Germinal Center B-cell-like Subtype Research Articles

High Level of IL-16 Secreted By Non-GCB DLBCL Facilitates Tumor Growth Though the Tumor-Promoting and Immunosuppressive Tumor Microenvironment

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is a highly heterogeneous non-Hodgkin's lymphoma. Based on cell origin, DLBCL is classified into two main molecular subtypes: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. ABC subtype is more aggressive and associated with a worse prognosis than GCB subtype. Previous studies have shown that cytokines play an important role in the growth of ABC subtypes, and IL-16 was supposed to be highly expressed in ABC-DLBCL, however it has not been explored in depth in DLBCL. In this study, we broadly assessed the expression and role of IL-16 on DLBCL. Method: Serum and tissues samples from patients with DLBCL and whole blood from healthy donors were collected. The characterization of protein expression, mRNA expression, and levels of cytokines in cells and tissues was detected by Western blotting, IHC/IHF, PCR, ELISA, RNA-seq, and Flow cytometry. Cells experiments such as cell proliferation, cell apoptosis, cell cycle, cell migration, and ADPC were detected by CCK-8, Flow cytometry, and Transwells. Gene knockdown or overexpression was conducted by lentiviruses and then verified by Western blotting and ELISA. We established subcutaneous tumors (human DLBCL cells and murine DLBCL cells) in BALB/c mice, BALC/c nude mice, and NOD SCID mice for the tumor size or the therapeutic evaluation. Results: First, we found that IL-16, secreted by the cleavage of active caspase-3 from the lymphoma cells under tumor burden, was highly expressed in ABC-DLBCL compared to GCB-DLBCL. The serum IL-16 levels were positively correlated with unfavorable prognosis in DLBCL patients. The dynamic change of serum IL-16 levels accurately predicted the treatment response in patients with DLBCL who received R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) or anti-CD19 Chimeric Antigen Receptor (CAR) T cells. Interestingly, bioactive IL-16 showed rare effect on DLBCL cells in vitro, but significantly promoted their growth in vivo. Mechanically, tumor-derived bioactive IL-16 promoted the migration of monocytes via CD4+ receptor, thereby inducing the enrichment of monocytes-derived tumor-associated macrophages in the tumor microenvironment (TME) of DLBCL, which accelerated the tumor growth primarily by expressing cytokines such as IL-6, inducing angiogenesis, and exerting immunosuppressive effects through the expression of cytokines like IL-10; thus, conferring growth advantage to the ABC- DLBCL. To translate the findings into clinical application, we design a novel treatment strategy to overcome the poor outcomes in ABC-DLBCL patients with high IL-16 expression by combination of high-dose cyclophosphamide (CTX) and the bispecific antibody targeting CD20 and CD47 (IMM0306). IMM0306 effectively reversed the tumor-promoting effects of macrophages in subsets of ABC-DLBCL, while CTX induced IL-16 and increased the infiltration of macrophages in the TME. The increasing effector cells/tumor cells ratio further improved the antitumor effect of IMM0306. The combination of high-dose CTX and IMM0306 was confirmed to significantly inhibit the growth of ABC-DLBCL with high IL-16 expression. A clinical trial is being designed to evaluate the potential for clinical applications. Conclusion: In conclusion, this study provides insight into the expression and role of IL-16 in DLBCL. IL-16 is proposed to be highly expressed in ABC-DLBCL, and serum IL-16 indicate the tumor burden, treatment response, and clinical prognosis of DLBCL patients. IL-16 may be a key driver of the TME of DLBCL, which can confer growth advantage to the ABC-DLBCL. Our data provides a theoretical basis for future clinical treatment. A novel treatment regimen (IMM0306 and CTX) could attenuate the pro-oncogenic roles of IL-16 to prevent ABC-DLBCL progression.

CT Based Tumor Radiomics with Machine Learning Classifiers for Molecular Subtyping of Diffuse Large B Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBL) has been classified into two distinct molecular subtypes based on their cell of origin- namely germinal center B-cell-like (GCB) and activated B-cell-subtype (ABC) based on Han's algorithm. These two entities are also prognostically distinct. We assessed CT based tumor radiomic features using machine learning classifiers to distinguish the two molecular subtypes. One hundred one patients were accrued in the study after institutional ethics committee approval. GCB subtype was diagnosed in 59 while 42 patients were ABC subtype. Lesion with maximum SUV was delineated using a semi-automated segmentation tool (3D slicer) and radiomic features were extracted using Pyradiomics. A total of 1030 features were extracted including Shape features, first, second, third, higher order and wavelet-based features. Recursive feature elimination was used for feature selection and optimal 10 features were selected. Five machine learning (ML) tools were used to build a model to sub- classify the two molecular subtypes. (Table -1) Model performance was assessed using Accuracy, Precision, Recall, F1 score and Area under curve of receiver operating characteristic curve. The models were constructed by two methods.1) A 5-fold internal cross validation strategy & 2) splitting the data into training (70%) and validation set (30%). Recursive feature elimination using a random forest algorithm method was used to select 7 radiomic features. These 7 features were then used to build the model using the 5 ML classifiers. Of the five classifiers, Random Forest Classifier (RFC) using a 70:30 training: test strategy was the best performer among all the ML models, with the highest accuracy of 80%, AUC of 0.87, recall(sensitivity), f1score(specificity) & precision (Positive Predictive value; PPV) or precision of 82% respectively. When an internal 5-fold cross validation strategy was used, RFC again performed better than other ML classifiers with an accuracy of 75%, AUC of 0.80, recall of 79%, PPV of 77% and specificity of 79%. The machine-learning based Radiomics features extracted from pre-treatment CT images can provide a simple and non-invasive method for the prediction of DLBL molecular subtypes with favorable predictive accuracy. The Random Forest Classifier was the most accurate in distinguishing GCB subtype from the ABC subtype.

CAR-T Therapy in Patients with Relapsed or Refractory Diffuse Large B Cell Lymphoma: Real World Outcomes Based on Tumor Biology

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30-40% of NHL cases with an incidence of 7 cases per 100,000 persons per year (Susanibar-Adaniya, 2021). There are two major subtypes of DLBCL based on the cell of origin (COO), germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Around 10-15% of cases are unable to be classified and are termed not otherwise specified (NOS). GCB is generally associated with a more favorable prognosis, showing 3-year progression free survival (PFS) rates of approximately 75% compared to 40-50% for ABC (Sehn, 2021). Chimeric antigen receptor (CAR)-T cell therapy has shown significant responses in relapsed or refractory diffuse large B-cell lymphoma (DLBCL).These treatments have shown objective response rates (ORR) of 52-83% and complete response (CR) rates of 39-58% in relapsed or refractory DLBCL (Abramson, 2020; Locke, 2019; Schuster, 2019; Schuster, 2021). While the CAR-T studies noted the number of patients in the GCB and ABC subtypes, differences in outcomes stratified by subtypes are less well defined. Methods: This is a retrospective, multi-center (Utah, Michigan, North Carolina) study evaluating outcomes of patients with DLBCL treated with CAR-T cell therapies. The primary outcome of this study is PFS at 12 months based on tumor biology (ABC, GCB, NOS). Secondary outcomes include PFS at 6 months, PFS, overall survival (OS) at 6 months, OS at 12 months, OS, and best response to treatment. Kaplan-Meier curves were created for OS and PFS stratified by DLBCL subtype. Patient characteristics and best response to treatment were summarized stratified by DLBCL subtype and compared by Wilcoxon Rank Sum Test or Chi-square test. Results: A total of 99 patients were included in this study. Of these, most were GCB subtype (n=50), followed by NOS (n=39), and ABC (n=10). There was no difference is baseline characteristics between each of these groups (Table 1). The median age was 65 years old, 87% were white, and 62% were male. Patients had received a median of 3 prior lines of treatment prior to CAR-T. The median PFS was not achieved for the ABC subtype, 4.6 months for the GCB subtype, and 3.5 months for those who were NOS (p=0.34) (Figure 1). Median OS was 14.8 months, 13.4 months, and 8 months for ABC, GCB, and NOS, respectively (p=0.083). The complete response rates were: ABC subtype (50%), compared to GCB (32%), and NOS (26%) groups (p=0.34). Progressive disease following CAR-T treatment rates were: NOS (51%), compared to ABC (20%) and GCB (32%) groups (p=0.34). Discussion: The data from this retrospective, multi-center study suggests there may be a trend towards a greater PFS benefit for those with the ABC subtype compared to the GCB and NOS groups. The ABC subtype also showed a numerically higher percentage of complete responses compared to the other groups, but neither were statistically significant differences. The limited number of patients in the ABC group limits the ability to determine a meaningful result currently. There are several limitations of this study. There was a limited sample size in this study, and as a result, statistical significance was unable to be achieved in this analysis. In addition, the percentage of patients in the NOS group in this study (~39%) was much higher than the number reported in the literature (~10-15%). This could be due to the retrospective nature of the study making it difficult to obtain cell of origin information from the time the diagnosis was made and leading to a patient being classified as NOS when they may have had a GCB or ABC subtype. Our study suggests a potential difference seen to treatment with CAR-T therapy based on the tumor biology of the disease. Future studies with a larger cohort to elucidate a difference in CAR-T therapy responses by cell of origin in DLBCL are warranted as this may influence patient treatment selection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LncRNA OR2A1-AS1 index predicts survival in germinal center-like diffuse large B-cell lymphoma.

BackgroundDiffuse large B‐cell lymphoma (DLBCL) is a highly aggressive form of non‐Hodgkin lymphoma. Long noncoding RNA (lncRNA) has been evaluated as prognostic markers in various carcinomas. However, the prognostic value of the lncRNA index in DLBCL has not been fully understood. Hence, this study aimed to identify the prognostic value of lncRNA olfactory receptor family 2 subfamily A member 1‐antisense RNA 1 (OR2A1‐AS1) in DLBCL.MethodsThe Gene Expression Omnibus (GEO) database was used to obtain the GSE97336 dataset comprising lncRNA expression profiles. Quantitative reverse transcription polymerase chain reaction (QRT‐PCR) was conducted to evaluate the expression of OR2A1‐AS1 in 98 cases of DLBCL.ResultsOR2A1‐AS1 expression was considerably reduced in DLBCL patients, reduced OR2A1‐AS1 expression was linked to a shorter overall survival (OS) and progression‐free survival (PFS) in DLBCL patients, especially those with the germinal center B‐cell‐like subtype (GCB). Multivariate analysis (MVA) revealed that the OR2A1‐AS1 index had prognostic significance. Patients with low OR2A1‐AS1 expression have a poor prognosis.ConclusionsOR2A1‐AS may represent an effective predictor of patients' outcomes with DLBCL.

Expression of programmed cell death ligand-1 protein in germinal center B-cell-like and non-germinal center B-cell-like subtypes of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma worldwide. However, there is still limited information on the expression of programmed cell death ligand-1 (PD-L1), a type 1 transmembrane protein of immunoglobulin B7/CD28 in the DLBCL subtypes. The present study aimed to identify the expression of PD-L1 in germinal center B-cell-like (GCB) subtype and non-germinal center B-cell-like (non-GCB) subtype of DLBCL. A total of 40 patient samples (formalin-fixed paraffin-embedded tissues) consisting of 20 cases of GCB subtype and 20 cases of non-GCB subtype of DLBCL were examined. The PD-L1 protein expressions were evaluated by using immunohistochemical staining in the tumor cells. The results showed a statistically significant difference (P=0.003) between the expression of PD-L1 in the GCB subtype and the non-GCB subtype of DLBCL. PD-L1 expression in the tumor cells were observed in 13 cases (65%) of non-GCB subtype and in three cases (15%) of the GCB subtype of DLBCL. In conclusion, it was found that the expression of PD-L1 protein in the tumor cells of the non-GCB subtype of DLBCL was significantly higher as compared with the tumor cells of the GCB subtype of DLBCL.

Systematic Analysis of the Prognostic Impact of Somatic Mutations in Diffuse Large B-Cell Lymphoma (DLBCL) with Evaluation of Cell-of-Origin Dependence: Results from the Phase 3 GOYA Trial in Previously Untreated DLBCL

Introduction: Diffuse large B-cell lymphoma (DLBCL) represents a biologically and clinically heterogeneous diagnostic category with well-defined cell-of-origin (COO) subtypes, each arising from different stages of normal B-cell development. While the prognostic impact of the distinct COO subtypes has been confirmed in several studies, including the GOYA trial (NCT01287741; Vitolo et al. J Clin Oncol 2017), the impact of the key genomic alterations and their dependence on COO are less clear. Using data from the GOYA study, the largest prospective randomized trial in previously untreated DLBCL, we characterized the mutational profile of DLBCL by targeted next-generation sequencing (NGS), and evaluated the prognostic impact of somatic mutations and its relation to COO.Methods: Diagnostic formalin fixed paraffin-embedded tissue biopsies collected from patients (pts) with previously untreated DLBCL were tested at central laboratories. COO was assessed using the NanoString Research Use Only Lymphoma Subtyping (LST) gene expression assay (NanoString Technologies, Inc., Seattle, WA, USA). COO was evaluable in 933 pts (66% of the GOYA intent-to-treat [ITT] population of 1418 pts). Targeted DNA NGS was performed using the Foundation One Heme™ platform (all coding exons of 465 genes, select rearrangements in 31 genes) in 499 pts (35% of the ITT population). NGS and COO were available for 482 pts (34% of the ITT population). Only genetic alterations/mutations (single nucleotide variants [SNVs], copy number abnormalities [CNAs], chromosomal translocations) with known somatic and functional status were included in the statistical analysis, which was conducted as previously described (Frampton et al. Nat Biotechnol 2013). Multivariate Cox-regression was used to evaluate the prognostic effect of gene alterations with multiple testing adjustment using the Benjamini-Hochberg false-discovery rate (FDR) procedure (“significance” <5% FDR).Results: Baseline patient and disease characteristics were similar between pts with COO and/or NGS available, and the overall ITT population, except for geographic region and race. According to COO, 272/482 (56.4%) pts had germinal center B-cell-like (GCB), 78/482 (16.2%) unclassified, and 132/482 (27.4%) activated B-cell-like (ABC) DLBCL, which was similar to the overall COO distribution in GOYA. Of the 465 genes analyzed, we identified 59 genes with mutations (13%) occurring in at least 10/499 pts (≥2% prevalence), with an additional 334/465 genes with mutations occurring in at least 1 pt. The median number of gene alterations per pt was 6 (range 0-17). Overall, 3% of pts had no identified mutation and 93% of pts had multiple alterations (≥2 gene mutations). SNVs were the most common mutation type, while CNAs were specific to a few genes, including CDKN2A/B and REL (Figure 1A). Genes with significant differences in mutation rate (<5% FDR) between the GCB and ABC subtypes are shown in Figure 1B.On multivariate analysis, only alterations in BCL2 were associated with shorter progression-free survival (PFS) independent of COO, International Prognostic Index (IPI), treatment arm and number of planned chemotherapy cycles (Table 1). This effect was observed for both BCL2 translocations and BCL2 SNVs (Table 1; Figure 2), although the vast majority of pts with BCL2 SNVs also harbored BCL2 translocations (n=29/39; 74%), possibly explaining the negative prognostic impact of BCL2 SNVs. BCL2 alterations were detected in 102/499 pts, with 92/102 having BCL2 translocations, 90% (83/92) of which had GCB subtype DLBCL. Of the 39 pts with BCL2 SNVs, 80% (31/39) had GCB subtype and 15% (6/39) had ABC subtype DLBCL. BCL2 alterations were also significantly correlated with BCL2 gene and protein expression levels.Conclusions: Our data confirm the molecular heterogeneity of DLBCL, with potential treatment targets harbored by the distinct COO subtypes. Only alterations in BCL2 were associated with clinical outcome independently of of other factors (COO, IPI). [Display omitted] DisclosuresBolen:Genentech: Employment, Equity Ownership. Klanova:F. Hoffmann-La Roche Ltd: Employment, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party editorial support, under the direction of Magdalena Klanova, was provided by Lynda McEvoy of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd. Trněný:BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Sehn:Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Vitolo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Gilead: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria. Oestergaard:F. Hoffmann-La Roche Ltd: Employment.

Prognostic significance of double expressor lymphoma subtype in patient with diffuse large B-cell lymphoma

Background: DLBCL is the most common type of non-Hodgkin lymphoma in Asia and Indonesia. DLBCL, based on cell of origin is divided into germinal center B-cell-like (GCB) and non-GCB subtypes. 20% of patients have a molecular profile double expressor lymphoma (DEL), which has a worse prognosis. The study aims to determine the relationship between DEL subtypes and cell of origin subtypes with clinical stage and 3-year overall survival of double large B-cell lymphoma (DLBCL) patients in Kariadi General Hospital Semarang.Methods: This study sample was 36 DLBCL patients in Kariadi General Hospital from January to September 2017. The data collection including age of diagnosis, location, stage, cell of origin subtype, DEL subtype and 3-year overall survival. Data analysis using chi-square test and Kaplan Meier curve.Results: DLBCL DEL subtype patients were significantly associated with advanced-stage (p: 0.028). DLBCL non-GCB subtype and DEL subtype patients had a 3-year overall survival that was significantly worse than GCB subtype and non-DEL subtypes (p: 0.026 and p: 0.006, respectively), with a 3-year survival rate of non-GCB subtypes was 38.9% and DEL subtypes were 33.3%. DLBCL patients with advanced stages also have a 3-year overall survival significantly worse than the early stage (p: 0.000), with a 3-year survival rate of 14.3%.Conclusion: DLBCL non-GCB subtype patients, DEL subtypes and advanced stages have a lower 3-year overall survival rate and thus have a worse prognosis

Ki67 Proliferation Index in Germinal and Non-Germinal Subtypes of Diffuse Large B-Cell Lymphoma.

IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell lymphoma. The 2016 World Health Organization (WHO) update on hematopoietic tumors suggested that all DLBCL cases should be subtyped into germinal and non-germinal center phenotypes. Ki67 immunohistochemistry is a maker of cell proliferation and thus is used as a prognostic and predictive marker in various tumors of human body. Only a few studies evaluated the proliferative index of DLBCL subtypes in our population. Therefore, in this study, we evaluated the frequency of subtypes of DLBCL in our population and K67 index in each subtype.MethodsA retrospective observational study was conducted in the Department of Histopathology, Liaquat National Hospital and Medical College, from January 2018 till December 2020, over a period of three years. A total of 101 cases with a histopathological diagnosis consistent DLBCL were included in the study. Immunohistochemical (IHC) stains CD10, B-cell lymphoma 6 (Bcl-6), and multiple myeloma oncogene 1 (MUM1) were applied for the further sub-categorization of DLBCL into germinal center B-cell-like (GCB) and non-GCB subtypes according to the Hans algorithm. The Ki67 index was interpreted in hot spots of the tumor and reported as an average percentage.ResultsOut of 101 DLBCL cases, 47.5% of DLBCL were GCB, while 52.5% were non-GCB subtypes. Bcl-2, Bcl-6, MUM1, c-Myc, CD10, and CD30 expression were noted in 62.4%, 45.5%, 42.6%, 44.6%, 39.6%, and 7.9% cases, respectively. The mean Ki67 index was 72.94±16.69%. The mean Ki67 index in non-GCB-type DLBCL was 77.67±14.80%, which was significantly higher than the mean Ki67 index in GCB-type DLBCL (67.70±17.22%) with a significant p-value (p=0.002). Cervical lymph node was the most common site of DLBCL, while the stomach was the most common extra-nodal site. A significant association of Ki67 index was noted with subtypes of DLBCL. A higher proportion of non-GCB-type DLBCL exhibited greater than 80% Ki67 index than GCB subtype DLBCL. Moreover, a significant association Ki67 index was noted with c-Myc positivity. A higher proportion of c-Myc-positive DLBCL had greater than 80% Ki67 index.ConclusionWe found that non-GCB-type DLBCL had a higher Ki67 index than GCB subtype DLBCL, portending a poor prognostic significance of non-GCB subtype of DLBCL. Moreover, c-Myc expression was associated with a higher Ki67 index.

TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents &amp; Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

Clinical characteristics and outcomes of patients with diffuse large B cell lymphoma treated with R-CHOP-like or CHOP-like regimens: an 8-year experience from a single center.

The CHOP regimen comprising cyclophosphamide, doxorubicin, vincristine, and prednisone is a basic chemotherapeutic regimen for diffuse large B cell lymphoma (DLBCL). Addition of rituximab (R) to chemotherapy has led to better efficacy than other regimens in clinical trials. However, data of clinical characteristics and outcomes of patients with DLBCL are scarce. Therefore, this study reports the clinical characteristics, treatment, and outcomes of patients with DLBCL in our hospital. We conducted a retrospective analysis of newly diagnosed DLBCL patients treated with CHOP-like or R-CHOP-like regimens at our hospital between 2011 and 2018. We analyzed the data on demography, clinical characteristics, treatment, treatment response, and survival time. Both univariate and multivariate analyses were applied. In total, 570 newly diagnosed DLBCL patients were included, of which 133 were treated with CHOP-like regimens and 437 with R-CHOP-like regimens. The overall response rate was 83.3%. Germinal center B-cell-like (GCB) subtype, R-CHOP-like treatment, Ann Arbor stage I-II, not more than 1 extranodal disease site, Eastern Cooperative Oncology Group (ECOG) score ≤1, normal serum lactate dehydrogenase (LDH) level, normal serum β2 microglobulin (β2-MG) level, absence of B symptoms, and lower International Prognostic Index (IPI) or National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) scores were associated with longer overall survival (OS) and progressionfree survival (PFS), and were favorable prognostic factors for OS and PFS. Only GCB subtype, R-CHOPlike treatment, absence of B symptoms, and lower IPI or NCCN-IPI scores were independent favorable prognostic factors for OS and/or PFS. Neither IPI nor NCCN-IPI could accurately and precisely predict the prognosis of high-risk DLBCL patients. This analysis of newly diagnosed DLBCL patients indicates that patients treated with R-CHOP-like regimens or with GCB subtype exhibited better outcomes. Further, IPI and NCCN-IPI have limited prognostic values in high-risk DLBCL patients.

Comparative molecular cell-of-origin classification of diffuse large B-cell lymphoma based on liquid and tissue biopsies

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a heterogenous blood cancer, but can be broadly classified into two main subtypes, germinal center B-cell-like (GCB) and activated B-cell-like (ABC). GCB and ABC subtypes have very different clinical courses, with ABC having a much worse survival prognosis. It has been observed that patients with different subtypes also respond differently to therapeutic intervention, in fact, some have argued that ABC and GCB can be thought of as separate diseases altogether. Due to this variability in response to therapy, having an assay to determine DLBCL subtypes has important implications in guiding the clinical approach to the use of existing therapies, as well as in the development of new drugs. The current gold standard assay for subtyping DLBCL uses gene expression profiling on formalin fixed, paraffin embedded (FFPE) tissue to determine the “cell of origin” and thus disease subtype. However, this approach has some significant clinical limitations in that it 1) requires a biopsy 2) requires a complex, expensive and time-consuming analytical approach and 3) does not classify all DLBCL patients.MethodsHere, we took an epigenomic approach and developed a blood-based chromosome conformation signature (CCS) for identifying DLBCL subtypes. An iterative approach using clinical samples from 118 DLBCL patients was taken to define a panel of six markers (DLBCL-CCS) to subtype the disease. The performance of the DLBCL-CCS was then compared to conventional gene expression profiling (GEX) from FFPE tissue.ResultsThe DLBCL-CCS was accurate in classifying ABC and GCB in samples of known status, providing an identical call in 100% (60/60) samples in the discovery cohort used to develop the classifier. Also, in the assessment cohort the DLBCL-CCS was able to make a DLBCL subtype call in 100% (58/58) of samples with intermediate subtypes (Type III) as defined by GEX analysis. Most importantly, when these patients were followed longitudinally throughout the course of their disease, the EpiSwitch™ associated calls tracked better with the known patterns of survival rates for ABC and GCB subtypes.ConclusionThis proof-of-concept study provides an initial indication that a simple, accurate, cost-effective and clinically adoptable blood-based diagnostic for identifying DLBCL subtypes is possible.

Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Young High Risk Patients with MYC/BCL2 Double Hit Lymphoma, BCL2+ and/or Germinal Centre B-Cell like Diffuse Large B-Cell Lymphoma Benefit from Dose-Dense Chemoimmunotherapy Including Early CNS Prophylaxis: Analysis of Data from the Nordic Lymphoma Group CRY-04 and Chic Trials

Background. Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression is relatively high. We investigated the efficacy of dose-dense chemoimmunotherapy and systemic CNS prophylaxis in two Nordic trials including patients less than 65 years with high-risk DLBCL. We combined individual patient data from these trials to compare clinical outcome and biological prognostic factors in patients treated with CNS prophylaxis given in the beginning (CHIC) versus at the end (CRY-04) of therapy.Patients and methods. In CRY-04 study, patients were treated with six courses of R-CHOEP14 followed by HD-Mtx and HD-Ara-C. In CHIC trial, treatment started with two courses of HD-Mtx in combination with R-CHOP14, followed by four courses of R-CHOEP-14 and one course of R-HD-AraC. In addition, liposomal AraC was administered intrathecally at courses 1, 3 and 5. For the correlative studies, formalin fixed paraffin embedded pretreatment tumor samples were analyzed by fluorescent in situ hybridization for BCL2 and c-MYC breakpoints and by immunochemistry for CD10, BCL6, MUM1, MYC and BCL2 expression. Germinal center B-cell-like (GCB)/non-GCB) subclassification was performed according to Hans algorithm.Results. Among 303 patients enrolled in the trials (CRY-04, n=160 and CHIC, n=143), 295 (CRY-04, n=154 and CHIC, n=139) were evaluable for baseline characteristics and outcome. Median age (54 and 56 years, p=0.222), male/female ratio, stage, and aaIPI scores were comparable in the two cohorts. CHIC regimen improved outcome over CRY-04; the findings included 4-year estimates of PFS (81% vs 66%, p=0.003), OS (83% and 79%, p=ns) and cumulative incidence rates of CNS progression (2.4% and 5.0%, p=ns). Treatment with the CHIC regimen reduced the risk of systemic progression (aaIPI adjusted RR=0.489, 95%CI 0.308-0.777, p=0.002). PFS benefit with CHIC over CRY-04 was observed across pre-specified subgroups, and particularly in patients less than 60 years old (p=0.008).In the entire study population, dual protein expression (DPE) of BCL2 and MYC was the only parameter to be significantly correlated with a worse PFS (4-y PFS 77% vs 50%, p=0.024; RR=2.300, 95% CI 1.088-4.860, p=0.029). Neither any single immunohistochemical marker nor the GCB/non-GCB subtype or MYC/BCL2-translocations significantly affected outcome. However, when treatment interaction was tested, MYC/BCL2 double hit status (DHL; 13%) predicted poor outcome among patients treated with CRY-04 regimen compared with patients who received CHIC regimen (4-y PFS; 38% vs 78%, p=0.086). GCB subtype and BCL2 positivity were also associated with better outcome in the CHIC cohort (4 y PFS; 63% vs 84%, p=0.011 and 61% vs 80%, p=0.007, respectively), whereas there were no significant survival differences between these regimens among the patients with non-GCB subtype, BCL2 negative DLBCL or DPE lymphomas.Conclusions. Our results derived from trial data with homogenous treatment support the use of HD-Mtx in the beginning rather than at the end of therapy. The survival benefit related to CHIC regimen over CRY-04 is due to better systemic control of the disease, and at least partly linked to improved survival among patients with GCB subtype, BCL2 positivity and DHL. DisclosuresLeppa:Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Holte:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Roche, Norway: Research Funding.

Baseline PET-Derived Metabolic Tumor Volume Metrics Predict Progression-Free and Overall Survival in DLBCL after First-Line Treatment: Results from the Phase 3 GOYA Study

Introduction: Quantitative 18fluorodeoxyglucose positron emission tomography (PET)/computed tomography assessment using total metabolic tumor volume (TMTV) and tumor lesion glycolysis (TLG) measurements has been found promising as an objective method to predict survival in diffuse large B-cell lymphoma (DLBCL) patients (pts). However, the methodology for PET-derived metrics is still evolving, and their predictive value is yet to be proven in large-scale, prospective, multicenter studies. We investigated the prognostic value of baseline maximum standardized uptake value (SUVmax), TMTV and TLG for progression-free survival (PFS) in a large pt cohort treated with obinutuzumab (GA101; G) or rituximab (R) combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in the Phase 3 GOYA study (NCT01287741; Vitolo et al. J Clin Oncol 2017). Methods: Pts aged ≥18 years, with previously untreated, CD20-positive DLBCL and an International Prognostic Index (IPI) score ≥2 and low-risk pts with IPI scores of 1 (not due to age alone) or 0 (with bulky disease) were randomized 1:1 to receive 8 x 21-day cycles of G (1000mg intravenous [IV] on Days [D] 1, 8, and 15 of Cycle [C] 1 and D1, C2-8) or R (375mg/m2 IV on D1, C1-8) plus 6 or 8 cycles of CHOP. All pts had a baseline and end of treatment (EOT) PET. PET images were segmented using an automated workflow program in MIM software, applying thresholds of 1.5 x liver background and a minimum volume of 1mL to the whole body PET images. The data were analyzed for the overall population and according to germinal center B-cell-like (GCB), unclassified, and activated B-cell-like (ABC) subtypes of DLBCL. TMTV, TLG, and SUVmax were split into 4 categories/levels according to the following quartiles: Q1, Results: Of 1418 enrolled pts, 1346 had a baseline PET scan and 1334 had detectable lesions. There was no statistical difference in PFS between the treatment arms (G vs R), thus the entire cohort was analyzed as a whole. Results of the predictive value of baseline TMTV for PFS are presented in quartiles in Figure 1, and results of the predictive value of TLG for PFS are presented in quartiles in Figure 2, for the overall PET intent-to-treat population. After a median follow-up of 29 months TMTV and TLG were highly predictive of PFS when comparing Q4 vs Q1: HR=2.21, 95% CI 1.48-3.29, p Three-year PFS for pts in TMTV Q1, 2, 3 and 4 was 86% (95% CI 81-89%), 84% (95% CI 78-88%), 78% (95% CI 72-83%) and 66% (95% CI 59-71%), respectively. TMTV also showed a trend for a better prediction of PFS (Figure 3) and OS in pts with the unclassified and ABC DLBCL subtypes when compared with those with the GCB subtype. Conclusions: This large prospective study confirms baseline TMTV and TLG as predictors of PFS and OS in DLBCL after first-line immunochemotherapy, while SUVmax may not be a predictor. Furthermore, TMTV and TLG appear to be better predictors of survival for pts with the unclassified and ABC subtypes of DLBCL than for those pts with the GCB subtype. Further analyses are underway comparing these results with the predictive value of percentage change from baseline to EOT PET, Deauville score-based analysis of EOT PET, and the various molecular DLBCL subtypes. Disclosures Kostakoglu: Roche: Consultancy, Other: GOYA is sponsored by F. Hoffmann-La Roche Ltd. Third-party editorial support, under the direction of Lale Kostakoglu and Denis Sahin, was provided by Helen Cathro of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd. Sehn: Celgene: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria. Chua: Lundbeck: Honoraria; Roche: Honoraria; Seattle Genetics: Honoraria; Gilead Sciences: Honoraria; Merck: Honoraria. Gonzalez-Barca: Gilead: Consultancy; Sandot: Consultancy; Janssen: Speakers Bureau; Roche: Speakers Bureau. Pinto: Millenium Takeda: Research Funding; Gilead: Honoraria; Roche: Honoraria; Bristol Myers Squibb: Honoraria; Merck Sharp Dome: Honoraria; Celgene: Honoraria; Helssin: Honoraria; Mundipharma EDO: Speakers Bureau. Fingerle-Rowson: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Knapp: Roche: Employment. Mattiello: Roche: Employment. Nielsen: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Sellam: Roche: Employment. Sahin: Roche: Employment, Equity Ownership. Vitolo: Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Gilead: Honoraria; Mundipharma: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Trněný: Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Abstract 666: CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma

Abstract PD-1 immune checkpoint blockade reconstituting antitumor immunity has changed the cancer treatment paradigm. PD-1 blockade has been successful in many types of solid tumors and Hodgkin lymphoma, but not yet for diffuse large B cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. We found several biomarkers including loss of CD37 tetraspanin expression in DLBCL correlated with increased PD-1 expression suggesting potential sensitivity of these DLBCL subsets to PD-1 blockade. CD37 (TSPAN26) is a member of the tetraspanin superfamily, widely expressed on normal and malignant mature B-cells and downregulated in plasma cells. It has been documented that CD37 plays important roles in T-cell-B-cell interactions, B-cell humoral response triggered by B-cell receptor cross-linking, and a balance between immune responses and tolerance. Interestingly, in a large cohort of DLBCL patients, we found that loss of CD37 expression in DLBCL predicts strikingly decreased overall and progression-free survival rates, and that PDCD1 gene expression was upregulated in CD37-negative activated B-cell-like (ABC) DLBCL by gene expression profiling, whereas the costimulatory molecule ICOSLG was upregulated in CD37+ germinal center B-cell-like (GCB) DLBCL. Using the new fluorescent multiplex technology, we further measured PD-1 and PD-L1 expression at the protein level on lymphoma cells and immune cells in the tumor microenvironment, and found that PD-1 protein levels were increased on both cytotoxic and helper T-cells infiltrating in CD37-negative DLBCL either of GCB or ABC subtype. These novel discoveries suggest that CD37 is important for sustained antitumor adaptive immunity, that immune dysregulation plays an important role for poor clinical outcomes in DLBCL, and that CD37-negative DLBCL may be sensitive to PD-1 blockade. In summary, loss of a CD37 tetraspanin was found to correlate with PD-1 overexpression in DLBCL clinical samples, and CD37 may serve as a novel biomarker for anti-PD-1 immunotherapy clinical trials in DLBCL. Note: This abstract was not presented at the meeting. Citation Format: Zijun Y. Xu-Monette, Ken H. Young. CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2017-666

Identification of prognostic factors in patients with diffuse large B-cell lymphoma.

The correlation between various clinicopathological factors and 5-year survival rate, the correlation between those factors with the International Prognostic Index, the concordance between Hans' and Choi's approach was evaluated. The survival in different subtypes as classified by Hans' or Choi's approach was mapped. The Eastern Cooperative Oncology Group (ECOG) performance score 2-5, positive Bcl-2 expression, negative CD10 expression or negative Bcl-6 expression significantly correlated with worse prognosis. The two algorithms showed good consistency (83% concordance, Kappa = 0.660, P < 0.001). By both classifications, the 5-year overall survival rate in germinal center B-cell-like subtype (GCB) lymphoma is significantly higher than that in the non-GCB subtype. There were 25 cases assigned to conflicting subtypes by the two approaches. Among these 25 cases, ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression significantly correlated with worse prognosis. ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression are independent markers for poor prognosis of DLBCL patients. There were 15% cases assigned to conflicting subgroups based on the two algorithms. For these cases, ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression still significantly correlate with poor prognosis.

Discovery and validation of immune-associated long non-coding RNA biomarkers associated with clinically molecular subtype and prognosis in diffuse large B cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive and complex disease characterized by wide clinical, phenotypic and molecular heterogeneities. The expression pattern and clinical implication of long non-coding RNAs (lncRNAs) between germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes in DLBCL remain unclear. This study aims to determine whether lncRNA can serve as predictive biomarkers for subtype classification and prognosis in DLBCL.MethodsGenome-wide comparative analysis of lncRNA expression profiles were performed in a large number of DLBCL patients from Gene Expression Omnibus (GEO), including GSE31312 cohort (N = 426), GSE10846 (N = 350) cohort and GSE4475 cohort (N = 129). Novel lncRNA biomarkers associated with clinically molecular subtype and prognosis were identified in the discovery cohort using differential expression analyses and weighted voting algorithm. The predictive value of the lncRNA signature was then assessed in two independent cohorts. The functional implication of lncRNA signature was also analyzed by integrative analysis of lncRNA and mRNA.ResultsSeventeen of the 156 differentially expressed lncRNAs between GCB and ABC subtypes were identified as candidate biomarkers and integrated into form a lncRNA-based signature (termed SubSigLnc-17) which was able to discriminate between GCB and ABC subtypes with AUC of 0.974, specificity of 89.6% and sensitivity of 92.5%. Furthermore, subgroups of patients characterized by the SubSigLnc-17 demonstrated significantly different clinical outcome. The reproducible predictive power of SubSigLnc-17 in subtype classification and prognosis was successfully validated in the internal validation cohort and another two independent patient cohorts. Integrative analysis of lncRNA-mRNA suggested that these candidate lncRNA biomarkers were mainly related to immune-associated processes, such as T cell activation, leukocyte activation, lymphocyte activation and Chemokine signaling pathway.ConclusionsOur study uncovered differentiated lncRNA expression pattern between GCB and ABC DLBCL and identified a 17-lncRNA signature for subtype classification and prognosis prediction. With further prospective validation, our study will improve the understanding of underlying molecular heterogeneities in DLBCL and provide candidate lncRNA biomarkers in DLBCL classification and prognosis.

Clinicopathological Analysis of 320 Cases of Diffuse Large B-cell Lymphoma Using the Hans Classifier.

The estimation of clinical prognosis for diffuse large B-cell lymphoma (DLBCL) with a quick, cost-efficient method is necessary because of the clinical heterogeneity of this disease, which leads to death, relapsed or refractory disease in approximately 40% of patients. We analyzed 320 cases diagnosed from 2007 to 2013 treated with R-CHOP therapy at Tokai University Hospital and associated institutions. DLBCL was classified according to the cell-of-origin using the Hans algorithm [germinal center B-cell-like (GCB) vs non-GCB subtypes], and into 6 subgroups derived from combinations of CD10, BCL6 and MUM1 markers. The percentage of GCB and non-GCB (NGCB) subtypes was 35% and 65%, respectively. GCB-DLBCL was characterized by lower BCL2 immunohistochemical expression, extranodal sites ＜1, better therapeutic response, and favorable overall survival (OS) and progression free survival (PFS) (P＜0.01). The most frequent subgroup was NGCB-1 (CD10-BCL6+MUM1+, 51%) followed by GCB-1 (CD10+BCL6+or-MUM1+, 21%), NGCB-2 (CD10-BCL6-MUM1+, 13%), GCB-2 (CD10+BCL6+or-MUM1-, 10%), GCB-3 (CD10-BCL6+MUM1-, 4%) and NGCB-3 (CD10-BCL6-MUM1-, 2%). In comparison with GCB-2 and GCB-3 (both MUM1-), the GCB-1 (MUM1+) was characterized by favorable PFS (5-year PFS 84% vs 65%, OR 0.368, P＜0.05), independent of high LDH (associated with unfavorable PFS, OR 7.04, P＜0.01) in the multivariate analysis. This predictive value of MUM1 was independent of CD10. Interestingly, triple-negative NGCB-3 tended to have a more favorable prognosis than the other NGCB subgroups. In conclusion, the Hans classifier is a valid method to evaluate the prognosis of DLBCL NOS. In the GCB subtypes, GCB subtypes, MUM1-positivity is associated with a more favorable outcome (PFS).

Integrated Analysis of IGHV Gene Status, Cell-of-Origin Signature and Genomic Features in Diffuse Large B-Cell Lymphoma

The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p<0.0001 for each). While IGHV gene usage generally reflected that of normal B cells, a marked increase in IGHV4-34 in ABC cases (30%) was confirmed and those cases tended to have less SHM. Acquisition of new N-glycosylation (N-Gly) sites, a hallmark of follicular lymphoma, was more prevalent in the GCB cases (48%) than in ABC (22%) (p=0.012) but was also observed in 5/7 (71%) PMBL cases with functional IGH rearrangement. This acquisition is correlated with a more frequent t(14;18)(q24;q32) in GCB subtype. As reported in FL, this suggests that lectin binding signals via surface Ig that contain N-Gly sites may provide an extrinsic antigen-independent signal in some DLBCL, and more specifically in t(14;18) DLBCL (Linley et al. Blood 2015 ). Finally, we correlated these immunogenetic features with the mutational status of some drivers genes frequently mutated in DLBCL. Within ABC subtype patients, MYD88 or TNFAIP3 mutated cases displayed a higher SHM rate (13.4% vs 8.4%, p <0.0001; 15.44% vs 11.13%, p=0.03), without any IGH family distribution bias. 9/56 (16%) GCB DLBCL harbored an EZH2 mutation. All expressed an IGHV3 rearrangement (100% vs 52% for the GCB-EZH2 wt, p=0.014). By contrast to MYD88 status, a similar IG SHM rate was observed in mutated and wt patients regarding EZH2, CARD11 or GNA13 genes. Conclusion: The IG characterization and the driver gene mutation analysis according to the cell of origin of the tumor allowed us to refine DLBCL subgroups. Our data suggest that there is a strong interaction between extrinsic mechanisms, including lectin-mediated antigen-independent activation or, in the IGHV4-34 cases, via N-acetyl lactosamine ligands, and intrinsic mechanisms to activate the BCR/NFKB pathway. These data could help us classify the different subgroups of DLBCL more accurately and, in particular, to identify new molecular features for BCR target therapy. DisclosuresHaioun:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Mundipharma: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Assessing the Feasibility of Pathology Informatics Approaches for Subtyping Diffuse Large B-Cell Lymphoma (DLBCL)

IntroductionPatients (pts) with DLBCL can experience marked difference in overall survival (OS) based on clinical characteristics, race, and biological subtype. Immunohistochemistry (IHC) algorithms can stratify pts into the germinal center B-cell-like (GCB) or activated B-cell-like (ABC) subtype, which is associated with worse OS with current standard of care therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP; Meyer et al. 2011). However, these algorithms can be challenging to perform in clinical practice. We evaluated the frequency of GCB and non-GCB subtypes in a retrospective cohort of patients and evaluated the feasibility of performing automated pathology informatics computer segmentation algorithms to assess DLBCL subtype.MethodsWe included all patients diagnosed with DLBCL at Emory University from 1995-2016. We collected demographic, clinical, laboratory and pathologic data on all patients. We evaluated GCB and non-GCB status using the Hans algorithm as performed during routine clinical care, collected IHC slides for H&E, CD10, CD20, CD30, BCL2, BCL6, C-MYC, and MUM1, and assessed the feasibility of performing automated computer segmentation algorithms to assess DLBCL subtype using these materials.ResultsOut of 364 patients with DLBCL in our database, 151 had available data for DLBCL classification by the Hans algorithm and 103 had available slides for computational algorithms. For the entire dataset the median age was 61 years, 51% were male, 31% had stage III/IV disease. Among patients who had classification data, the median age was 61 years, 53% were male, 45% had stage III/IV disease, and 47% GCB DLBCL and 53% had non-GCB. Among patients with slides available for computational algorithms the median age was 64 years, 53% were male, 48% had stage III/IV disease.ConclusionsIn this retrospective cohort study, the group of patients with available slides for performing computer segmentation algorithms had similar demographics and disease characteristics to patients in the general population of DLBCL at an academic medical center. This dataset and associated pathology images provides a useful resource to evaluate whether computational algorithms can aid in defining the prognosis for DLBCL patients. Future directions for this work will involve the establishment of an online DLBCL digital archive to develop a more precise, repeatable and objective image-analysis based scoring of DLBCL tissues to improve the prognostic accuracy and classification of DLBCL. DisclosuresFlowers:NIH: Research Funding; Genentech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; Mayo Clinic: Research Funding; Infinity: Research Funding; Millenium/Takeda: Research Funding; ECOG: Research Funding; AbbVie: Research Funding; Acerta: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Research Funding.