Cohort Of Diffuse Large B-cell Lymphoma Patients Research Articles

Cytoplasmic Lipid Droplets Predict Worse Prognosis in Diffuse Large B-Cell Lymphoma: Next-Generation Sequencing Deciphering Lipogenic Genes.

Burkitt lymphoma is characterized by high cell turnover and numerous cytoplasmic vacuoles that are demonstrated to be lipid droplets (LDs) decorated by adipophilin. By contrast, cytoplasmic vacuoles are variably observed in diffuse large B-cell lymphoma (DLBCL) and less well characterized. In this study, we first validated in DLBCL that cytoplasmic vacuoles are indeed LDs by Oil-red-O stain, Bodipy fluorescent stain, and electron microscopy. Second, in a cohort of DLBCL patients (n=52) we showed that LDs in effusional lymphoma cells were associated with a poorer prognosis ( P =0.029, log-rank test) and higher International Prognostic Index (IPI) score (94% vs. 66%, P =0.026) than those without. Moreover, using adipophilin as a surrogate marker for LDs, we found in another cohort of biopsy specimen (n=85) that expression of adipophilin by lymphoma cells predicted a poorer prognosis ( P =0.007, log-rank test) and higher IPI score (63% vs. 30%, P =0.005). In addition, whole exome sequencing of effusional DLBCL cells showed LD-positive DLBCL shared genetic features with the MCD ( MYD88 and CD79B mutations) subtype and highlighted OSBPL10 and CUBN as the most frequently mutated genes involved in lipogenesis. Whole transcriptome analysis by comparing effusional DLBCL cells with versus without LDs showed upregulation of EHHADH , SLC1A1 , CD96 , INPP4B , and RNF183 relevant for lymphoma lipogenesis and upregulation of epithelial-mesenchymal transition and KRAS signaling pathways. Higher expression of EHHADH and CD96 were validated in LD-positive clinical samples and LD-rich cell lines than LD-poor cells along with the known lipogenic gene, FASN . Our findings highlight the roles of LDs and adipophilin expression in DLBCL, suggest that these markers may predict prognosis and show that lipogenic genes may be potential therapeutic targets.

Validating the Halp Score (Hemoglobin,Albumin, Lymphocytes, and Platelets) and the Neutrophil/Lymphocyte Ratio ( NLR) As Prognostic Factors for Overall Survival in Patients with Diffuse Large B-Cell Lymphoma. Retrospective Analysis By the Grupo De Estudio De Latino America De Linfoproliferativos (GELL)

Introduction. Diffuse large B-cell lymphoma ( DLBCL) is the most common type of malignant lymphoid neoplasm. The international Prognostic Index (IPI) and its variants are the main prognostic tools used in DLBCL with value in the rituximab era. Certain molecular biomarkers and genetic signatures in DLBCL have been identified, but the cost and unavailability in Latin America ( LATAM) are an issue. Therefore, using accesible tools in LATAM is a potencially unmet need. Vlatka (2021), reported a new score (HALP: hemoglobin, albumin, lymphocytes, and platelets) for overall survival (OS) in DLBCL. These parameters are easy to access in LATAM centers. Thus, we aim to validate this novel OS score in the GELL database, and expanding database from LATAM countries. Methods. This was a retrospective analyses of patients with de novo DLBCLtreated with curative intent between 2010 and 2018. The classic 5 international prognostic index variables [age, ECOG, Extranodal (EN) involvement, Lactate dehydrogenase (LDH), and advanced stage] and low serum albumin defined as ≤3.5mg/dL(divided in low, 3.4-2.5 mg/dL, and very low, ≤2.4 mg/dL), as previously described by our group ( Villela,2018&2019). We also calculated the NLR considering an adverse prognostic factor >4, as previously published by us ( Beltran&Villela,2020). The HALP score was calculated using the following formula: hemoglobin(g/L) x albumin (g/L) x absolute lymphocyte count (k/μL) divided by platelets (k/μL). The ROC method was used to calculate the HALP cut-off. Demographic characteristics are reported using descriptive statistics. Cox proportional-hazard regression model was used to evaluate parameters associated with OS, and survival curves were estimated with the Kaplan-Meier (KM) method. Outcomes. 1407 patients were included, who were treated with standard RCHOP (n=1112,79%), RminiCHOP (n=93,7%), REPOCH (n=111,8%), and CHOP (n=91,6%). The median follow-up was 36 months (IQR: 7 to 56). NLR >4 was observed in 18.5%. The median HALP score was 24 (IQR, 12 to 40), and the cut-off of ≤13 (AUC 0.58;95%CI 0.56 to 0.61; p<0.0001) was considered an adverse prognostic factor, which was observed in 451 patients (32.7%). Female sex, ECOG >1, EN >1. high LDH, advance stage, low and very low albumin, and NLR>4 were associated with HALP≤13, but age was not ( Table 1). Patients with HALP ≤13 had a lower 3-years OS rate than HALP>13 (48% vs. 66%, respectively; p<0.001). Table 2 shows the univariate & multivariate analysis of the variables with independent influence on OS, including HALP & NLR. In multivariate analysis, HALP and EN involvement were left out of the model ( Harrell´s C-Index, 0.73;95%CI 0.69 to 0.76). Conclusion: NLR>4, but not HALP ≤13, could prognosticate inferior OS in LATAM patients with DLBCL treated with curative intent. The adverse prognostic value of NLR>4 should be validated prospectively in other cohort of DLBCL patients.

Tumor Mutational Burden As a Prognostic Factor in Diffuse Large B-Cell Lymphoma

Background: Tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase (Mb) in the coding regions of a tumor genome, has been associated with improved response to immune checkpoint blockade treatments and more broadly with overall survival (OS) in many cancers. Limited prior studies have found that TMB is higher in diffuse large B-cell lymphoma (DLBCL) compared to many other cancers. However, only two studies have evaluated TMB and prognosis in newly diagnosed DLBCL patients. The prognostic role of TMB in diffuse large B-cell lymphoma (DLBCL) remains unclear as prior studies have been limited to panel sequencing of cancer genes and/or lacked matched germline-tumor samples for filtering. Methods: We characterized TMB from whole exome sequencing (WES) in newly diagnosed DLBCL patients with matched diagnostic tumor-normal samples prior to treatment with standard immunochemotherapy (IC). Tumor DNA was extracted from formalin-fixed, paraffin embedded tissue sections and germline DNA from peripheral blood. We evaluated the association between TMB and event-free (EFS) and OS using Cox regression adjusted for the international prognostic index (IPI) and other factors including cell of origin (COO), MYC double hit (DH) with BLC2 or BCL6 by FISH, and TP53 mutation status. EFS and OS were defined as time from diagnosis to earliest of treatment failure (EFS only), death, or last follow-up; EFS at 24 months (EFS24) was also modeled as a marker of early aggressive DLBCL. An optimal high/low TMB cutpoint was determined based on EFS using maximally selected rank statistics; sensitivity analyses used an 20/80 percentile to define the threshold. We also evaluated the association of driver tumor mutational load (driverTML) with EFS and OS, defined as the total number of mutations in 292 DLBCL driver genes. Results: In the analysis cohort of 394 DLBCL patients, the median age at diagnosis was 65 years (interquartile range: 56-73), 43% were female, 62.2% had IPI 0-2, and 57.4% germinal center B-cell COO. MYC was DH in 7.5% and TP53 was mutated in 16.2% of patients. Through a median follow-up of 7.5 years, there were 141 events and 122 deaths. EFS24 was achieved in 75.1% of the cohort; 5-year EFS was 69% (95% CI 64-74%) and 5-year OS was 75% (95% CI 71-80%). Median TMB in our cohort of DLBCL patients was 3.6 mutations/Mb, which was the fifth highest compared with 32 cancers in The Cancer Genome Atlas. As a continuous variable, increasing TMB (mutation/Mb) was associated with superior EFS (HR=0.89, 95% CI 0.81-0.98) and OS (HR=0.89, 95% CI 0.81-0.99) after adjustment for IPI. Spline analyses confirmed linear associations of TMB with EFS and OS. The optimal modeled cutpoint based on EFS was 4.5 mutations/Mb, with 31% classified as high TMB (>4.5). No significant ( P >.05) differences were observed between high and low TMB patients with clinical (i.e., age, sex, IPI category) or molecular (cell of origin, MYC DH, TP53 mutated) factors. In Cox regression, high-TMB was significantly protective for EFS and for OS compared to low-TMB after adjustment for each clinical factor ( Figure 1), with simultaneous adjustment for all these factors associated with superior EFS (HR=0.52, 95% CI 0.33-0.84) and OS (HR=0.59, 95% CI 0.36-1.00). TMB (high/low) combined with IPI (0-2, 3-5) was significantly ( P =.001) associated with EFS in Kaplan-Meier analysis ( Figure 2). Among patients with IPI 3-5, high TMB was significantly associated with superior EFS (HR=0.47, 95% CI 0.27-0.81) and OS (HR=0.57, 95% CI 0.33-0.99). Among patients with IPI 0-2, we also observed longer EFS (HR=0.62, 95% CI 0.34-1.11) and OS (HR=0.62, 95% CI 0.32-1.20) in high TMB patients, although the associations were not statistically significant ( P >.05). In sensitivity analyses, a 20/80 percentile TMB threshold found similar results for EFS (HR=0.64, 95% CI 0.41-1.03) and OS (HR=0.69, 95% CI 0.42-1.12) as in our main analyses. TMB and driverTML were significantly correlated ( R=0.65, P <10 -16) and we found similar effect sizes for EFS (HR=0.45, 95% CI 0.22-0.91) and OS (HR=0.55, 95% 0.27-1.13) as in our main analyses. Conclusions: In a large and well-defined cohort of IC treated DLBCL patients, we found DLBCL patients with higher TMB had superior EFS and OS after adjustment for other clinical and molecular prognostic factors, and the prognostic association for TMB was robust to threshold and gene set.

Circulating Tumor DNA Clearance during Treatment As a Predictive and Prognostic Biomarker for Diffuse Large B-Cell Lymphoma Patients Receiving First-Line Therapy

Background: Circulating tumor DNA (ctDNA) dynamics has been proven to be a reliable outcome prediction biomarker in a plenty of solid tumors. However, its clinical utility in response assessment and prognosis prediction for diffuse large B-cell lymphoma (DLBCL) has not been well established. This study was aimed to explore the association between ctDNA dynamics and clinical benefits in a cohort of DLBCL patients. Methods: A total of 37 newly diagnosed DLBCL patients treated with first-line therapy were enrolled retrospectively in this study. Tumor biopsy tissues were collected at diagnosis (n = 17) and peripheral blood samples were obtained before treatment (n = 31) as well as after receiving the initial two treatment cycles (C2, n = 37). Tumor genomic DNA and cell-free DNA (cfDNA) were extracted from tumor tissues and plasma samples, respectively. Targeted next generation sequencing of 188 lymphoma associated genes (Onco-LymScan, Genetron Health) was performed in all of the tumor samples to identify disease-associated mutations. Genomic DNA extracted from the paired peripheral blood mononuclear cells were also sequenced and analyzed to filter out germline variants for each patient. Results: All of the 37 patients were identified at least one somatic mutation from their pretreatment tumor and/or plasma samples. We sought whether these basal mutations could be traced in the C2 plasma of each patient. Mutations that were only detected in C2 plasma were considered uninformative and were removed from analysis. Accordingly, 12 patients were identified at least one basal mutation at C2 and 25 patients showed a rapid clearance of these basal mutations. Based on the ctDNA clearance status at C2, patients were classified into two groups: the ctDNA clearance group (n = 25) and the ctDNA persistence group (n = 12). There were no significant difference in serval clinical factors such as age, B symptoms, LDH (Lactate Dehydrogenase) level, stage and IPI (International prognostic Index) between the two groups. In response to the first-line treatment, patients with a ctDNA clearance showed a significantly higher complete remission rate (62.5% vs. 23.1%, p = 0.038; Figure A) than the patients with a persistence of ctDNA. With a median follow-up visit period of 26.0 mouths, patients with a ctDNA clearance at C2 presented a favorable progression-free survival (median PFS, unreached vs. 5.45 months; HR 2.919; p = 0.0039; Figure B) and overall survival (median OS, 43.6 vs. 25.87 months; HR 2.758; p = 0.0393; Figure C) compared with those without a ctDNA clearance. These results indicated that patients achieving an early ctDNA clearance during treatment were more likely to achieve a superior later clinical benefit. Conclusions: Early ctDNA clearance during treatment could effectively serve as a predictive and prognostic biomarker for DLBCL patients receiving first-line therapy. Integrating ctDNA profiling into clinical disease management may help to identify patients at high-risk and contribute to individualized treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

18F-FDG PET Maximum-Intensity Projections and Artificial Intelligence: A Win-Win Combination to Easily Measure Prognostic Biomarkers in DLBCL Patients.

Total metabolic tumor volume (TMTV) and tumor dissemination (Dmax) calculated from baseline 18F-FDG PET/CT images are prognostic biomarkers in diffuse large B-cell lymphoma (DLBCL) patients. Yet, their automated calculation remains challenging. The purpose of this study was to investigate whether TMTV and Dmax features could be replaced by surrogate features automatically calculated using an artificial intelligence (AI) algorithm from only 2 maximum-intensity projections (MIPs) of the whole-body 18F-FDG PET images. Methods: Two cohorts of DLBCL patients from the REMARC (NCT01122472) and LNH073B (NCT00498043) trials were retrospectively analyzed. Experts delineated lymphoma lesions from the baseline whole-body 18F-FDG PET/CT images, from which TMTV and Dmax were measured. Coronal and sagittal MIP images and associated 2-dimensional reference lesion masks were calculated. An AI algorithm was trained on the REMARC MIP data to segment lymphoma regions. The AI algorithm was then used to estimate surrogate TMTV (sTMTV) and surrogate Dmax (sDmax) on both datasets. The ability of the original and surrogate TMTV and Dmax to stratify patients was compared. Results: Three hundred eighty-two patients (mean age ± SD, 62.1 y ± 13.4 y; 207 men) were evaluated. sTMTV was highly correlated with TMTV for REMARC and LNH073B datasets (Spearman r = 0.878 and 0.752, respectively), and so were sDmax and Dmax (r = 0.709 and 0.714, respectively). The hazard ratios for progression free survival of volume and MIP-based features derived using AI were similar, for example, TMTV: 11.24 (95% CI: 2.10-46.20), sTMTV: 11.81 (95% CI: 3.29-31.77), and Dmax: 9.0 (95% CI: 2.53-23.63), sDmax: 12.49 (95% CI: 3.42-34.50). Conclusion: Surrogate TMTV and Dmax calculated from only 2 PET MIP images are prognostic biomarkers in DLBCL patients and can be automatically estimated using an AI algorithm.

Screening for Prognostic microRNAs Associated with Treatment Failure in Diffuse Large B Cell Lymphoma.

Simple SummaryAround 30–40% of patients with diffuse large B cell lymphoma suffer early relapse after standard chemotherapy, but today no prediction whether a patient belongs to this group is possible. MicroRNA are small nucleotide sequences that regulate cellular functions via post-transcriptional modification of gene expression and can serve as prognostic biomarkers. A novel two-step strategy first used a small patient discovery group to identify possible microRNA candidates by comparing their levels in chemosensitive and chemoresistant patients via microarray. Overexpression of these microRNA was then analyzed in a large patient cohort and, as a result, three new microRNA biomarkers with prognostic potential could be identified. Early identification of those patients being at risk of failure with standard therapy is a prerequisite to develop more efficient treatments and a step towards precision medicine.Diffuse large B cell lymphoma (DLBCL) treatment with R-CHOP regimen produces 5-year progression-free survival and overall survival of around 60–70%. Our objective was to discover prognostic biomarkers allowing early detection of the remaining 30–40% with poor long-term outcome. For this purpose, we applied a novel strategy: from a cohort of DLBCL patients, treated with standard therapy, a discovery group of 12 patients with poor prognosis (advanced stage III–IV, R-IPI > 2) was formed, consisting of six chemoresistant (refractory/early relapse < 12 months) and six chemosensitive (complete remission > 3 years) subjects. By using microarray assays, the most differentially expressed miRNAs were defined as an initial set of prognostic miRNA candidates. Their expression was then analyzed in a validation cohort of 68 patients and the three miRNAs with the most significant impact on event-free and overall survival were selected. In the DLBCL cell line U-2932 the transfection with miR-1244 and miR-193b-5p, but not miR-1231, blocked the effect of CHOP on cell viability. A subsequent gene set enrichment analysis in patients revealed the implication of the first two miRNAs in cell cycle control and chemoresistance-related pathways, whereas the last one was involved in immunological processes. In conclusion, this novel strategy identified three promising prognostic markers for DLBCL patients at high risk of failure with standard therapy.

Tumor-collagen digital proximity signature to indicate prognosis in diffuse large B-cell lymphoma.

e19073 Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous tumor that originates from normal B-cells. Despite the use of combination chemotherapy, around 40% of DLBCL patients die (de Jonge, et al. European Journal of Cancer, 2016). Limited studies have investigated the role of collagen in the acellular tumor microenvironment. In this study, we present a novel digital signature of the proximity of tumor cells and collagen-VI (COL6) that can predict overall survival (OS) in DLBCL patients. To the best of our knowledge, this is the first study of its kind to employ automated image analysis. Methods: The proposed digital proximity signature (DPS) aggregates summary-level statistics from the entire whole slide image (WSI) and serves as a marker of regions, categorizing weak, moderate, significant, and strong tumor-collagen proximity and can be described as a surrogate for signaling. To accomplish this, we developed a novel artificial intelligence (AI) based multi-task model for simultaneous detection and classification of tumor cells and another bespoke method for automatically identifying COL6 fiber. The tumor-collagen proximity analysis was then performed by aggregating tumor cell statistics within the vicinity of COL6 fibres. Finally, the prognostic significance of DPS for OS in DLBCL was investigated with Kaplan-Meier analysis, stratifying patients into two groups based on the median of the DPS values. Results: We took WSIs of DLBCL tissue slides for 32 cases immunohistochemically stained with COL6 and Hematoxylin counterstain. The AI model for tumor cell identification achieved a high F1-score of 0.84, outperforming recent single-task learning models. Our results show that strong proximity of COL6 and tumor cells is linked to better OS in DLBCL patients ( p = 0.03). Conclusions: Our novel digitally computed COL6-tumor proximity signature shows prognostic significance for overall survival on a pilot dataset of 32 DLBCL patients. We are further validating the utility of this novel signature as a prognostic biomarker in larger cohorts of DLBCL patients.

Deep Phenotypic Analysis Reveals a Monocyte Subpopulation Predictive of Relapse/Refractory Diffuse Large B-Cell Lymphoma

Background: Diffuse large B cell lymphoma (DLBCL) is the most frequent lymphoma subtype with up to two thirds of patients achieving cure following standard immuno-chemotherapy. However, approximately 10-15% will be primary refractory and a further 20-30% relapse. There is an urgent need for improved biomarkers to identify these poor risk patients at diagnosis who are destined to fail standard therapy so that novel approaches can be considered. The diagnostic peripheral blood monocyte count is known to be predictive of outcome in DLBCL, with higher counts identifying patients with worse outcome but mechanistic understanding is currently lacking. The aim of this study was to investigate the peripheral blood monocyte population driving this phenomenon and we present here a deep phenotypic analysis of the monocyte compartment at diagnosis in patients with extremes of outcome following immuno-chemotherapy.Methods: We identified 112 immuno-chemotherapy treated DLBCL patients with viably cryopreserved peripheral blood mononuclear cells (PBMCs) in our tissue bank. The diagnostic absolute monocyte (AMC) and lymphocyte counts (ALC) were examined as prognostic variables. For phenotypic analysis a subset of 20 patients, 10 with relapse/refractory disease within 12 months of therapy (Poor risk) and 10 with continued complete remission >24 months post therapy (Good risk), were selected. PBMCs from these patients were stained with a panel of 29 metal-tagged antibodies designed to identify immune populations and characterize the monocyte compartment and analyzed using cytometry by time-of-flight (CyTOF2TM). Samples were acquired in batches run with the same healthy donor control PBMCs to ensure consistency of staining. Normalized data were subjected to traditional Boolean gating in Cytobank (www.mrc.cytobank.org) to identify CD45+ live single cells and further analyzed using FlowSOM and CITRUS.Results: In the full 112 patient cohort, a higher diagnostic AMC (≥0.6 x 109/L) predicted worse outcome (5-year OS, 59% vs 74%, p=0.047*). The lymphocyte to monocyte ratio (LMR) was more discriminatory, with a low LMR (<2.6) predicting worse outcome (5-year OS, 49% vs 81%, p=0.0009*). CyTOF analysis of the CD45+ events from all 20 DLBCL patients were clustered in an unsupervised manner with FlowSOM and visualized according to the tSNE algorithm. This identified all expected immune populations with further subdivision into multiple clusters / sub-populations based on protein marker expression. Repeating the FlowSOM analysis by outcome revealed clusters with clear differences between the 2 groups, including striking changes to the monocyte compartment (Figure 1). CITRUS, an algorithm for automated biomarker discovery, was used to further assess the differences in the monocyte compartments between poor risk and good risk DLBCL patients. This identified a monocyte subset, representing approximately 10% of non-lymphoid CD45+ PBMCs, with increased abundance in poor risk patients with relapsed/refractory disease (p=0.0028*, Figure 2). This subset expressed markers including HLA-DR, CD11b, CD13, CD14, CD33, SIRPα (CD172a), CCR2 (CD192, monocyte chemo-attractant protein receptor), CD206 and PD-L1 (CD274).Conclusions: We confirmed the previous finding that the diagnostic AMC is predictive of outcome in our cohort of DLBCL patients and have identified differences in the peripheral blood monocyte compartment between patients with good and poor risk disease. Unsupervised analysis using FlowSOM identified monocyte clusters with greater abundance in poor risk patients. A second high-dimensional single cell data analysis algorithm, CITRUS validated this finding by revealing a stratifying monocyte sub-population that was enriched in poor risk patients. This phenotype of peripheral blood monocytes may account for the adverse nature of a higher AMC at diagnosis. We are now functionally characterizing these monocytes and evaluating their relationship to differences between the lymphoid populations of good and poor risk patients. [Display omitted] DisclosuresSeymour:Celgene: Research Funding. Gribben:Janssen: Honoraria, Research Funding; Pharmacyclics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Acerta Pharma: Honoraria, Research Funding; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; NIH: Research Funding; Unum: Equity Ownership; Roche: Honoraria; Novartis: Honoraria; Cancer Research UK: Research Funding; Medical Research Council: Research Funding; TG Therapeutics: Honoraria.

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

Addition of Intermediate-Dose Methotrexate Is Beneficial in Terms of Both Progression-Free Survival and Overall Survival for Patients with Diffuse Large B Cell Lymphoma Receiving Either R-CHOP or CHOP

Introduction: Patients with diffuse large B cell lymphoma (DLBCL) and high International Prognostic Index (IPI) or extra-nodal disease are at an increased risk of systemic and central nervous system (CNS) relapse. Presently, the management of DLBCL patients at the Rambam Health Care Campus (Haifa, Israel) and Hadassah Medical Center (Jerusalem, Israel) includes 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) along with 4 doses of intrathecal (IT) methotrexate (MTX), which may be followed by 2 additional cycles of an intermediate dose (3 g/m2) of intravenousMTX (ID-MTX). The current study compared progression-free survival (PFS), overall survival (OS) and CNS relapse rate in a cohort of DLBCL patients with risk factors for CNS relapse who did or did not undergo prophylactic therapy with ID-MTX.Methods: Four hundred and eighty DLBCL patients treated at two tertiary care centers (Rambam and Hadassah) between the years 1991 and 2013 were retrospectively evaluated. The cohort included 224 females and 256 males at a median age of 58 years (range18-76). OS, PFS and CNS involvement at relapse were analyzed. The study incorporated all patients with the primary diagnosis of DLBCL and risk factors for CNS relapse, i.e., Waldeyer ring (2%), breast (1%), testicular (3%) or orbita (1%) involvement, bone marrow involvement (21%), stage IV disease (64%), LDH higher than normal (70%), ≥1 extra-nodal site (28%), IPI ≥3 (36%). Eight percent of patients had stage I-IE disease, 17% - stage II-IIE, 11% - stage III, 64% - stage IV, 29% had B symptoms. Patients with CNS involvement at diagnosis were excluded from the analysis.Results: Four hundred and seventy three patients (99%) were treated with either CHOP or CHOP-like regimen, and 384 (80%) patients received rituximab (R). In the R-CHOP cohort, 23% of patients with IPI 0/1, 28% with IPI 2 and 42% with IPI ≥3 were given prophylaxis. One hundred and thirty patients received ID-MTX (27%), 35 patients (7%) received IT-MTX only, while no prophylaxis was administered to 315 patients (66%). The cumulative incidence of CNS relapse at 5 years was 6%, with no difference in the incidence between the patients who received prophylaxis with ID-MTX, IT-MTX or were given no prophylaxis at all. Patients with IPI ≥3 treated with ID-MTX had a decreased overall relapse rate and significantly superior OS and PFS (Table 1; Figure 1).Conclusion: In the current study, the addition of two cycles of ID-MTX to the standard R-CHOP regimen resulted in improved 5-year PFS and OS of DLBCL patients with IPI ≥3. These data need to be further confirmed in a randomized controlled study. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transcriptome Sequencing Reveals Thousands of Novel Long Non-Coding RNAs in B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of lymphoma. Roughly 40% of patients do not respond or relapse after current therapy. Gene expression profiling of DLBCL has revealed broad gene expression deregulation compared to normal B Cells and categorized DLBCLs into two main subtypes: germinal center B-cell like and activated B-cell like (GCB-DLBCL and ABC-DLBCL), both associated with distinct clinical outcomes. Recent RNAseq studies have demonstrated that large portion of the genome consists of long non-coding transcripts (lncRNAs) that exhibit cell type specific expression patterns and are shown to play critical roles in disease specific epigenetic gene regulation. We sought to discover and characterize novel lncRNAs expressed in DLBCL that are responsible for the disease phenotypes. We performed a systematic de novo lncRNA discovery analysis on RNAseq data obtained from 116 primary DLBCL tumors (dbGaP, phs000235.v6.p1), and 8 normal B-cell counterparts (4 Naïve B-cell and 4 Germinal Center B-cell samples). We identified 2,632 novel expressed lncRNAs in DLBCL. These novel lncRNAs exhibit characteristics similar to known lncRNAs, such as low coding potential, > 200 nt in length, and exon counts (>=2 exons). Further comparison between tumors and normal B-cells revealed that 942 lncRNAs were expressed in DLBCL only. We noticed that many of these lncRNAs were located nearby coding-genes that are important in B-cell biology, such as BCL6, IKZF1, IL10, IRF4, PRDM1, TNFAIP3, etc. Indeed, using a computational tool GREAT, we found that the coding-genes located nearby DLBCL-specific lncRNAs were significantly enriched in immune cell activation and differentiation pathways (FDR < 0.05), suggesting that novel DLBCL lncRNAs are preferentially located near key B-cell and lymphoma genes perhaps contributing to the expression regulation of these genes. Furthermore, through a systematic approach, we found that 88% of these lncRNAs correlated significantly with at least one coding gene expression (Spearman correlation, FDR < 0.2), and collectively these co-expressed coding genes were enriched in B-cell specific functional pathways, including CD40-regulated pathways. Interestingly, we observed that 81 lncRNA were located within 17 DLBCL locus control regions (LCRs or super enhancers), representing a significant enrichment of lncRNAs at LCRs (p < 0.001). This suggests that super-enhancers may coordinate the co-expression of lncRNAs and their neighborhood coding-genes. Moreover, through supervised analysis, we derived a lncRNA expression signature that can differentiate ABC and GCB subtypes of DLBCL. There were 465 lncRNAs significantly differentially expressed between ABC and GCB DLBCLs (FDR < 0.05). When we performed pathway analysis on the protein coding genes significantly co-expressed with these 465 subtype-specific lncRNAs, we found they were enriched in signatures that can define the two subtypes, including Germinal_center_B-cell_DLBCL and ABC_gt_GCB_Affy signatures.To understand the regulation of lncRNA expression in DLBCL, we examined the histone mark profiles at lncRNA loci. First, we found in any given DLBCL cell line, there was a significant overlap of lncRNA promoters and H3K4me3 peaks (p < 2.2e-16), suggesting like coding genes, lncRNA promoters are marked with H3K4me3. Second, we identified that expression of 323 lncRNAs were negatively correlated with BCL6 expression (FDR < 0.2) in this cohort of DLBCL patients, among which 104 exhibited increased expression upon BCL6 knocking down. BCL6 is a transcription repressor and a master regulator of germinal center transcription programming. Taken together, our data suggest that expression of novel DLBCL lncRNAs is potentially under the control of B-cell specific transcription factors.Finally, we performed single nucleotide variant analysis on these novel lncRNAs by using VarScan. We identified 9,713 SNVs within the exonic regions of these lncRNAs. We calculated the Minimum Free Energy Scores (MFEs) by using RNAfold to examine whether these SNVs would have an impact on the stability of the lncRNA secondary structure. We observed a significantly higher than background shift towards the stabilization of the secondary structure with the SNVs (p < 2.2e-16, Wilcoxon test), indicating a potential genetic mechanism of aberrant expression of lncRNA in DLBCL. DisclosuresMelnick:ROCHE: Other: Research; Genentech: Speakers Bureau; Janssen: Other: Research; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy.

The B Cell Receptor Antagonist R406 Alters The Balance Between Anti-Apoptotic and Pro-Apoptotic Signals To Strongly Synergize With ABT-199 In Diffuse Large B Cell Lymphoma Cells

SYK plays an essential role in B cell receptor (BCR) mediated survival in Diffuse Large B Cell Lymphoma (DLBCL). In a previous clinical investigation, fostamatinib, a potent Syk inhibitor, showed evidence of anti-tumour activity with an overall response rate of ∼20% in a cohort of DLBCL patients (JW Friedburg et al Blood. 2010, 115:2578-85). This finding prompted us to investigate the mechanism of action of fostamatinib in DLBCL. Our previous work demonstrated R406, the metabolic active form of fostamatinib, functions as a BCR antagonist in DLBCL cells. Treatment with R406 reduced MCL1 and down-regulated Bfl-1 expression, leading to apoptosis induction in sensitive cells. Due to R406's impact on apoptosis machinery, here we explored the ability of R406 to combine with a selective Bcl2 inhibitor, ABT-199 in DLBCL cells.Drug combinations were tested in DLBCL cells and synergy scores were determined using a 6x6 dose matrix followed by analysis via Chalice software (Zalicus). ABT-199 strongly synergized with R788, the prodrug of R406, in multiple R406 sensitive, but not resistant cell lines. In contrast, little effect was observed in combinations of R788 with other reagents, including ibrutinib (PCI-32765), bortezomib, CAL-101 and dasatinib. Flow-cytometry apoptosis analysis confirmed that R406 enhanced ABT-199-induced apoptosis in both ABC subtype and GCB subtype DLBCL cells. Further studies demonstrated R406 not only decreased anti-apoptotic proteins, but also activated pro-apoptotic proteins. Specifically, R406 suppressed ERK-dependent phosphorylation of BIM on S69, resulted in stabilization of BIM. In addition, R406 induced dephosphorylation of BAD at S136 in sensitive but not resistant cells, likely through inhibition of AKT kinase activity. Furthermore, treatment of R406 also upregulated other pro-apoptotic BH3-only proteins such as HRK1 and BMF1, which may partially contribute to the observed synergy.In conclusion, we have demonstrated R406 strongly synergizes with ABT-199 in vitro, likely by altering the balance between anti-apoptotic and pro-apoptotic signals in DLBCL cells. This may provide a potential combination therapy to be tested in DLBCL. Disclosures:Zhang:AstraZeneca: Employment, Possible shareholder Other. Francoise:AstraZeneca: Employment, Possible shareholder Other. Devereaux:AstraZeneca: Employment, Possible shareholder Other. Passino:AstraZeneca: Possible shareholder Other. Parmentier:AstraZeneca: Possible shareholder Other. Byth:AstraZeneca: Employment, Possible shareholder Other.

Exploring the Immune Microenvironment of Diffuse Large B Cell Lymphoma in a Tissue Microarray: Predicting Survival with a Score That Incorporates Macrophages, Cytotoxic and Regulatory T Cells

Abstract 951Gene expression profiling studies in Diffuse Large B cell Lymphoma (DLBCL) have described signatures enriched in genes representing the immune microenvironment. These signatures, together with the cell of origin classification have prognostic significance. However, a comprehensive functional understanding of the role of inflammation and the immune response in DLBCL is still lacking. The use of immunohistochemistry (IHC) to validate and elaborate upon molecular results is appealing. Our objective was to assess the cellular composition of the non-malignant population in DLBCL quantitatively and thoroughly in a single centre TMA of patients with DLBCL with corresponding clinical and survival data. Based on reported findings and known functional interactions a panel of antibodies were selected that included CD68, FOXP3 and TIA-1.IHC was performed on TMAs from high quality formalin-fixed paraffin-embedded diagnostic tissue biopsies of DLBCL from 1977 to 2009. Triplicate cores were made from areas of representative tumour tissue, arrayed onto glass slides and stained for a panel of antibodies, aiming to characterize immune cell subsets. The TMA included tissue from 218 patients: 128 males, 90 females, with a median age of 55 (18-94) years; 23.2% high-int/high risk IPI; with median follow up of 3,1 years. 33% of patients were treated in the rituximab era. Absolute numbers of positive cells were counted across all intact cores using an automated image analysis system (Ariol), confirmed by histopathologists, and means calculated and corrected to a 1 mm2 area. A training-validation set method was used to generate optimal cutoff values discriminating prognostic groups and comparisons performed using the chi-square test.Among nine antibodies examined, statistically significant prognostic information was provided using staining for FOXP3, TIA-1 and CD68. Using single cut-offs of positive cells/mm2 to define low and high marker density, two prognostic groups were defined for each marker. Patients with a more favourable prognosis had the highest cell density for FOXP3, TIA-1 and CD68.To clarify whether there was an additive prognostic relationship we developed a score that incorporated these three markers. This scoring system allowed us to discriminate two subgroups of patients with divergent survival parameters (fig 1).Marker (cutoff value high vs low)Overall Survival at 3yr (high vs low)Progression Free Survival at 3yr (high vs low)Disease Specific Survival at 3yr (high vs low)FOXP3 (450+ cells/mm2)64% vs 44% (p 0.005)55% vs 36% (p 0.001)69% vs 54% (0.01)CD68 (900+ cells/mm2)58% vs 28% (p 0.023)50% vs 20% (p 0.05)66% vs 36% (p 0.005)TIA-1 (2500+ cells/mm2)76% vs 45% (p 0.002)66% vs 41% (p 0.01)80% vs 56% (p 0.005)Combined Score68% vs 39% (p 0.0002)60% vs 32% (p 0.0003)73% vs 49% (p 0.0008) [Display omitted] A Cox regression model was developed, incorporating age, IPI, rituximab treatment and IHC score. The score results were validated as independent prognostic markers for OS, PFS and DSS, together with age, IPI (for OS and DSS) and rituximab (for PFS).We confirm that there is heterogeneity in immune cell infiltration at diagnosis in DLBCL, and we provide correlative prognostic data to suggest a corresponding biological relevance. Attributing single cut-off values for immune cell markers is feasible, attractive as a clinical tool, and appears to provide robust prognostic information. In our single centre cohort we found that higher numbers of macrophages, Tregs and cytotoxic T cells correlate independently with improved patient survival. CD68 has been associated with an adverse prognosis in lymphoid malignancies but phenotypic diversity of macrophages may account for this study's finding and will be explored using macrophage subset specific markers.Our multivariate analysis revealed that the IHC results have an independent impact on survival on this cohort of DLBCL patients. We are currently validating this approach in an independent TMA of R-CHOP treated patients. Combining these three different immune cell parameters allowed us to discriminate prognostic subgroups providing further evidence of the impact of the non-malignant immune cells in the biology of DLBCL and validating their role as potential therapeutic targets for intervention. Disclosures:Gribben:Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria.

Vitamin D Deficiency Is Associated with Inferior Event-Free and Overall Survival in Diffuse Large B-Cell Lymphoma.

Abstract 1952Poster Board I-975 Background:Vitamin D is a naturally occurring steroid hormone. In addition to its well-established role in maintaining serum calcium homeostasis, vitamin D has effects on cellular differentiation, proliferation, apoptosis, and angiogenesis. Vitamin D3 is naturally produced in skin in response ultraviolet-B (UVB) radiation from the sun. Several recent studies, however, have shown that a high proportion of community-dwelling subjects in both tropical and temperate climates are deficient in vitamin D, and that subjects in northern latitudes often require dietary supplementation to maintain vitamin D sufficiency. Further, several reports now suggest that vitamin D sufficiency is protective against the development of several cancers, including non-Hodgkin lymphoma (NHL). However, it is not known whether vitamin D impacts prognosis in diffuse large B-cell lymphoma (DLBCL), the most common NHL subtype. Methods:We evaluated serum vitamin D concentrations in two cohorts of DLBCL patients. The first cohort consisted of 374 patients newly diagnosed from September 2002-February 2008 who were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource, an observational study in which neither therapeutic nor diagnostic management is prescribed. All serum was obtained within 120 days of diagnosis, and was prior to treatment in 221 (59%). In the second study, 62 patients newly diagnosed from February 2006-August 2007 were enrolled on NCCTG clinical trial N0489, and all serum was obtained before the first course of therapy. Central pathology review was performed on all patients to confirm the diagnosis of DLBCL. Serum 25-hydroxyvitamin (25-OH-VitD) levels were measured by the gold standard method for vitamin D determination: liquid chromatography tandem mass spectroscopy (LC-MS/MS). Vitamin D deficiency was defined as total serum 25-OH-VitD < 25 ng/mL (62.5 nmol/L). SPORE patients were followed per a standard protocol for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively); N0489 patients were followed in a similar way per the clinical trial protocol. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CI). Results:SPORE patients were primarily treated with immunochemotherapy (83%); all N0489 patients were treated with epratuzumab + R-CHOP21. The median age at diagnosis was 62 years (range, 19-93) for SPORE patients and 61 years (range, 21-82) for N0489 patients. Median follow-up for SPORE patients was 36 months (range, 1-77) with 95 deaths and 131 events; median follow-up for N0489 patients was 24 months (range, 7-39) with 13 deaths and 18 events. Vitamin D deficiency was identified in 188 SPORE patients (50%) and 15 N0489 patients (24%). There were no differences in the prevalence of vitamin D deficiency by age, IPI, or whether serum was obtained prior to starting treatment (all p > 0.28). Vitamin D deficiency was associated with inferior overall (HR=2.33, 95% CI 1.53-3.57, logrank p = 0.0001) and event-free survival (HR=1.71, 95% CI 1.20-2.44, logrank p = 0.003) in the SPORE cohort. These associations remained significant after adjusting for IPI and treatment: OS HR=1.97, 95% CI 1.27-3.07; EFS HR=1.47, 95% CI 1.02-2.21. These results were similar for the subset of SPORE patients who were treated with R-CHOP. The HRs for vitamin D deficiency and event-free survival were consistent in the N0489 patients (HR=1.63, 95% CI 0.61-4.35, IPI adjusted HR=1.43, 95% CI 0.53-3.85), although these results lacked precision due to a smaller sample size. We were unable to evaluate OS in N0489 due to the small number of deaths. Conclusions:Approximately 50% of all DLBCL patients in this northern US latitude population are vitamin D deficient at the time of diagnosis and treatment. Vitamin D deficient patients have an inferior event-free and overall survival compared to patients with vitamin D levels within the normal range. Vitamin D supplementation during treatment of DLBCL patients with vitamin D deficiency should be evaluated in a clinical trial setting.CohortSerum 25-OH-VitD levels (ng/mL)<10 (severe deficiency)10 – 24 (mild to moderate deficiency)25 – 80 (optimal levels)SPORE29 (8%)159 (43%)186 (50%)N04892 (3%)13 (21%)47 (76%)All DLBCL31 (7%)172 (39%)233 (53%)▪▪ Disclosures:No relevant conflicts of interest to declare.