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines with an IC50 of ~300 nM, including ABC, GCB, double-hit and triple-hit lymphoma lines. BTM-3566 has > 50% of oral bioavailability and 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. In a dose-finding study using the DLBCL line SUDHL-10, once daily oral treatment with 20 mg/kg BTM-3566 for 21 days resulted in complete regression in all tumor-bearing animals. Importantly, no subsequent tumor growth was observed for 2 weeks after cessation of therapy. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1). Transcriptome analyses revealed that BTM-3566 activates the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and upregulation of the stress transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined that HRI (EIF2AK1) was uniquely required for BTM-3566 eIF2a phosphorylation, activation of the ATF4-ISR and induction of apoptosis. HRI is activated by mitochondrial-related stress, including heme depletion, ROS generation or blockage of mitochondrial ATP synthesis which all result in the activation of the mitochondrial protease OMA1. We found that BTM-3566 activates OMA1 in a manner unrelated to changes in mitochondrial ATP synthesis, reactive oxygen species generation or electron transport chain inhibition. OMA1 activation was required for the therapeutic effect and CRISPR-Cas9 depletion of OMA1 eliminated BTM-3566’s apoptotic activity. Substrates of OMA1 include the mitochondrial GTPase OPA1 and DELE1, a mitochondrial protein recently shown to act as a sensor of mitochondrial dysfunction and to signal through HRI kinase and ATF4. We show that downstream of OMA1 the apoptotic stress signal is relayed to HRI by DELE1, whereas OMA1-dependent OPA1 cleavage and mitochondrial fragmentation were dispensable for the therapeutic effect. Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a poorly characterized mitochondrial membrane protein, negatively correlated with response to BTM-3566. Overexpression of FAM210B prevented OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis and cell cycle arrest. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation. Image:Summary/Conclusion: We describe a novel antitumor mechanism in DLBCL, where BTM-3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An IND application in DLBCL has been completed with initiation of first in human clinical trials planned in 2022.

Abstract 5377: Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax

Abstract Introduction: Despite recent advances in the therapy of diffuse large B cell lymphoma (DLBCL), many patients are still not cured, and therefore new therapeutic combinations are needed. The anti-apoptotic B cell lymphoma 2 (BCL-2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32.3;q21.3), copy number alterations and gene amplifications; however, targeting BCL-2 with a selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a novel combination partner for venetoclax to improve response to therapy. Previously with dynamic BH3 profiling, we found that DNA hypomethylating agents (HMA) could increase BCL2 dependence in DLBCL cells. Here, we investigate the activity of the HMA decitabine with venetoclax in DLBCL. Methods: We utilized BH3 profiling, a functional assay to assess the mitochondrial priming of cells for apoptosis and the dependence of cells on various anti-apoptotic BCL-2 family proteins. Other standard techniques included Western blot, cell cycle (BrdU/7-AAD), CellTiter-Glo, and Seahorse XF cell mito stress test. The DLBCL cell lines (TMD8, HBL1, OCI-Ly3, OCI-Ly1, Karpas 422, SUDHL4, Toledo, OCI-Ly7) and double hit lymphoma (DHL) patient-derived xenograft cell lines were used to investigate the in vitro anti-cancer activity of decitabine and venetoclax. Results: Through BH3 profiling, we found heterogenous dependence of DLBCL, consistent with the variable expression of multiple anti-apoptotic proteins in DLBCL assessed by Western blot. DLBCL cells were less sensitive to single BH3 mimetic treatment (venetoclax, S63 (MCL-1i) and A133 (BCL-xLi)). Using dynamic BH3 profiling (which assesses the change in priming induced by ex vivo drug treatment), we found that decitabine primes DLBCL cells for apoptosis and increases BCL-2 dependence. Consequently, decitabine increases the sensitivity of DLBCL cells to venetoclax, as the combination of decitabine and venetoclax synergistically induces apoptosis and suppresses cell proliferation. Decitabine has previously been shown to increase TGF-β signaling in DLBCL by restoring the expression of a downstream target, SMAD1. Interestingly, we found that inhibition of TGF-β signaling only partially antagonizes the anti-cancer activity of decitabine, implicating other anti-cancer mechanisms of decitabine. Indeed, we found that decitabine also induces DNA damage and leads to cell cycle arrest in DLBCL cells. Furthermore, decitabine promotes activation of the key apoptotic effector proteins, BAK and BAX. Additionally, we found that this combination also suppresses oxidative phosphorylation, thus restricting the energy supply for DLBCL cells. Conclusions: The HMA decitabine sensitizes DLBCL cells to venetoclax in vitro through increasing apoptotic priming and BCL-2 dependence. This combination is worthy of further study in in vivo model systems. Citation Format: Matthew S. Davids, Fen Zhu, Stephen J. Chong, Jennifer Crombie, Liam Hackett, May C. Collins. Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5377.

Abstract 2639: KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers

Abstract MYC is an early-response gene downstream of many receptor complexes, and a component that promotes several pro-growth signal transduction pathways. As a result, MYC expression is highly regulated, but once dysregulated in cancer, MYC promotes tumorigenesis via activation of cell growth, proliferation, and angiogenesis. CDK9 is both an upstream and downstream cofactor of MYC. CDK9 promotes MYC expression through its recruitment to super enhancers at the MYC amplicon and acts as a cofactor to drive gene expression programs. Previously, we profiled pan-cancer sensitivity to KB-0742, a potent, selective, and orally bioavailable small-molecule inhibitor of CDK9, and identified MYC-amplified cancers as being especially sensitive to KB-0742. To better understand the connection between MYC and KB-0742 sensitivity, 11 different tumor types were evaluated preclinically using immortalized cell lines, patient-derived cell lines (PDCs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). The 11 indications included head and neck, esophageal, gastric, liver, non-small cell lung cancer (NSCLC), pancreatic, ovarian, triple-negative breast cancer (TNBC), prostate, colon, and lymphoma. For 5 of the indications (head and neck, esophageal, gastric, liver, and colon), little activity was observed in cell lines and in vivo with weak correlations to MYC amplification. TNBC, ovarian cancer, and lymphoma showed good responses to KB-0742 treatment. For TNBC and ovarian, immortalized and patient-derived cell lines indicated lower IC50 values with increased MYC amplification or expression, but the results were not statistically significant. However, in vivo assessments using 15 PDX models showed good correlation of tumor growth inhibition (TGI) and MYC amplification/expression, with some tumor regressions observed in several ovarian models. Two PDO models of TNBC with different treatment histories were also treated with KB-0742 and compared to 4 standard-of-care (SOC) compounds. KB-0742 showed much greater activity in the models, with maximal inhibition rates of 100% and 89%. Cell-line screens of blood cancer-derived cell lines showed that lymphocyte-derived cell lines were the most sensitive to KB-0742 as compared to all other cancer types. We tested several PDX models for response to our compound. Treatment with KB-0742 resulted in TGIs of over 50% in several lymphoma models, including a TGI of 56% in 1 model of double-hit diffuse large B-cell lymphoma. These data support the continual development of KB-0742, which is currently being tested in a phase 1/2 clinical trial (NCT04718675). The dose-escalation phase is opened to relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommend phase 2 dose is determined, we plan to open dedicated expansion arms enrolling patients with MYC over-expression/amplification within the tumor types supported by these studies. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2639.

Plasmablastic lymphoma with MYC::IGH fusion and BCL2 rearrangement.

A 50-year-old male with history of nodal stage III diffuse large B-cell lymphoma (DLBCL), refractory to R-CHOP, presented with vomiting, abdominal pain, and an umbilical mass. Abdominal computed tomography (CT) scan showed bulky retroperitoneal adenopathy and possible bowel obstruction. Histologic examination of the umbilical mass needle biopsy demonstrated diffuse atypical cells with medium to large nuclei, prominent central nucleoli, and eccentrically placed amphophilic cytoplasm (Figure 1, top left panel, objective 100×). The original lymphoma was of germinal center B-cell phenotype, was MYC-negative by immunohistochemistry (IHC), and had BCL2 rearrangement (BCL2-R) without MYC-R by interphase fluorescence in situ hybridization (FISH). In contrast, the umbilical mass neoplastic cells expressed plasma cell markers CD138 (Figure 1, top right, objective 60×) and MUM1 (Figure 1. Bottom left, objective 60×) and MYC (90%), were lambda light chain restricted, had a very high Ki-67 proliferation index (>90%), and were negative for all B-cell markers, germinal center cell markers, and Epstein-Barr virus. Interphase FISH studies identified BCL2-R and IGH::MYC fusion (Figure 1, bottom right, Ch 8 centromere: aqua probe, MYC [8q24]: red probe, IGH [14q32]: green probe; yellow fusion indicates IGH::MYC) without BCL6-R. These findings are characteristic of plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. This likely represents transformation of the patient's prior DLBCL, with clonal evolution characterized by acquisition of MYC-R. Transformation of follicular lymphoma to plasmablastic lymphoma with MYC-R and BCL2-R has been previously described, but to our knowledge this is the first reported case of transformation of DLBCL to plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. The “double-hit” cytogenetic features may portend an aggressive clinical course, similar to what has been observed in “high-grade B-cell lymphomas with MYC-R and BCL2-R and/or BCL6-R” (so-called double-hit lymphoma). The authors would like to thank the Mayo Clinic Genomics Laboratory for their continuous assistance and support of our service. The authors declare they have no conflict of interest. The authors received no specific funding for this work. Mohammad Barouqa wrote the manuscript. Patricia Greipp reviewed the article and cytogenetic probes. Rebecca King reviewed the article and stains. Ellen D. McPhail supervised the manuscript and finalized it.

Clinical laboratory mutation analysis performed on aggressive B cell non-Hodgkin lymphoma patient biopsies.

e19561 Background: Results from comprehensive genomic analysis performed on aggressive B cell non-Hodgkin lymphomas (aBNHL) patient (pt) biopsies (bx) have identified mutations (mut) which may be predictive of pt survival. However, results from mut analysis (MA) performed on aBNHL pt bx in clinical laboratories are not well-described. Methods: Bx diagnostic of non-Burkitt aBNHL obtained from 7/2015-2/2021 with MA performed in the Penn Center for Personalized Diagnostics clinical laboratory using 3 consecutive versions of lymphoma sequencing panels were retrospectively analyzed. Mut tested varied by panel. Mut detected with variant allele frequency (VAF) ≥10% were included. Results: MA was performed on 293 aBNHL pt bx with a successful assay for 270 (7.8% failure rate). Request for MA was made by interpreting hematopathologist 93% and treating clinician 7%. Median turnaround time (TAT) from receipt of bx to result report was 18 (range 8-53) days. Notable pt characteristics included prior/concurrent indolent lymphoma (IL) 31% and prior systemic therapy received for IL and/or aBNHL 38%. Notable bx characteristics included bx type paraffin-embedded tissue 85% and bone marrow 12%, histologic classification diffuse large B cell lymphoma 86% and high grade B cell lymphoma 13%, cell of origin (COO) by Hans algorithm non-germinal center B (non-GCB) 51% and GCB 44%, known double expressor lymphoma 30%, known MYC rearrangement 18%, known double hit lymphoma 9% and any mut detected with VAF ≥10% 82%. Number and percentage of mut occurring in ≥10% of bx (minimum tested bx for mut ≥92) with associated COO are listed in the table. Subset analysis of 147 bx obtained from pts with newly-diagnosed aBNHL (15% with previously-untreated IL) had similar bx characteristics to the entire population were analyzed for the presence of mut reported in poor-prognosis COO-associated classifications: Cluster 5 for non-GCB and Cluster 3 for GCB (PMID: 29713087), double-hit signature (DHITsig) for GCB (PMID: 30523716) and molecular high grade (MHG) for GCB (PMID: 30523719). For 77 non-GCB bx, 19% had Cluster 5 mut. For 59 GCB bx, 56% had at least one of Cluster 3, DHITsig or MHG mut. Conclusions: Clinical laboratory MA performed on nearly 300 aBNHL pt bx was highly successful with a short TAT and demonstrated recurrent gene mut associated with aBNHL development. In the subset of bx from previously-untreated pts, MA identified mut associated with poor-prognosis genetic subtypes of aBNHL.[Table: see text]

FrontMIND: A phase III, randomized, double-blind study of tafasitamab + lenalidomide + R-CHOP versus R-CHOP alone for newly diagnosed high-intermediate and high-risk diffuse large B-cell lymphoma.

TPS7590 Background: Up to 40% of patients relapse or are refractory (R/R) to R-CHOP, the current standard of care for diffuse large B-cell lymphoma (DLBCL), indicating an unmet need, particularly in patients with high-intermediate and high-risk disease (IPI 3–5). POLARIX (NCT03274492) evaluated a modified regimen substituting polatuzumab vedotin for vincristine (pola-R-CHP), demonstrating modest improvement in PFS with no OS benefit between treatment arms. The addition of lenalidomide (LEN) to R-CHOP improved both PFS and OS in the ECOG-ACRIN E1412 trial but not in the Phase III ROBUST study, in which a lower dose of LEN was used, despite a positive trend toward 2-year PFS in a subgroup analysis. The chemo-free immunotherapy tafasitamab + LEN has received accelerated approval in the United States (2020), and conditional marketing authorization in Europe and Canada (2021) in adult patients with R/R DLBCL ineligible for ASCT based on L-MIND (NCT02399085). The primary analysis of First-MIND (NCT04134936), a Phase Ib randomized safety study of R-CHOP + tafasitamab ± LEN in patients with previously untreated and newly diagnosed DLBCL, demonstrated that adding tafasitamab + LEN does not impair dosing and scheduling of R-CHOP, with toxicities similar to those expected with R-CHOP alone (ASH 2021; #3556). frontMIND (NCT04824092) will investigate the efficacy and safety of R-CHOP + tafasitamab + LEN vs R-CHOP alone in previously untreated patients with high-intermediate and high-risk DLBCL. Methods: frontMIND is a Phase III, multicenter, randomized, double-blind, placebo-controlled study. Approximately 880 patients are planned to be randomized at ̃350 centers in North and South America, Europe, and Asia-Pacific. Eligible patients aged 18‒80 years with previously untreated local biopsy-proven, CD20+ DLBCL with IPI score 3–5 (age-adjusted IPI 2–3 if ≤60 years), and ECOG PS 0–2 will be enrolled. Patients with transformed lymphoma (except double or triple hit lymphoma) are excluded. Patients will be randomized 1:1 to receive six 21-day (D) cycles of either R-CHOP + tafasitamab (12 mg/kg intravenously, D 1, 8, and 15) + LEN (25 mg orally, D1–10) or R-CHOP + tafasitamab and LEN placebos. Patients will be followed for up to 5 years after the end of treatment. The primary endpoint is investigator-assessed PFS. Secondary endpoints include investigator-assessed event-free survival, OS, safety, and tafasitamab serum concentration (trough and Cmax levels). Sensitivity and specificity of minimal residual disease for early detection of disease progression is an exploratory endpoint; further MRD parameters may also be investigated. The study is funded by MorphoSys AG and conducted with the scientific support of members of the Fondazione Italiana Linfomi and the German Lymphoma Alliance. Clinical trial information: NCT04824092.

Double-drug loading upconversion nanoparticles for monitoring and therapy of a MYC/BCL6-positive double-hit diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a systemic hematological malignancy. Herein, through whole exome sequencing (WES), we found that DLBCL genome changes and expression characteristics are associated with various immune cells. Lenalidomide (Len) is a leading candidate for the immunomodulatory treatment of multiple myeloma in the clinic. Inspired by lenalidomide as an immunomodulatory drug for the treatment of multiple myeloma, we constructed a multifunctional nanoplatform with therapeutic and imaging properties for DLBCL by co-loading lenalidomide and dexamethasone (Dex) with upconversion nanoparticles using a GSH-sensitive linker (named as UCNPs-Len-Dex). In vitro cell experiments proved that the UCNPs-Len-Dex had good biocompatibility and obvious antitumor efficacy. UCNPs-Len-Dex also exhibited excellent anti-tumor efficacy and imaging properties in vivo. RNA sequencing showed that UCNPs-Len-Dex targeted and activated the E3 ligase of CRBN, resulting in IKZF1/3 degradation, which inhibited MYC/BCL6-positive DLBCL and maintained the stability of the immune microenvironment. Therefore, this study provided a new monitoring and therapeutic synergetic strategy for DLBCL.

Dose-adjusted EPOCH-R is a safe and well tolerated outpatient treatment regimen in double-hit lymphoma.

Double-hit lymphoma (DHL) is an aggressive subtype of high-grade B-cell lymphoma with inferior prognosis using standard dose chemotherapy. Controversy remains whether more intensive chemotherapy regimens such as dose-adjusted etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R) provide better outcomes in this cohort. To review consecutive cases of DHL treated with DA-EPOCH-R at our institution in comparison to available literature. We conducted a retrospective study of 13 consecutive patients with DHL treated with DA-EPOCH-R at our institution. Primary endpoints included complete response (CR), event-free survival (EFS) and overall survival (OS). CR rate with DA-EPOCH-R in DHL was 69% in our cohort. Median EFS and OS duration was 61 months (95% CI: 41-86 months) and 64 months (95% CI: 42-86 months) respectively. One patient discontinued DA-EPOCH-R due to recurrent febrile neutropenia and there were no treatment or infection-related deaths during the study. This study suggests that DA-EPOCH-R is a well tolerated outpatient regimen for DHL and should be considered for initial treatment in medically fit patients. Further prospective studies are warranted to confirm these findings.

Unusual presentation of extra-nodal double-hit follicular lymphoma: a case report

BackgroundTo the best of our knowledge, this case represents the first report of an extranodal double-hit follicular lymphoma (DH-FL) as an intestinal polypoid lesion.Case presentationA 72-year-old woman presents with constipation. Colonoscopy reveals a sessile polypoid lesion of the colon bearing morphological, immunohistochemical and molecular hallmarks of DH-FL. Complete clinical staging and bone marrow biopsy showed no signs of disseminated disease. The patient, after two years of follow-up is still free of disease confirming the indolent behaviour of this limited lesion.ConclusionsA synoptic view at all the features of the patient and not merely at the molecular hallmarks of a disease are essential to establish the correct clinical approach.

Targeting MYC and BCL2 by a natural compound for "double-hit" lymphoma.

Concurrent translocations of MYC and BCL2 lead to abnormal expression of both oncoproteins, which contribute to the aggressive clinical characteristics of double-hit lymphoma (DHL). An effective therapy for DHL remains an unmet clinical need. In this study, we showed that both Ca2+/calmodulin-dependent protein kinase II δ (CAMKIIδ) and γ (CAMKIIγ) were highly expressed in DHL. Both isoforms of CAMKII stabilize c-Myc protein by phosphorylating it at Ser62, increase BCL2 expression, and promote DHL tumor growth. Inhibition of CAMKIIδ and CAMKIIγ by either berbamine (BBM) or one of its derivatives (PA4) led to the down regulation of c-Myc and BCL2 proteins. BBM/PA4 also exhibited anti-tumor efficacy in DHL cell lines and NSG xenograft models. Altogether, CAMKIIδ and CAMKIIγ appear to be critical for DHL tumor development and are promising therapeutic targets for DHL.

Clinical Features and Immunophenotypes of Double-Hit Diffuse Large B-Cell Lymphoma.

Double-hit (DH) genetics induces a reduction in the complete remission (CR) and, consequently, in poor overall survival (OS) in diffuse large B-cell lymphoma (DLBCL) patients. Unfortunately, DH identification is time-consuming. Here, we retrospectively reviewed 92 newly diagnosed DLBCL patients, stratified them into the DH (n = 14) and non-DH groups (n = 78), and compared their clinical features and outcomes. The results revealed that the DH group had a higher percentage of bulky disease than the non-DH group (64.3% vs. 28.2%; p = 0.013). More patients in the DH group tested positive for double expresser (DE) (50.0% vs. 21.8%; p = 0.044). The three-year OS rates of patients with and without DH were 33.3% and 52.2%, respectively (p = 0.016). Importantly, advance stage and multiple comorbidities were correlated with a high mortality rate in multivariate analysis. Furthermore, by combining DE and the bulky disease, a specificity of 89.7% for DH prediction was achieved. In summary, DH genetics, not DE immunopositivity, could be a factor for an inferior OS in DLBCL. A combination of bulky disease and a positive DE immunophenotype could facilitate DH genetics prediction in newly diagnosed DLBCL patients.

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.

Primary cardiac diffuse large B-cell lymphoma presenting with cardiac tamponade: a case report

BackgroundPrimary cardiac tumours are extremely rare with an autopsy incidence of 0.05%. They can present with a variety of symptoms, including life-threatening arrhythmia and cardiac tamponade. In this case report, we focus on the diagnostic process and management of a primary cardiac lymphoma (PCL) presenting with cardiac tamponade.Case summaryWe report on a 71-year-old male presenting with a large pericardial effusion, tamponade, and a mass in the right atrioventricular groove. Multimodality imaging was performed, including transthoracic echocardiography, computed tomography, magnetic resonance imaging, positron emission tomography, and computed tomography-guided transthoracic biopsy. The final diagnosis of a double-hit diffuse large-cell B-cell lymphoma was made, for which treatment consisting of a combination of chemotherapy and immunotherapy was initiated. Low-dose colchicine was also added to the treatment.DiscussionPrimary cardiac lymphoma remains a very rare diagnosis and this case highlights the need for multimodality imaging and imaging-guided biopsy to differentiate cardiac masses. First-line treatment for PCL remains a combination of chemotherapy with immunotherapy, with the addition of low-dose colchicine to prevent recurrence of malignant pericardial effusion.