Germinal Center B-cell Phenotype Research Articles

Detection of Occurrent Somatic Hypermutation and Associated Gene Expression Profile in Single B Cells

Introduction Somatic Hypermutation (SHM) is an endogenous mutator mechanism in B lymphocytes that preferentially targets the immunoglobulin V(D)J coding region and facilitates antibody affinity maturation. The exact frequency and impact of SHM in individual B cells remains elusive, as previous quantifications only considered cumulative outcomes at population level. We therefore employed single-cell RNA sequencing on primary follicular lymphoma (FL) cells, a B-cell malignancy retaining the germinal center (GC) B-cell phenotype, to identify and quantify individual SHM events. This approach allowed us to detect neovariants, defined as single nucleotide differences that originate from recent SHM events, and to explore the gene expression profiles of the B lymphoma cells with neovariants, particularly focusing on genes and pathways effectuating SHM. Methods We collected and processed samples from 12 follicular lymphoma (FL) and five chronic lymphocytic leukemia (CLL) patients as non-germinal center controls. Single cell whole cDNA libraries were prepared by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with semi-nested amplification using 3′ constant domain primers followed by 10X Genomics prep. Both single cell libraries were sequenced in paired-end mode on Illumina HiSeq 4000. We developed a customized method to preprocess scBCR data designed specifically for highly mutated FL BCR sequences. To ensure data quality and validity of putative neovariants, we applied a comprehensive filtering system, including stringent thresholds in terms of numbers of individual immunoglobulin (IG) mRNA molecules sequenced as defined by their UMI, and reads per position obtained from the median of the estimator's distribution, base quality scores, exclusion of cell doublets, position within the V(D)J region, and proportion of cell-level variants. Based on a reported IG mRNA half-life of 3 h and assuming a first-order decay, the filtering criteria implied that detected neovariants were created between 1 and 7 hours prior to the time of sampling. Results A median of 1112 cells (range: 215-16432) per case were analyzed. 1239 IG neovariants were detected in a total of 334 FL cells in 7 of the 12 FL cases. Neovariants were undetectable in control CLL cells. A remarkably high proportion (95,4%) of neovariants were found within known AID motifs (WRCY, WA, and RCG), suggesting active AID involvement in the hypermutation process. The observed fractions of FL cells carrying recent SHM events indicated initiation of FL pathophysiology at least 11,2 months prior to sampling. To further evaluate the role of AID, we compared gene expression profiles between FL cells with and without neovariants. The results demonstrated not only higher expression of AID but as well of pathways involved in SHM. Mismatch repair (hsa03430, 9 enriched genes with p.adjust < 0.001) and base excision repair (hsa03410, 17 enriched genes with p.adjust = 0.01), together with DNA replication (hsa03030, 14 enriched genes with p.adjust < 0.001), were the three most prominent pathways with upregulated genes. In cells with detectable neovariants, the MLH1/MSH2 genes, associated with the recognition of the deamination process, as well as POLH gene, which is involved in error-prone mutations in MMR, were highly expressed. Overall, we observed a more prominent expression of genes related to MMR than BER in cells with neovariants supported by a higher normalized enrichment score (NES) in MMR (MMR:1.93 and BER:1.6). This suggests not only an active SHM process but also potential interplay between AID and MMR in driving SHM in FL. Conclusions Here we report for the first time quantitative detection of ongoing somatic hypermutation at single cell level in FL. Based on the lifespan of IG mRNA, simultaneous detection of two IG mRNA transcripts differing by one (or a few) nucleotides only within a single cell permits to estimate the timing when this SHM event occurred. The detection of this phenomenon in 58% of FL samples and its association with AID expression suggests that AID-induced mutagenesis might be acting at a much higher rate than expected. Quantification of SHM at the single-cell level should facilitate the understanding of the cellular heterogeneity and dynamics in physiological GC reactions but also may offer novel insights into the AID-dependent shaping of the genomic landscape of germinal center B-cell neoplasms.

Value of GCET1, HGAL (GCET2), and LMO2 in the Determination of Germinal Center Phenotype in Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous disease that is classified into germinal center B-cell (GCB) and non-GCB subtypes, which are prognostically different. The Hans algorithm is the most widely used tool based on CD10, BCL6, and MUM1 expression, but some cases with the non-GCB phenotype are still known to be misclassified. In this study, we investigate the extent to which GCET1, HGAL, and LMO2 protein expressions reflect GCB phenotype together with their roles in determining the GCB phenotype of DLBCL and their contributions to the performance of the Hans algorithm. Sixty-five cases of DLBCL-not otherwise specified, 40 cases of follicular lymphoma (FL), and 19 non-GC-derived lymphoma cases were included in this study. The DLBCL cases were grouped as CD10+ (Group A) or only MUM1+ (Group B), and the remaining cases constituted the intermediate group (Group C). GCET1, HGAL, and LMO2 expressions were evaluated. In the FL group, GCET1, HGAL, and LMO2 were positive in 85%, 77.5%, and 100% of the cases, respectively. Among the non-GC-derived lymphoma cases, all three markers were negative in cases of small lymphocytic lymphoma, plasmablastic lymphoma, peripheral T-cell lymphoma, and anaplastic large cell lymphoma. GCET1 and HGAL were negative in cases of marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). Two of the 3 MZL and 2 of the 4 MCL cases were positive for LMO2. In the DLBCL group, the number of cases with GCET1, HGAL, and LMO2 positivity was 18 (90%), 17 (85%), and 20 (100%), respectively, in Group A and 0 (0%), 2 (13.3%), and 2 (13.3%), respectively, in Group B. Considering these rates, when the cases in the intermediate group were evaluated, it was concluded that 13 cases typed as non-GCB according to the Hans algorithm may have the GCB phenotype. GCET1, HGAL, and LMO2 are highly sensitive markers for determining the germinal center cell phenotype and can increase the accuracy of the subclassification of DLBCL cases, especially for cases that are negative for CD10.

Transformation of FL into DLBCL with a PMBL gene expression signature.

We investigated the clinicopathologic features of 5 follicular lymphomas (FLs) that transformed (tFL) morphologically to diffuse large B-cell lymphomas (DLBCLs) and had a primary mediastinal large B-cell lymphoma (PMBL)-like gene expression profile (tFL-PMBLsig-pos). None of the tFL-PMBLsig-pos cases arose in the mediastinum, all cases tested had a germinal center B-cell phenotype, 20% were CD30+, 60% CD23+, 80% MAL+, 20% CD200+, and 0% CD273/PDL2+. Whole-exome sequencing detected alterations in genes associated with both FL/DLBCL (CREBBP, KMT2C, KMT2D, ARID1A, HIST1 members, and TNFRSF14) and PMBL (JAK-STAT pathway genes, B2M, and CD58). Copy number (CN) analysis detected gains/amplification of REL and STAT6 in 60%, gains of SOCS1 in 40%, and gains of chromosome 16, including IL4R, in 40% of the cases. CN gains/amplification of BCL6 and MYC and loss of TNFRSF14 and TNFAIP3 were identified in 20% of the cases. Three of 5 cases lacked a BCL2 rearrangement. Despite having some features that are less common in DLBCL (MAL and CD23 expression and JAK-STAT activation), these tFL-PMBLsig-pos cases lack the most characteristic CN alteration seen in PMBL (9p24.1 gain/amplification). This cohort expands the biologic heterogeneity of tFL, illustrating a subset with gene expression and some genetic features reminiscent of PMBL, with potential treatment implications that include the use of novel targeted therapies.

Primary Large B-Cell Lymphoma of Immune-Privileged Sites of the Cerebellum: A Case Series and Review of the Literature.

We reported 3 cases of cerebellar IP-LBCL followed at our hospital and reviewed the medical literature to unravel the peculiarities of this poorly studied entity. Analyzing our cases and reviewing the literature, we could collect and study 26 cases of cerebellar IP-LBCL. To the best of our knowledge, this is the largest cohort of such patients currently published. Cerebellar IP-LBCL presents more often in adult females with cerebellum-related focal neurological signs such as ataxia, headache, and nausea. Histological confirmation is mandatory for a correct diagnosis and treatment and all cases feature diffuse large B-cell lymphoma histopathology. Compared to other encephalic IP-LBCL, cerebellar cases seem to include a higher number of cases with germinal center B-cell phenotype and better survival. These differences may be related to a different immune microenvironment and especially immunoregulation that distinguishes the cerebellum from other areas of the CNS.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Polyostotic DLBCL Is Characterized By a NF-Κb Pathway Affecting Molecular Profile and Superior Survival

Introduction Polyostotic diffuse large B-cell lymphoma (DLBCL) is a rare extranodal disease with only multifocal bone localizations that has not extensively been studied. Other known bone DLBCL comprises primary bone (PB-)DLBCL with a single bone localization with(out) locoregional lymphadenopathy and disseminated-DLBCL with ≥1 bone lesion(s) and ≥1 (extra) nodal localization(s). Recently (PMID: 34478526, de Groen et al.), we demonstrated a germinal center B-cell (GCB)-subtype with a centrocyte-like phenotype and a unique corresponding molecular profile, reflecting favorable prognosis for PB-DLBCL compared to non-bone DLBCL with a GCB-subtype. Here, the molecular characteristics of polyostotic-DLBCL were assessed and compared to PB-DLBCL, disseminated-DLBCL, and nodal DLBCL-GCB, using an extensive targeted next-generation sequencing (tNGS) panel. Methods This retrospective multicenter study analyzed 14 polyostotic-DLBCL, 44 PB-DLBCL, 26 disseminated-DLBCL, and 36 nodal DLBCL-GCB cases (selection based on PET/CT-scan), including well-annotated histological and clinical data. Cases were diagnosed with immunohistochemistry following the revised WHO classification of 2016. Cell-of-origin (COO) was determined by Hans' algorithm. Presence of MYC/BCL2/BCL6-rearrangements were identified through fluorescent in situ hybridization (FISH) and break-apart probes. High-grade B-cell lymphomas (double/triple hit) were excluded. As described (PMID: 34478526), tNGS was performed, using an extended panel consisting of 128 (LYMFv2, mostly used) or 52 (LYMFv1) B-cell lymphoma relevant genes. Results Our cohort consisted of 120 patients (78 males, 65%) with a median age at diagnosis of 61 (range 13-91). Half of the polyostotic-DLBCL cases belonged to a GCB phenotype (8/14, 57%), while a GCB phenotype was more frequently identified in PB-DLBCL (31/44, 70%) and disseminated-DLBCL (18/26, 69%). Using tNGS with LYMFv2 and LYMFv1, respectively 57 and 36 cases were successfully analyzed, elucidating (in)activating pathogenic mutations in 124 different genes. In polyostotic-DLBCL, frequent mutations in f.e.CARD11 and MYD88 demonstrated a unique mutational profile affecting the NF-κB pathway characteristic of ABC-genotypes (Figure 1, P=0.018), compared to PB-DLBCL, disseminated-DLBCL and nodal DLBCL-GCB. In contrast, an exclusive immunomodulatory profile with frequent mutations in B2M, IRF8 and TNFRSF14 was identified for PB-DLBCL (P=0.029). Focusing on GCB-associated epigenetic genes (CREBBP, EP300, EZH2, KMT2D, etc.), no significant difference was found between the mentioned DLBCL subgroups. Mutational profiles of disseminated-DLBCL revealed a broad spectrum of abnormalities (BCL2, CREBBP, TNFRSF14, and TP53), partially consistent with either an immunomodulatory or an epigenic genotype. Nodal DLBCL-GCB was specifically characterized by frequent aberrations in BCL2 and MYC and lacked an immunomodulatory or NF-κB activating genotype. Given >1 extranodal localizations, polyostotic-DLBCL is classified as high-risk disease with Ann Arbor stage IV. Compared to disseminated-DLBCL with similar stage III/IV disease, polyostotic-DLBCL demonstrated a superior 3-year overall survival (Figure 2, log-rank, p<0.001, HR: 4.76, CI: 1.4-15.9). Conclusion Polyostotic-DLBCL showed a unique mutational profile with frequent NF-κB-activating mutations, in contrast to PB-DLBCL, disseminated-DLBCL and nodal DLBCL-GCB. Despite this ABC-associated profile of polyostotic-DLBCL and its high Ann Arbor stage, a favorable prognosis was identified especially compared to similar high Ann Abor stage disseminated-DLBCL. To study COO and tumor microenvironment in more detail to evaluate polyostotic-DLBCL as a separate biological disease entity, gene-expression profiling with the BLYM777 panel (PMID: 35454765) will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Special Anatomical Subtypes of Diffuse Large B-Cell Lymphoma Have Distinct Tumor Microenvironments

Background The heterogeneity of diffuse large B-cell lymphoma (DLBCL) and its diverse clinical course encouraged the World Health Organization (WHO) to recognize specific subtypes based on anatomical location. These anatomical subtypes are accompanied by specific mutational profiles and gene-expression patterns. For example, the poor-prognostic primary DLBCL of immune-privileged sites (IP-DLBCL) originating in the central nervous system or testis have frequent NF-κB-activating mutations and a predominantly activated B-cell (ABC) phenotype. In contrast, we recently showed that good-prognostic bone DLBCL (B-DLBCL) have frequent mutations in immune-modulating and epigenetic regulating genes and a germinal center B-cell (GCB) phenotype (PMID: 34478526). We hypothesize that these remarkable anatomical site-specific enrichments for tumors with a distinct molecular signature are associated with different local anti-tumor immune responses. Here, IP-DLBCL and B-DLBCL could be considered opposing ends of a spectrum and we compared the composition of the tumor microenvironment (TME) of these entities to each other and to DLBCL with nodal localization only (nodal-DLBCL). Method This retrospective study used formalin fixed and paraffin embedded biopsy specimens that were selected from clinically, histologically and molecularly well-annotated cases of IP-DLBCL (n=29), B-DLBCL (n=21) and nodal-DLBCL (n=18). All cases were diagnosed according to the revised 2016 WHO classification. High grade B-cell lymphomas (double/triple hit) were excluded. Imaging mass cytometry (Hyperion) was used to define the TME at single-cell resolution and with intact spatial relationships. The panel comprised 41 antibody-markers for deep phenotyping of four major cellular compartments: tumor cells, tumor-infiltrating lymphocytes, myeloid- and stromal cells. Image output was processed to create single-cell data, as described before (PMID: 34196108). Cell phenotyping was performed by supervised clustering after dimension reduction using opt-SNE algorithm (OMIQ software). Cluster abundances were depicted as median (±IQR) per thousand of total cells per patient(‰). Kruskal-Wallis testing was used to determine statistical differences between abundances. Results In total 1,7M single cells (range 7-49k) were obtained and 90 unique cellular phenotypes were defined. Differences in composition of the four cellular compartments were compared between IP-DLBCL and B-DLBCL (Figure 1). The lymphocyte compartment of IP-DLBCL was significantly enriched with granzyme B expressing lymphocytes, mainly cytotoxic T cells (IP 9,4‰ ± 8,8 versus B-DLBCL 2,7‰ ± 3,8; p<0,01), including activation related markers like CCR4 and Ki-67. In contrast, B-DLBCL was characterized by a substantial influx of T cells, more specifically T-helper cells (B-DLBCL 49,9‰ ± 32,5 versus IP 35,4‰ ± 24,6; p<0,01), including T-regulatory cells (B-DLBCL 2,7‰ ± 4,4 versus IP 0,9‰ ± 1,5; p<0,01) and cells expressing ICOS. T-helper 1 cells, expressing TBET, were less abundant (B-DLBCL 0,3‰ ± 0,5 versus IP 1,0‰ ± 0,6; p<0,01). The myeloid compartment was generally increased in B-DLBCL and comprised of HLADR and/or CD11c expressing dendritic cells (B-DLBCL 84,3‰ ± 39,8 vs IP 32,5‰ ± 31,9; p<0,01). In the stromal compartment, a significantly higher expression of activation markers related to the "stromal1" gene signature (PMID: 34597589) in B-DLBCL was identified (B-DLBCL 15,4‰ ± 12,4 vs IP 10,98‰ ± 9,0; p=0,04). Immune checkpoint markers like PD1, LAG3, TIM3, TIGIT and VISTA were generally low in expression within the entire cohort. In nodal-DLBCL both IP- as well as B-DLBCL-like phenotypes were identified. Conclusion This study shows significant differences in the tumor microenvironment of DLBCL subtypes based on anatomical site. In IP-DLBCL, substantial presence of cytotoxic T cells-with activated phenotypes-was identified. In contrast, in bone-DLBCL the TME was generally non-cytotoxic and exhibited immune regulatory elements. Altogether, knowledge on these distinct tumor microenvironments may help understanding heterogeneity between these subtypes and advocates correlation with efficacy of emerging treatments like bispecific antibodies (for the cytotoxic TME) and CAR T-cell therapy (for the non-cytotoxic/immune regulatory TME). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Multi-Scale Modeling Recapitulates the Effect of Genetic Alterations Associated With Diffuse Large B-Cell Lymphoma in the Germinal Center Dynamics

Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin’s lymphoma. It is a germinal center (GC)–derived, aggressive, and heterogeneous disease. Several transcription factors and signaling pathways that play a central role in the progression of the GC reaction and B-cell differentiation have been shown to play an oncogenic role in diffuse large B-cell lymphoma. B-cell lymphoma 6 (BCL6) is a transcriptional repressor that induces the GC B-cell phenotype and blocks plasma cell (PC) differentiation, while interferon regulatory factor 4 (IRF4) and B lymphocyte-induced maturation protein 1 (BLIMP1), a transcriptional promoter, both mediate PC differentiation and exit from the GC (1). Computational models are useful alternatives to trial-and-error experimental investigation. Ordinary differential equation (ODE) models have been used to study different known mechanisms of lymphomagenesis and suggest candidate tumorigenic alterations (2). Furthermore, multi-scale models (MSMs) have been used to study the role of cellular and molecular mechanisms involved in tumor growth (3–6). In this study, we used an existing MSM of PC differentiation in the GC to simulate eight different models with several candidate genetic alterations of the BCL6-IRF4-BLIMP1 regulatory network that lead to transcription factor deregulation and could explain the onset of diffuse large B-cell lymphoma and recapitulate the GC dynamics observed in such conditions. We observed that models with loss of BLIMP1 function (BLIMPloss and BLIMPlossIRFinc) result in an accumulation of B cells in the GC and a block of PC differentiation and thus correctly recapitulate the observed GC and transcription factor dynamics. Models with constitutive activation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-kB) pathway alone and in codominance or co-expression with the enforced BCL6 expression (IRFinc and BCLincIRFinc) result in a decrease of GC B cells and unaltered PC production at early stages of the GC reaction, as observed experimentally. Interestingly, we also found that in IRFinc and BCLincIRFinc models, an increase in PC production could happen at later stages of the GC reaction. Nevertheless, models with enforced BCL6 expression (BCLauto and BCLinc) result in an expansion of GC B cell population and a block in the PC production that was not observed experimentally. Finally, models with loss of IRF4- and BLIMP1-mediated silencing of BCL6 (IRFsil and BLIMPsil) did not affect GC and transcription factor dynamics.

Predictors of Relapse and Survival Following Autologous Stem Cell Transplant in Patients with Diffuse Large B-Cell Lymphoma

Background: Although the majority of patients with diffuse large B-cell lymphoma (DLBCL) can be cured with intensive chemotherapy and rituximab, 30-40% of patients will be refractory to or relapse after first line treatment. For these patients, the current standard of care is salvage chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT). Prior studies have largely examined clinical risk factors associated with higher risk of relapse after ASCT; however, there is limited data integrating both pathologic and molecular features. Thus, we aimed to identify high-risk features associated with relapse and survival after ASCT using a combination of clinical, molecular, pathologic, and transplant characteristics.Methods: We retrospectively analyzed the medical records of all adult patients with DLBCL who underwent ASCT at our two institutions from 2010 to 2020. Patients with primary CNS lymphoma, primary mediastinal B-cell lymphoma, or Burkitt lymphoma were excluded. We analyzed demographics, clinical characteristics, cell of origin (COO) by immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH) testing, and treatment/transplant characteristics. The primary endpoints were 3-year progression-free survival (PFS) and overall survival (OS) from ASCT. The Kaplan-Meier method was used to estimate survival with univariate and multivariate Cox proportional hazards regression performed to identify factors associating with PFS and OS, summarized using hazard ratios (HR) with 95% confidence intervals (CI).Results: A total of 235 DLBCL patients underwent ASCT from 2010 to 2020. Median age at ASCT was 61 years (range: 25-75) and 63% were male. At DLBCL diagnosis, 80% had advanced stage disease, 74% had extranodal involvement, 13% had poor performance status, and 65% had elevated lactate dehydrogenase (LDH). 71 patients (30%) had a prior or concurrent indolent lymphoma diagnosis indicating transformed disease. Of the patients with available COO and molecular data, 115 (60%) had germinal center B-cell (GCB) phenotype by IHC, 10 (6%) had a single MYC rearrangement by FISH, and 35 (22%) had MYC plus BCL2 and/or BCL6 rearrangements (DHL/THL). After first-line treatment, 12% remained refractory and 62% later relapsed at a median of 13 months (range: 1-240). Patients received a median of 2 (range: 1-5) lines of treatment pre-ASCT. At time of ASCT, 66% were in complete response (CR) and 32% were in partial response (PR) by standard of care imaging and response criteria.With median follow-up of 5.2 years from time of ASCT, 98 patients (42%) relapsed and 78 (33%) died. 3-year PFS and OS were 58% (95% CI 51-64%) and 74% (95% CI 67-79%), respectively. In univariate analysis, factors associated with worse PFS and worse OS included 3 or more lines of treatment pre-ASCT (p<0.01 for both) and non-CR at ASCT (p<0.01 for both) (Figure 1A and B). Transformed disease was also associated with worse PFS (p=0.03). In multivariate analysis, non-CR at ASCT remained significant (HR 2.22, 95% 1.26-3.90, p<0.01) for worse OS, along with non-GCB COO (HR 1.81, 95% CI 1.03-3.18, p=0.04) and age >60 at ASCT (HR 1.92, 95% CI 1.06-3.47, p=0.03) (Figure 1C). Stratifying by COO and disease status at transplant, 3-year OS was best in the GCB/CR group (84%, 95% CI 73-90%), while worse but similar in the GCB/non-CR and non-GCB/CR groups (68%, 95% CI 51-80% and 71%, 95% CI 56-83%, respectively) (Figure 1D). The non-GCB/non-CR group had the worst 3-year OS (48%, 95% CI 27-67%). No individual factors beyond CR/non-CR at ASCT were associated with worse 3-year PFS. Notably, DHL/THL patients (77% of whom were in CR at time of ASCT) had similar PFS (p=0.08) and OS (p=0.30) to non-DHL/THL patients, suggesting that response to therapy may be more prognostic than high-risk molecular features alone.Conclusions: This analysis indicated that factors associated with OS after ASCT were disease status at time of transplant and COO, with non-GCB patients not in CR having the poorest outcomes. GCB patients not in CR were still able to be cured by ASCT at a high rate. Molecular rearrangements including DHL/THL were not prognostic, although interpretation may be limited by the modest number of DHL/THL patients. These findings may inform which patients should undergo ASCT, while the highest risk group may be better treated with alternatives including novel targeted agents or chimeric antigen receptor cell therapy. [Display omitted] DisclosuresBachanova: FATE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; KaryoPharma: Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fehniger: Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; OrcaBio: Other; Kiadis: Other; HCW Biologics: Research Funding; Indapta: Other. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding.

PET-CT Quantitative Parameter Delta Suvmax As a Predictor of Early Relapse in Patients with Difuse Large B-Cell Lymphoma

Introduction: About one third of patients with diffuse large B-cell lymphoma (DLBCL) fail frontline treatment or have early relapse. Baseline quantitative parameters derived from positron emission computed tomography (PET/CT) at baseline may predict outcome in DLBCL patients. However, their prognostic value in interim scans is unclear. Recently, the role delta maximum standardized uptake value (ΔSUVmax) has been tested as a predictor of PFS. In this retrospective study we aimed to analyze the role of ΔSUVmax between baseline and interim PET-CT as a predictor of progression of disease within 1 year in DLBCL patients and, to compare this parameter to the standard validated method to evaluate disease response: Deauville scale (DS).Methods: This is a single-center retrospective cohort study from São Paulo, Brazil. We included patients with DLBCL who had baseline (b-PET) and interim (i-PET, after 2-3 cycles of treatment) PET-CTs and who have been treated with 6 cycles of R-CHOP given every 21 days. All PET-CTs were analyzed by the same physician and blinded for the clinical features. Delta maximum standardized uptake value (ΔSUVmax) was calculated by the difference in % between initial PET SUVmax and iPET SUVmax. The objective was to assess if ΔSUV performed better than Deauville scale (DS) in predicting early disease progression. Cut-off values were defined by receiver operating characteristic (ROC) analysis. Analysis was performed with Cox model. Model comparison was based on the lowest AIC.Results: A total of 83 patients with DLBCL were available for analyses. The median age was 60 years and 66% of patients had a germinal center B-Cell (GCB) phenotype. With median follow-up of 15 months, patients with a cut-off ΔSUVmax <85% had a higher risk of disease relapse (49% vs 2%, p<0.001) at 12 months (figure 1). Patients with DS >3 in the iPET were also at a higher risk of disease progression (p<0.001) (figure 2). The model with ΔSUVmax was better adjusted (AIC=86) than the one with DS (AIC=92). The median maximum uptake at baseline PET/CT, MYC and/or BCl2 expression were not predictive of relapse at 12 months. International prognostic index (IPI) was also predictive of relapse (p=0.003), but again performed poorer than ΔSUVmax (AIC=99).Discussion/Conclusion: We showed that ΔSUVmax quantitative PET-CT model has superior discriminatory power than the qualitative DS model and the clinical IPI. Although we have a small sample, this finding corresponds with PETAL trial, which also showed that the ΔSUVmax method is superior to the commonly used Deauville five-point scale to identify patients at high risk of treatment fails. Although, the ΔSUVmax cut-off value calculated in our study was 85%, while in the PETAL study a reduction off 66% in the baseline SUV was the considered cutoff. Maybe, combing ΔSUVmax method in a with DS and IPI can improve prognostic stratification and early identification of high risk DLBCL. The ΔSUVmax is an easily reproducible method, that can be easily assessed by PET-CT, differing from other quantitative measures like the total metabolic tumor volume. In conclusion, further validation in a large sample of patients should be performed before the incorporation of quantitative PET-CT methods in clinical practice however it is a promising prognostic marker. [Display omitted] DisclosuresPerini: Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Astra Zeneca: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau.

High-Grade B-Cell Lymphoma, Not Otherwise Specified (HGBL, NOS): Characteristics, Treatment, and Outcomes from 17 Academic US Centers

High-Grade B-Cell Lymphoma, Not Otherwise Specified (HGBL, NOS): Characteristics, Treatment, and Outcomes from 17 Academic US Centers

Frequent mutated B2M, EZH2, IRF8, and TNFRSF14 in primary bone diffuse large B-cell lymphoma reflect a GCB phenotype

AbstractPrimary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma subtype. This retrospective study elucidates the currently unknown genetic background of a large clinically well-annotated cohort of DLBCL with osseous localizations (O-DLBCL), including PB-DLBCL. A total of 103 patients with O-DLBCL were included and compared with 63 (extra)nodal non-osseous (NO)-DLBCLs with germinal center B-cell phenotype (NO-DLBCL-GCB). Cell-of-origin was determined by immunohistochemistry and gene-expression profiling (GEP) using (extended)-NanoString/Lymph2Cx analysis. Mutational profiles were identified with targeted next-generation deep sequencing, including 52 B-cell lymphoma-relevant genes. O-DLBCLs, including 34 PB-DLBCLs, were predominantly classified as GCB phenotype based on immunohistochemistry (74%) and NanoString analysis (88%). Unsupervised hierarchical clustering of an extended-NanoString/Lymph2Cx revealed significantly different GEP clusters for PB-DLBCL as opposed to NO-DLBCL-GCB (P < .001). Expression levels of 23 genes of 2 different targeted GEP panels indicated a centrocyte-like phenotype for PB-DLBCL, whereas NO-DLBCL-GCB exhibited a centroblast-like constitution. PB-DLBCL had significantly more frequent mutations in four GCB-associated genes (ie, B2M, EZH2, IRF8, TNFRSF14) compared with NO-DLBCL-GCB (P = .031, P = .010, P = .047, and P = .003, respectively). PB-DLBCL, with its corresponding specific mutational profile, was significantly associated with a superior survival compared with equivalent Ann Arbor limited-stage I/II NO-DLBCL-GCB (P = .016). This study is the first to show that PB-DLBCL is characterized by a GCB phenotype, with a centrocyte-like GEP pattern and a GCB-associated mutational profile (both involved in immune surveillance) and a favorable prognosis. These novel biology-associated features provide evidence that PB-DLBCL represents a distinct extranodal DLBCL entity, and its specific mutational landscape offers potential for targeted therapies (eg, EZH2 inhibitors).

Screening Strategy for Detecting Double-Hit Lymphoma in a Resource-Limited Setting

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements [double-hit lymphomas (DHL)] are aggressive lymphomas. Current literature recommends fluorescent in-situ hybridization analysis (FISH) in all cases of diffuse large B-cell lymphoma (DLBCL) to identify cases of DHL. However, this approach is not feasible in a resource-limited setting. We analyzed cases of de novo high-grade B-cell non-Hodgkin lymphoma using histomorphology, immunohistochemistry and FISH to identify which cases need to undergo FISH testing in a resource-limited setting. Cases of de novo high-grade B-cell non-Hodgkin lymphoma that included DLBCL, not otherwise specified and B-cell lymphoma unclassifiable (BCLU) with features intermediate between DLBCL and Burkitt lymphoma diagnosed over a period of 5 years were analyzed by Hans algorithm, MYC, BCL2, and Ki67. MYC, BCL2, and BCL6 break apart FISH was tested in selected cases. One hundred and nine cases were obtained, of which 102 had DLBCL morphology and 7 had BCLU/blastoid morphology. BCL2 expression was noted in 48 cases (44%), MYC in 33 cases (30.3%) and MYC/BCL2 co-expression in 24 cases (22%). FISH testing could be done in 42 consecutive cases, of which 5 cases had MYC and BCL2 co-rearrangement (11.9%) (double-hit) and 2 cases showed rearrangement for only MYC (4.7%) (single-hit). Single-hit lymphoma/DHL showed significant independent positive correlation with BCLU/blastoid morphology, CD10 expression, germinal center B-cell phenotype, and MYC/BCL2 co-expression. The sensitivity and specificity of each parameters include BCLU/blastoid morphology (42% vs. 94%), CD10 positive (50% vs. 88%), germinal center B-cell phenotype (57% vs. 82%), MYC/BCL2 co-expression (85% vs. 80%). Selected candidates for FISH (any one of the above parameters) using this strategy showed a sensitivity and specificity of 100% and 68%, respectively (P=0.001). We propose a highly sensitive screening strategy for detection of MYC/BCL2 rearrangement in high-grade B-cell lymphoma in a resource-limited setting (pending validation in a larger cohort).

Cell of Origin Impacts Δsuvmax Cutoff for Prediction of Relapse in Patients with Diffuse Large B-Cell Lymphoma

Introduction:The role of interim positron emission tomography (PET) as a predictor for progression free survival (PFS) in patients with diffuse large B-cell lymphoma (DLBCL) remains controversial. The incorporation of volumetric and quantitative parameters aims to improve the identification of patients with high risk for treatment failure. Recently, the role of ΔSUVmax, defined as the reduction between the maximum uptake at initial PET and interim-PET (iPET) has been tested as a predictor for PFS. In most studies, a cutoff of 66% reduction successfully identified patients at high risk for treatment failure. We aimed to analyze the role of ΔSUVmax in predicting relapse in a cohort of Brazilian patients with DLBCL, and the impact of COO in this analysis. Methods:We retrospectively analyzed a cohort of patients with DLBCL from Sao Paulo, Brazil. All diagnosis were made by the same hemepathologists, and COO was defined using Hans algorithm. PET scans were obtained at baseline and after 2-3 cycles (iPET) of CHOP-like chemoimmunotherapy. All PET-CTs were read by the same physician, blinded for COO and patient's clinical features. ΔSUVmax was calculated by the difference in % between initial PET SUVmax and iPET. Initially, a cutoff of 66% reduction was used for analysis, but due to low number of patients not achieving a 66% reduction in the activated B-cell (ABC) subgroup, the optimal cutoff was selected based on the ROC curve. ResultsA total of 54 patients were available for analysis. Median age of all patients was 61 years old, and 61% of patients had a germinal center B-Cell (GCB) phenotype. The median maximum uptake at baseline was 31 for the germinal B-cell (GCB) and 34.1 for the activated B-Cell (ABC) phenotype. For all patients, ΔSUVmax &amp;lt;66 was strongly related to relapse (HR=0.065, p=0.0005). In the GCB cohort (n=33), ΔSUVmax &amp;lt;66 was also strongly related to relapse (H=0.02, p=0.0004). In the ABC cohort (n=21), only one patient failed to achieve ΔSUVmax&amp;gt;66. A ROC curve identified a ΔSUVmax&amp;gt;90 as a better cutoff for relapse, and patients not achieving 90% reduction had a higher risk for relapse (p=0.03). MYC/Bcl-2 expression was also compared, and ΔSUVmax &amp;lt;66 was related to events in both double-expressors (DE, p=0.0003) and in non-DE(p=0.036) ConclusionA reduction &amp;lt;66% in SUVmax from baseline PET to iPET was strongly related to the risk of relapse in a cohort of 54 patients with DLBCL. However, the cutoff of 66% was only significant in patients with GCB DLBCL, mostly due to the low number of ABC patients who did not reach a 66% in SUVmax. For ABC patients, a ΔSUVmax&amp;gt;90 improved the identification of patients at high risk for relapse. Larger cohort of patients are needed to validate our exploratory findings. Figure Disclosures No relevant conflicts of interest to declare.

Molecular Subtypes and Genomic Profile of Primary Central Nervous System Lymphoma.

Primary central nervous system lymphomas (PCNSL) are aggressive non-Hodgkin lymphomas affecting the central nervous system (CNS). Although immunophenotyping studies suggested an uniform activated B-cell (ABC) origin, more recently a spectrum of ABC and germinal center B-cell (GC) cases has been proposed, with the molecular subtypes of PCNSL still being a matter of debate. With the emergence of novel therapies demonstrating different efficacy between the ABC and GC patient groups, precise assignment of molecular subtype is becoming indispensable. To determine the molecular subtype of 77 PCNSL and 17 secondary CNS lymphoma patients, we used the NanoString Lymphoma Subtyping Test (LST), a gene expression-based assay representing a more accurate techniqueof subtyping compared with standard immunohistochemical (IHC) algorithms. Mutational landscapes of 14 target genes were determined using ultra-deep next-generation sequencing. Using the LST-assay, a significantly lower proportion (80% vs 95%) of PCNSL cases displayed ABC phenotype compared with the IHC-based characterization. The most frequently mutated genes included MYD88, PIM1, and KMT2D. In summary, we successfully applied the LST-assay for molecular classification of PCNSL, reporting higher proportion of cases with GC phenotype compared with IHC analyses, leading to a more precise patient stratification potentially applicable in the diagnostic algorithm of PCNSL.

Molecular features of a large cohort of primary central nervous system lymphoma using tissue microarray

AbstractThe objective of this study was to evaluate the distribution and prognostic impact of a broad range of molecular attributes in a large cohort of immunocompetent patients with primary central nervous system lymphoma (PCNSL) by using tissue microarray. Patients diagnosed with PCNSL were initially identified in the BC Cancer Lymphoid Cancer clinical and pathology databases. Tissue microarrays were constructed by using archival formalin-fixed paraffin-embedded diagnostic biopsy tissue. Immunohistochemistry and fluorescent in situ hybridization studies were performed. A total of 115 patients with PCNSL with diffuse large B-cell lymphoma (DLBCL) histology were identified. The majority of cases (≥75%) had a non–germinal center B-cell phenotype according to immunohistochemistry algorithms, but cell of origin did not affect progression-free or overall survival. MYC (40%), BCL2 (75%), and programmed death-ligand 1 (29%) protein expression were common, but their corresponding gene rearrangements were rare (≤1% each), suggesting that alternate mechanisms were driving expression. There were no dual rearrangements involving MYC and BCL2. Only 22% of cases had membranous expression of major histocompatibility complex class II, suggesting a mechanism for escape from immune surveillance. Epstein-Barr virus–encoded RNA was positive in 1 immunocompetent patient. BCL6 protein expression (77%) and BCL6 rearrangements (31%) were frequent; the latter was the only factor associated with a poor prognosis in the overall cohort and in the subgroup of 52 patients treated with high-dose methotrexate–based regimens. This large population-based study shows that prominent molecular features of PCNSL are unique and different from those of systemic DLBCL. These results may better inform drug development in PCNSL.

High Frequencies of Mutated EZH2 and IRF8 and Other Epigenetic Genes in Primary Bone Lymphomas Are Indicative of GCB-Phenotype

Introduction Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma comprising 1-2% of all malignant lymphomas. This study aims to elucidate the genetic background of a homogeneous cohort of PB-DLBCL. Methods This retrospective study consists of primary DLBCL-patients with bone localization(s) of which pretreatment fresh frozen or formalin-fixed paraffin-embedded bone tissue samples were available. Patients were diagnosed (2003-2019) at Leiden University Medical Center (LUMC), a center of expertise for bone tumors, Amsterdam University Medical Center (AUMC), Erasmus MC and affiliated Dutch hospitals. Based on strict definitions regarding radiological assessment of anatomical disease localizations at diagnosis three subgroups were categorized: solely osseous involvement (single or multiple bone lesions; PB-DLBCL), osseous involvement and locoregional lymphadenopathy (locoregional disease), and osseous and (multiple) extra-osseous localizations (disseminated disease). Cell-of-origin (COO) was determined by immunohistochemistry (BCL6, CD10, and MUM1) and classified according to the Hans' algorithm. Additionally, COO was confirmed with NanoString and the Lymph2Cx assay (Scott et al., Blood 2014), in a subset of patients. With similar procedures (Vermaat et al., Haematologica 2019), molecular profiles were determined with an in-house developed and validated targeted next-generation sequencing (tNGS) panel, comprising 52 DLBCL-specific genes, for sequencing with the Ion S5TM System. Obtained results were compared to sequencing data of (1) an independent 'in-house' cohort of 23 primary GCB (Germinal Center B-Cell)-DLBCL patients without bone localization ('non-osseous') and (2) pooled data of 651 GCB-DLBCL patients from literature (Chapuy et al., Nature Medicine 2018, Karube et al., Leukemia 2018, Reddy et al., Cell 2017, Schmitz et al., NEJM 2018). Results Our cohort contained 56 patients (males, N=33, (59%)) with a median age at diagnosis of 62 years (range 13-92). Twenty-four patients had PB-DLBCL (45%), 8 had locoregional disease (14%), and 23 had disseminated disease (41%). In general, immunohistochemistry and Lymph2Cx identified a GCB subtype for the majority of all DLBCL with bone localizations (Figure-1A) and these results for the hitherto unperformed cases will follow shortly. tNGS identified 48 genes with 'pathogenic' mutations, with on average four mutated genes per patients (range 0-10; Figure-1A). Overall, high mutation frequencies were observed in TNFRSF14 (33%), KMT2D (27%), EZH2 (25%), CREBBP (22%), B2M (22%), and TP53 (20%) in DLBCL with bone localizations and mainly genes involved in epigenetic machinery. In PB-DLBCLs, high frequency of mutated EZH2 (38%) and IRF8 (25%) were identified. Both are epigenetic genes that regulates tumor suppression and type I interferon, respectively. In four PB-DLBCLs EZH2 and IRF8 were concomitantly mutated. Locoregional disease showed a similar molecular profile as PB-DLBCL. Association with clinical characteristics will be performed shortly. Compared to our cohort of non-osseous GCB-DLBCL (Figure-1B) and pooled data of GCB-DLBCL in large sequencing studies (Figure-1C), EZH2 (Chi-square; P=0.046 and P=0.005, respectively) was significantly more frequently mutated in PB-DLBCL, though IRF8 did not attain this significance (Chi-square; P=0.121 and P=0.111, respectively; Figure-1D). Conclusion This study is the first that provides integrative analyses of immunohistochemistry, Lymph2Cx, and tNGS of a homogeneous cohort of PB-DLBCL, demonstrating the importance of epigenetic genes in lymphomagenesis. In contrast to (non-osseous) GCB-DLBCLs, the molecular profile of PB-DLBCL is characterized by significantly frequent mutations in EZH2 and frequent mutations in IRF8 and other epigenetic genes, which is indicative for a GCB phenotype (Scherer F. et al., Sci Transl Med 2016) and supported by immunohistochemistry and Lymph2Cx data. These results suggest that PB-DLBCL is a specific DLBCL-entity with a unique molecular profile and provide a rationale for exploration of novel targeted treatment with EZH2 (and IRF8) inhibitors for PB-DLBCL patients. Disclosures Lugtenburg: Genmab: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; Celgene: Consultancy, Honoraria; Janssen Cilag: Honoraria; Takeda: Consultancy, Honoraria, Research Funding. Kersten:Gilead: Honoraria; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda Oncology: Research Funding; Miltenyi: Honoraria; Kite Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Burkitt-like lymphoma with 11q aberration: a germinal center-derived lymphoma genetically unrelated to Burkitt lymphoma.

Burkitt-like lymphoma with 11q aberration is characterized by pathological features and gene expression profile resembling those of Burkitt lymphoma but lacks the MYC rearrangement and carries an 11q-arm aberration with proximal gains and telomeric losses. Whether this lymphoma is a distinct category or a particular variant of other recognized entities is controversial. To improve the understanding of Burkitt-like lymphoma with 11q aberration we performed an analysis of copy number alterations and targeted sequencing of a large panel of B-cell lymphoma-related genes in 11 cases. Most patients had localized nodal disease and a favorable outcome after therapy. Histologically, they were high grade B-cell lymphoma, not otherwise specified (8 cases), diffuse large B-cell lymphoma (2 cases) and only one was considered as atypical Burkitt lymphoma. All cases had a germinal center B-cell signature and phenotype with frequent LMO2 expression. The patients with Burkitt-like lymphoma with 11q aberration had frequent gains of 12q12-q21.1 and losses of 6q12.1-q21, and lacked common Burkitt lymphoma or diffuse large B-cell lymphoma alterations. Potential driver mutations were found in 27 genes, particularly involving BTG2, DDX3X, ETS1, EP300, and GNA13. However, ID3, TCF3, or CCND3 mutations were absent in all cases. These results suggest that Burkitt-like lymphoma with 11q aberration is a germinal center-derived lymphoma closer to high-grade B-cell lymphoma or diffuse large B-cell lymphoma than to Burkitt lymphoma.

Targeting BCL2 with venetoclax is a promising therapeutic strategy for "double-proteinexpression" lymphoma with MYC and BCL2 rearrangements.

The so-called “double-hit” and “double-protein-expression” lymphoma with MYC and BCL2 rearrangements is a rare, mature B-cell neoplasm characterized by a germinal center B-cell phenotype, abundant protein expression of MYC and BCL2, rapid disease progression, and a poor prognosis. In this study, we showed the potential benefit of the BCL2 inhibitor venetoclax in the treatment of this disease. Immunohistochemical studies of the lymphoma tissues confirmed that overexpression of MYC and BCL2 was observed more frequently in this subtype than in other germinal center B-cell-like diffuse large B-cell lymphomas. In contrast, another pro-survival protein MCL1 was less expressed in this subtype, even when compared with its expression in the non-“double-hit” and “double-protein-expression” type. Furthermore, in vitro studies using two “double-hit” and “double-protein-expression” lymphoma-derived cell lines, Karpas231 and OCI-Ly8, clearly showed that a low concentration of venetoclax, but not the MCL1 inhibitor S63845, was sufficient to induce apoptosis in the two lines, compared with in other germinal center B-cell-derived cell lines, BJAB and SU-DHL10. These results indicate that the survival of this type of lymphoma depends predominantly on BCL2 rather than on MCL1. Unexpectedly, we found that venetoclax not only disrupts the interaction between BCL2 and the pro-apoptotic protein BIM, but also leads to dephosphorylation of BCL2 and further downregulates MCL1 protein expression, probably through modulation of the protein phosphatase 2A B56α activity in Karpas231 and OCI-Ly8. Indeed, a low concentration of venetoclax induced substantial apoptosis in the primary lymphoma cells, regardless of high protein expression of MCL1 associated with venetoclax resistance. Venetoclax clearly triggers the signal transduction related to BCL2 and MCL1 in “double-hit” and “double-protein-expression” lymphoma cells.