Diffuse Large B-cell Lymphoma Group Research Articles

Analysis of microsatellite aberrations and loss of heterozygosity in follicular lymphoma, diffuse large B-cell lymphoma, and high-grade B-cell lymphoma patients

Background . The study of genetic predictors of non-Hodgkin’s lymphomas prognosis is one of the most relevant areas of oncohematology. It is extremely interesting to search for integral markers that reflect the most important stages of tumor pathogenesis. DNA repair system plays one of the key roles in genomic instability. Aberrations of microsatellite repeats such as microsatellite instability (MSI), in particular elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is characteristic for mismatch repair system and loss of heterozygosity (LOH) is an integral feature of genomic instability. Objective . Analysis of MSI, EMAST, LOH significance in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and high-grade B-cell lymphoma (HGBL) patients. Materials and methods . The study was performed by multiplex PCR and fragment analysis with diagnostic panels COrDIS Plus and COrDIS MSI in 85 FL patients, 32 DLBCL patients, and 37 HGBL patients. Results . The frequency of LOH in the general FL group was 40/81 (49.4 %), MSI – 10/82 (12.2 %), EMAST – 15/81 (18.5 %). The frequency of LOH in the HGBL group was 21/31 (67.8 %), MSI – 11/37 (29.7 %), EMAST – 13/31 (41.9 %). The frequency of LOH in the DLBCL group was 18/29 (62.0 %), MSI – 5/32 (15.6 %), EMAST – 14/32 (43.8 %). When considering the morphological types of FL, it was noted that a higher frequency of genetic aberrations was characteristic of lymphomas with a more aggressive morphology (p <0.05). LOH identifies FL and HGBL patients with an unfavorable prognosis. The EMAST analysis allows identifying additional patients in the LOH+ cohort with early events and low EFS. Conclusion . LOH and EMAST have a prognostic value for FL and HGBL. No associations of LOH and EMAST with the survival were observed in DLBCL. Changes in mononucleotide repeats in FL, DLBCL and HGBL did not correspond to the MSI-H characteristic of solid tumors. For this reason, the clinical consequences of MSI-H in solid neoplasms, in particular the efficacy of immune checkpoint inhibitors, in lymphomas cannot be expected to be replicated solely on the basis of microsatellite aberrations detection.

Analysis of the Correlation of Basic Fibroblast Growth Factor in Serum of Patients with Diffuse Large B-Cell Lymphoma with Clinicopathological Efficacy and International Prognostic Index.

The correlation of basic fibroblast growth factor (bFGF) in serum of patients with diffuse large B-cell lymphoma (DLBCL) with clinicopathological efficacy and International Prognostic Index (IPI) is analyzed. 115 DLBCL patients admitted to our hospital for treatment from June 2020 to June 2021 are selected as the DLBCL patient group, 65 healthy subjects who received physical examination in our hospital during the same period are selected as the healthy control group, and the serum bFGF levels of DLBCL group and healthy control group are observed before treatment. The experimental results show that the serum bFGF expression of DLBCL patients is decreased significantly after chemotherapy, and the serum bFGF expression of DLBCL patients is closely related to the treatment effect, disease progression, tumor invasion, and prognosis, which has important clinical significance for judging the disease, treatment effect, and prognosis of patients.

Upregulation of CD47 Expression in De Novo Diffuse Large B-Cell Lymphoma Is More Frequent in Activated B-Cell Type

CD47 is a marker of self that provides a “don't-eat-me-signal” through activation of signal-regulatory protein alpha (SIRPa), a cell surface receptor expressed on monocytes/macrophages and granulocytes. This interaction negatively regulates effector functions such as, phagocytosis, migration, and superoxide production. Upregulation of CD47 expression in cancer, including diffuse large B-cell lymphoma (DLBCL), has emerged as a mechanism to escape innate immune surveillance. Using conventional immunohistochemical detection, we assessed CD47 expression in DLBCL and interrogated its association with clinicopathologic features.Patients with de novo DLBCL were identified from two large institutions and were uniformly treated with R-CHOP and had sufficient material for study. Immunohistochemical stains (IHC) were performed on FFPE tissue (Hans algorithm, BCL2, MYC, and CD47) and scored semi-quantitatively from no reactivity (0) to strong (2 and 3; Figure 1). Mutational analysis using a 334 gene target sequencing panel, gene expression profiling using Lymph2Cx to determine the cell of origin (COO), and FISH analysis for MYC, BCL2, and BCL6 translocations, were performed. The Lymphgen tool (Wright et al, 2020) was also used to determine the DLBCL group. Fisher's exact test and Kaplan-Meier survival analysis for overall survival (OS) were performed and P <0.05 was considered significant.CD47 expression was assessed by IHC in a cohort of 152 cases of de novo DLBCL, including 107 cases of germinal center B-cell (GCB) type (70%), 37 cases of activated B-cell (ABC) type (24%), and 8 cases of intermediate type (5%). A total of 17 cases (11%) showed strong and diffuse CD47 expression with IHC scores of 2 or above (CD47hi). CD47hi cases were significantly more frequent in ABC DLBCL (24%, 9/37) than GCB DLBCL (6%, 6/107; P=0.003). The remaining 2 CD47hi cases were in the intermediate DLBCL group (25%, 2/8). ABC DLBCL with CD47hi showed more frequent mutations with TET2 (33% vs 7%; P=0.08) and ZFP36L1 (22% vs 0%; P=0.05) compared to cases with low expression of CD47 with IHC scores of less than 2 (CD47low). ABC DLBCL with CD47low showed more frequent mutations of NOTCH2 (18% vs 0%; P=0.31) and MYD88 (29% vs 11%; P=0.4) compared to CD47hi. GCB DLBCL with CD47hi showed frequent mutations of TP53 (67% vs 21%; P=0.026) and CCND1 (33% vs 0%; P=0.003) compared to CD47low. None of the 13 cases with double- or triple-hit for MYC, BCL2 and/or BCL6 showed CD47 expression. The Lymphgen tool showed that cases of DLBCL with CD47hi were mostly in the ‘other’ group (50%), with other groups represented such as ST2 (21%), EZB (14%), MCD and BN2 (1 case each). There was no difference in overall survival (OS) between CD47hi and CD47low DLBCL (5-year OS, 75% vs 72%; P=0.57), or with the GCB or ABC subtypes.Strong expression with CD47 is more frequent in ABC DLBCL and is seen in a subset of GCB DLBCL with mutations in TP53 and/or CCND1. The level of CD47 expression does not appear to predict OS in patients with DLBCL treated with R-CHOP. This study demonstrates that conventional immunohistochemical methods can readily identify DLBCL with high CD47 expression, and these patients may benefit from the use of anti-CD47 therapy. [Display omitted] DisclosuresHerrera: Gilead Sciences: Research Funding; Takeda: Consultancy; Tubulis: Consultancy; Karyopharm: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seagen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding.

Diagnosis and staging of diffuse large B-cell lymphoma using label-free surface-enhanced Raman spectroscopy

Non-invasive diagnosis and staging of diffuse large B-cell lymphoma (DLBCL) were achieved using label-free surface-enhanced Raman spectroscopy (SERS). SERS spectra were measured for serum samples of DLBCL patients at different progressive stages and healthy controls (HCs), using colloidal silver nano-particles (AgNPs) as the substrate. Differences in the spectral intensities of Raman peaks were observed between the DLBCL and HC groups, and a close correlation between the spectral intensities of Raman peaks with the progressive stages of the cancer was obtained, demonstrating the possibility of diagnosis and staging of the disease using the serum SERS spectra. Multivariate analysis methods, including principal component analysis (PCA), linear discriminant analysis (LDA), support vector machine (SVM) classifier, and k-nearest neighbors (kNN) classifier, were used to build the diagnosis and staging models for DLBCL. Leave-one-out cross-validation was used to evaluate the performances of the models. The kNN model achieved the best performances for both diagnosis and staging of DLBCL: for the diagnosis analysis, the accuracy, sensitivity, and specificity were 87.3%, 0.921, and 0.809, respectively; for the staging analysis between the early (Stage I & II) and the late (Stage III & IV) stages, the accuracy was 90.6%, and the sensitivity values for the early and the late stages were 0.947 and 0.800, respectively. The label-free serum SERS in combination with multivariate analysis could serve as a potential technique for non-invasive diagnosis and staging of DLBCL.

Programmed cell death-ligand 1 (PD-L1)+ tumour cells and low-reacting programmed cell death 1 (PD1)+ tumour-infiltrating lymphocytes predict poor prognosis in Epstein-Barr virus+ diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV)+ diffuse large B-cell lymphoma (DLBCL) has specific tumour cell characteristics, and these patients have worse outcomes than EBV-negative DLBCL patients. We compared 38 EBV+ DLBCL patients with 43 methotrexate-associated EBV+ B-cell lymphoproliferative disorders (MTX+/EBV+ BLPDs) and 30 non-germinal centre (GC) subtype DLBCL. Lymphoma cells of the EBV+ DLBCL group were positive for BCL2 in 17 patients (44.7%), CMYC in 23 patients (60.5%), and p53 in 33 patients (86.8%), which was significantly higher than in the MTX+/EBV+ BLPD group (P < 0.05), and were positive for CD30 in 29 patients (76.3%), compared with two in non-GC subtype DLBCL (6.7%) (P < 0.0001). Significantly more EBV+ DLBCL patients (n = 16, 42.1%) had programmed cell death-ligand 1 (PD-L1)+ tumour cells than patients with non-GC subtype DLBCL (n = 5, 16.7%; P = 0.024), and PD-L1+ tumour cells were more common in advanced stages than in early stages (P = 0.048). Twenty-five EBV+ DLBCL patients (69.4%) had few reactive PD1+ tumour-infiltrating lymphocytes (TILs), compared with 12 patients with MTX+/EBV+ BLPDs (37.5%) (P = 0.008). In the EBV+ DLBCL group, CD30, BCL2, CMYC, and p53 expression was not related to patient prognosis. Poor outcomes were associated with PD-L1+ tumour cells (P = 0.001) and low-reacting PD1+ TILs (P = 0.02), while their combination conferred a worse outcome (P < 0.0001). Immune evasion by PD-L1+ tumour cells and exhaustion of PD1+ TILs may occur in EBV+ DLBCL patients, and PD-L1/PD1 interactions may influence tumour progression and poor prognosis.

Predictive Factors of Event-Free Survival at 24 Months in Patients with Peripheral T-Cell Lymphoma: A Retrospective Study.

PurposeEvent-free survival at 24 months (EFS24) is known to be a surrogate marker for overall survival (OS) for patients with peripheral T-cell lymphoma (PTCL). We examined the role of EFS24 in PTCL compared to diffuse large B-cell lymphoma (DLBCL), and then assessed the clinical predictive factors of achieving EFS24.Materials and MethodsPatients with newly diagnosed PTCL treated with anthracycline-based chemotherapy were included. Subsequent OS was defined as the time elapsed from 24 months after diagnosis until death from any cause in those who achieved EFS24.ResultsOverall, 153 patients were evaluated, and 51 patients (33.3%) achieved EFS24. Patients who achieved EFS24 showed superior OS compared to patients who did not (p < 0.001). EFS24 could stratify the subsequent OS although it did not reach to that of the general population. After matching the PTCL group to the DLBCL group based on the international prognostic index, the subsequent OS in patients who achieved EFS24 was similar between the two groups (p=0.094). Advanced stage was a significant factor to predict the failing EFS24 by multivariable analysis (p < 0.001).ConclusionPatients with PTCL who achieve EFS24 could have a favorable subsequent OS. Since advanced disease stage is a predictor of EFS24 failure, future efforts should focus on developing novel therapeutic strategies for PTCL patients presenting with advanced disease.

The Incidence of HBV Reactivation in Patients Receiving First-Line R-CHOP Chemotherapy: Three-Year Follow-up Results from a Prospective, Observational Study

Background:Due to the high rate of chronic hepatitis B virus (HBV) infection in China, hepatitis B surface antigen (HBsAg) can be detected among 25-61% of patients with diffuse large B-cell lymphoma (DLBCL) and 20-40% with follicular lymphoma (FL), while HBsAg- negative (neg) /HBcAb-positive (pos) can be detected among 20-44% of patients with DLBCL (Chinese Society of Hematology, 2013). When these patients receive immune-suppressing treatment, they are at an increased risk of HBV reactivation which may lead to hepatic mortality and interrupt the rituximab-based chemotherapy. For patients and their physicians, HBV infection management plays a crucial role for achieving optimal survival outcomes without compromising safety. This multicenter, single-arm, prospective, non-interventional study was conducted at 24 centers in China between January 17, 2011 and October 31, 2016. Previously untreated CD20-positive DLBCL patients who were eligible to receive rituximab plus chemotherapy (R-chemo) (CHOP or non-CHOP) as first-line treatment were enrolled with no specific exclusion criteria. We investigated the risk of HBV reactivation in patients with CD20+ DLBCL or FL which received R-chemo as first-line treatment.Methods:Patients who received at least one dose of R-chemo were included in the analysis. Data were collected and analyzed from medical records for 3 years since the last R dose was administered. The evaluation of HBV infection management included HBV infection and liver function screening before R-chemo, viral replication monitoring during and after R-chemo, antiviral prophylaxis use and HBV reactivation rates. HBsAg-pos patients had HBV reactivation if HBV DNA increased ≥1 log10 from baseline, if HBV DNA appeared (above the lower limit of detection) or if HBeAg appeared in HBeAg- neg patients. HBsAg-neg/HBcAb-pos patients had HBV reactivation if there was appearance of either HBsAg or HBV DNA (above the lower limit of detection). The HBV reactivation was determined by SAP and investigator's judgment. This study was registered in clincialtrials.gov (NCT01340443).Results:In total, 282 DLBCL patients and 31FL patients were included in the analysis. HBV screening data were available from 271 enrolled patients. At baseline, 26 patients were HBsAg-pos, 79 were HBsAg- neg/HBcAb-pos and 166 were double neg. HBV infection status was undefined for 42 patients. During the study, HBV reactivation was detected in 8 patients, which was only in the DLBCL group. Seven cases were observed in treatment period, while 1 case occurred during 3-year follow-up. None of them led to study termination, temporary termination, or dose adjustment.Cox univariate regression analysis in DLBCL patients showed that baseline type of hepatitis B correlated with overall response rate (ORR) and complete response (CR) but had no effect on progression-free survival (PFS).In term of HBV infection management, in DLBCL group, 25 patients with HBsAg-pos or HBsAg-neg/ HBcAb-pos receiving antiviral prophylaxis. In FL group, only 3 patients received antiviral treatment. Lamivudine was mostly administered by Chinese investigators. Details on treatment, duration and infection status are listed in Table 1.Summary:In this prospective, observational study, we followed the patients with DLBCL or FL for 3 years to investigate the risk of HBV reactivation after they received R-chemo. Our study showed that, in real-world setting, the incidence of HBV reactivation in patients with a history of hepatitis, particularly those positive for HBsAg is acceptable, baseline type of hepatitis B had no effect on PFS. Close monitoring and antiviral prophylaxis would be beneficial to the patients with DLBCL or FL combined with hepatitis B infection while they receive R-chemo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Downregulation of the Tumor Suppressor SAMHD1 in Classical Hodgkin Lymphoma

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. Therefore, a line of evidence suggests that SAMHD1 may play a role in oncogenesis as a tumour suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, and thus, it may lead to resistance to certain chemotherapeutic agents with cytarabine in acute myeloid leukemia being a recent example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of classical Hodgkin lymphoma (HL) are not yet known.Methods: SAMHD1 protein levels were assessed by Western blot analysis in 20 human lymphoma and leukemia cell lines including 5 classical HL (L1236, L428, HDML2, HDMyZ, MDA-V) and 3 diffuse large B-cell lymphoma (DLBCL) (RC-K8, OCI-Ly3, U2932) cell lines. As our findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, HL cell lines were treated with selective MYC inhibitors to assess possible MYC-associated regulation of SAMHD1 expression. The patient cohort included 212 cases of classical HL diagnosed and treated at the University of Texas MD Anderson Cancer Center (USA), University of Athens (Greece) and University of Crete (Greece). A control group of 4 reactive lymph nodes and 76 B-cell non-Hodgkin lymphomas (58 DLBCLs) was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on TMAs with duplicate tumor cores from each case or full tissue sections using a monoclonal antibody (Bethyl Laboratories, San Antonio, TX). An arbitrary 10% cutoff was used to define SAMHD1 positivity in the neoplastic cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models.Results: In 4 out of 5 HL cell lines, SAMHD1 protein levels were dramatically reduced as compared to all 3 DLBCL and other lymphoma and leukemia cell lines tested. Inhibition of MYC activity resulted in a substantial increase in the SAMHD1 protein level suggesting a possible MYC-associated gene regulation. Similarly, the neoplastic Hodgkin-Reed Sternberg (HRS) cells of classical HL were SAMHD1-negative in 132 of 212 (62.3%) patients. Among the SAMHD1-positive cases, the percentage of positive HRS cells ranged from 10 to 95% with a median 40%. By contrast, SAMHD1 was negative in only 15% of the control group of DLBCLs. In addition, most of the germinal center (GC) cells of the reactive lymph nodes were SAMHD1-positive with consistently higher intensity of immunostaining as compared to HRS cells of classical HL. No significant associations between SAMHD1 levels and clinicopathologic parameters were observed in the HL cohort. In a subset of 56 HL patients with long follow up period (median 195 months), FFS at 10 years was 79% vs. 71% for patients with SAMHD1-negative vs. SAMHD1-positive HL, respectively. Similarly, OS at 15 years was 71% vs. 63% for SAMHD1-negative vs. SAMHD1-positive HL, respectively. These differences did not reach statistical significance (p>0.05, log-rank) likely because of the relatively small number of patients included in this preliminary survival analysis.Conclusions: SAMHD1 is shown for the first time to be downregulated in HRS cells of classical HL further supporting its role as a tumor suppressor and its regulation may involve MYC. Preliminary survival analysis shows a statistical trend towards inferior FFS and OS for patients with SAMHD1-positive HRS cells, however, analysis of the entire cohort of HL patients is underway to draw definite conclusions. DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Kiromics: Research Funding; Stemline Therapeutics: Research Funding.

Relationship between Polymorphism of miR-155 and Its Target Gene MyD88 and Clinicopathological Features of Diffuse Large B-cell Lymphoma

To investigate the relationship between the polymorphism of miR-155 and its target gene MyD88 and clinicopathological features of diffuse large B-cell lymphoma (DLBCL). 135 cases of DLBCL patients in our hospital from March 2015 to August 2017 were selected, and 90 cases of reactive hyperplasia of lymph nodes were selected as the control group. The relative expression of miR-155 and MyD88 gene polymorphism were detected in the two groups, and the relationship between miR-155 and MyD88 gene polymorphism and clinicopathological characteristics of DLBCL was analyzed. The relative expression of miR-155 in DLBCL patients was significantly higher than that in the control group (P<0.05). The mutation rate of MyD88 L265P in DLBCL group was significantly higher than that in control group (P<0.05). The relative expression of miR-155 in patients with MyD88 L265P mutation was significantly higher than that in patients with wild-type DLBCL (P<0.05). The relative expression of miR-155 and the polymorphism of MyD88 L265P were associated with lesion location, stage, BCL-2 protein expression and MyD88 protein expression in DLBCL patients (t=7.461、8.804、6.487、10.812； χ2=10.681、8.599、7.251、23.008；P<0.05). The survival of DLBCL patients with low miR-155 expression was significantly better than that with high miR-155 expression (P<0.05). The survival of wild-type DLBCL patients with MyD88 L265P locus was significantly better than that of mutant DLBCL patients (P<0.05). There was a significant positive correlation between the relative expression of miR-155 and the expression of MyD88 protein (r=0.428, P=0.000). The abnormal expression of miR-155 and the mutation rate of MyD88 gene in DLBCL patients are increased, and the expression of miR-155 and the mutation of MyD88 gene affect the disease progression and prognosis of patients, which may be potential biological indicators for the diagnosis, treatment and prognosis of DLBCL.

Gut Microbiota in Untreated Diffuse Large B Cell Lymphoma Patients.

Intestinal microecology plays an important role in the development and progression of hematological malignancies. However, characteristics of gut microbiota in diffuse large B cell lymphoma (DLBCL) have not been reported. The microbiota composition of fecal samples from 25 untreated DLBCL patients and 26 healthy volunteers was examined by 16S rRNA gene sequencing. On α-diversity analysis, there was no significant difference in species diversity and abundance between the two groups. However, a significant difference was observed on β-diversity analysis. The intestinal microbiota in patients with DLBCL showed a continuous evolutionary relationship, which progressed from phylum, proteobacteria, to genus, Escherichia-Shigella. Their abundance was significantly higher than that of the control group. At the genus level, Allisonella, lachnospira, and Roseburia were more abundant in patients with DLBCL than in the control group. Functional prediction by PICRUSt indicated that thiamine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis were significantly lower in the DLBCL group than in the control group. In conclusion, our results clearly demonstrate that the gut microbiota was changed significantly in DLBCL. The study highlights fundamental differences in the microbial diversity and composition of patients with DLBCL and paves the way for future prospective studies and microbiome-directed interventional trials to improve patient outcomes.

Clinicopathological analysis of 34 Japanese patients with EBV-positive mucocutaneous ulcer

Epstein–Barr virus (EBV)-positive mucocutaneous ulcer (EBVMCU) is a unifocal mucosal or cutaneous ulcer that is histologically characterized by proliferating EBV-positive atypical B cells. While EBVMCU demonstrates a histology similar to that of EBV-positive diffuse large B-cell lymphoma (DLBCL), their clinical behavior differs. Thus, characterizing distinguishing features of EBVMCU and EBV-positive DLBCL is critical. To identify unique characteristics between EBVMCU and lymphoma, we analyzed the clinicopathological and genetic features of 34 Japanese patients with EBVMCU and compared them to those of 24 EBV-positive DLBCL patients and 25 EBV-negative DLBCL patients. All patients with EBVMCU had localized ulcerative lesions, and 31 patients (91%) were using immunosuppressants, such as methotrexate (MTX) or hydroxycarbamide. All patients that were followed up with exhibited good prognosis following immunosuppressant reduction or chemotherapy. In addition, 17 EBV-positive DLBCL patients, and 15 EBV-negative DLBCL patients, received chemotherapy (P < 0.001, P < 0.001, respectively). Our data showed that EBVMCU did not increase indicators associated with lymphoma prognosis, such as soluble interleukin 2 receptor (sIL-2R) and lactate dehydrogenase (LDH) compared to those in the EBV-positive DLBCL or EBV-negative DLBCL groups (sIL-2R, P < 0.001, P = 0.025; LDH, P = 0.018, P = 0.038, respectively). However, histologically, EBVMCU exhibited EBV-positive, variable-sized, atypical B-cell proliferation. Thus, EBVMCU was histologically classified as: (1) polymorphous; (2) large cell-rich; (3) classic Hodgkin lymphoma-like; and (4) mucosa-associated lymphoid tissue lymphoma-like. Moreover, genetic analysis showed that immunoglobin heavy chain (IGH) gene rearrangement did not differ significantly between EBVMCU and EBV-positive DLBCL (44% vs. 32%; P = 0.377), or between EBVMCU and EBV-negative DLBCL (44% vs. 58%; P = 0.280). Therefore, it is difficult to distinguish EBVMCU from EBV-positive DLBCL using only pathological and genetic findings, suggesting that clinical information is important in accurately distinguishing between EBVMCU and EBV-positive DLBCL.

Clinical Characteristics and Outcome of Diffuse Large B Cell Lymphoma Among HTLV-1 Carriers in Peru: A Matched Cohort Study

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common variant of non-Hodgkin lymphoma (NHL) accounting for approximately 30% of the NHL cases worldwide. Previous reports have associated certain viral infections with the development of DLBCL such as HIV and EBV, both infections related with an aggressive clinical course and worse outcome. The human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus regarded as the pathogenic agent for adult T-cell lymphoma/leukemia. HTLV-1 is endemic in Japan, the Caribbean basin, South America, and parts of Africa. In Peru, up to 3% of the healthy adult population carries HTLV-1. As data on the impact of HTLV-1 infection in DLBCL outcomes is scarce, we aim to describe the clinical features and outcomes of HTLV-1-positive patients with a pathological diagnosis of DLBCL. Methods: We retrospectively reviewed medical records of patients diagnosed and managed for DLBCL at the National Institute of Neoplastic Diseases in Lima-Peru between 2007 and 2019. Patients were evaluated for HTLV-1 infection at the time of diagnosis. Positive HTLV-1 cases were matched to negative HTLV-1 controls based on age, sex, and cancer staging. Treatment responses were assessed according to the Lugano criteria. Overall survival (OS) and event-free survival (EFS) curves were estimated using the Kaplan-Meier method and compared with the Log-rank test to determine the impact of HTLV-1 infection. Multivariate Cox regression models were reported with adjusted Hazar Ratios (aHR) with a 95% confidence interval (95% CI). Results: A total of 192 patients with DLBCL were identified and had sufficient data for analysis. Seventy (37%) cases were positive for HTLV-1 infection and 122 (63%) were not. Table 1 summarizes the clinical features and outcomes of DLBCL patients according to HTLV-1 status. Overall, the majority of patients were ≥65 years (59%), had ECOG performance status ≤2 (95%) and were stage III-IV (51%) at diagnosis. One third (n=64) of patients had extranodal involvement with 71 affected sites of which bone marrow involvement was frequently found in HTLV-1-negative DLBCL cases (55% vs. 7%, p&amp;lt;0.001) and liver/gastrointestinal tract in HTLV-1-positive cases (48% vs. 9%, p&amp;lt;0.001). There was no difference among DLBCL groups regarding risk stratification based on NCCN-IPI score (p=0.394). With a median follow-up of 6.5 years, we found that in DLBCL patients, HTLV-1 infection had no significant impact in 5-year OS (HTLV-1-positive 40% versus HTLV-1-negative 42%, p=0.930) and EFS rates (HTLV-1-positive 33% versus HTLV-1-negative 32%; p=0.890) (Figure 1). Multivariate cox regression analysis could not identify HTLV-1 infection as a risk factor for higher mortality or disease progression (Figure 1). Conclusion: To the best of our knowledge, this is the largest case series describing the clinical characteristics and outcome of HTLV-1-positive DLBCL patients. A study from Japan on early stage localized (head and neck) B-cell-NHL (n=198, HTLV-1 seropositive n=21 and with DLBCL n=12) treated with radiotherapy and/or multi-agent chemotherapy found poorer prognosis on HTLV-1 carriers compared to non-carriers (5-year OS: HTLV-1-positive n=21, 49% vs. HTLV-1-negative n=177, 78%, p=0.007; Hiroaki et al BJH 2003). In this study, we included DLBCL patients with both early and advanced stage disease along with localized and extranodal involvement. We found that HTLV-1 infection had no significant impact on 5-year OS and EFS rates when using conventional therapy for DLBCL. Moreover, we did not find differences in relapsed and mortality rates. Further investigation is needed to confirm the potential impact of HTLV-1 infection in DLBCL outcome. Disclosures No relevant conflicts of interest to declare.

Diffuse Large B Cell Lymphoma and Programmed Death-Ligand 1 Expression. a Clinical and Pathological Study of Patients Seen in Tawam Hospital UAE

Introduction: Diffuse Large B Cell Lymphoma (DLBL) is the most common type of Non-Hodgkin's Lymphoma (NHL) seen in Tawam hospital UAE. DLBL is a heterogenous disease arising either from a Germinal center (GC) or non- GCB cells. Molecular signature has identified 3 different groups of DLBL with significantly different prognosis (Primary mediastinal, Germinal center and Activated B Cell type). The therapy for DLBL has been without innovation since introduction of Rituximab more than 20 years ago and using R-CHOP. Attempts at improving the outcome has been uniformly unsatisfactory either by adding additional drugs, increasing the dose of drugs or deceasing the frequency of each cycle. New biomarkers and or targets are needed to improve the prognosis of patients with DLBL. Programmed Death-Ligand 1 (PDL1) has been shown to protect tumor cells by inducing T cell apoptosis (Dong et al., 2002). PDL1 targeting agents have been approved for many different types of malignancies including Hodgkin's Lymphoma. The role of PDL1 in DLBL is still under intense study. We report our findings of PDL1 expression in DLBL patients in UAE. Methods: Patients diagnosed with DLBL between December 1, 2019 till July 15th 2020. Patient records were reviewed in this study. Data was collected regarding patient demographics, clinical stage, International prognostic index (IPI), pathology and cell of origin {using immunohistochemistry (IHC) and Han's criteria}. PDL1 testing was performed by using FDA approved PharmaDx Dako Ab 22C3 kit for IHC on the gold standard Link 48 Dako platform. PDL1 expression was reported as Tumor proportion score (TPS). TPS in this assay was the percentage of viable tumor cells showing any perceptible partial or complete linear membranous (or membranous &amp; cytoplasmic) staining, in the large lymphoma cells. The specimen was considered PDL1 positive if the TPS was greater than or equal to 30%(Xu-Monette et al Blood 2018). All scores were reported by single pathologist Results Twenty-six patients were diagnosed with DLBL in the study period. The median age was 44 years (range 18-90 years). Majority of patients (75%) were less than sixty years of age. Advanced stage disease was seen in preponderance (66%) as compared to early stage (34%) while the LDH was elevated in 65 % patients (institutional normal &amp;lt; 225 IU per liter). Majority of patients (60%) were poor risk with IPI 3 or higher. Pathology review showed a uniformly elevated Ki 67 with the majority of specimens showing greater than 80 %. Molecular classification showed GCB type 50 % (n=13) as compared to non-GCB 42 % (n=11), data was not available for 2 patients. Epstein-Barr virus encoded small RNA (EBER) CISH was positive in only 3 patients (data was not available for 8 patients). PDL1 expression was tested in eighteen (GCB n=8, ABC n=10) patients. PDL1 expression was elevated in ABC type (90%) as compared GCB type (50 %) Fig 1. Conclusion The median age (44 years) is at least 2 decades less than the reported western cohort. Majority of the patients had high risk disease with advanced stage and IPI. Ki 67 was uniformly elevated (&amp;gt;80 % in 90 % of patients). There was a slight preponderance of GCB type (50 % vs 42%). PDL1 expression in ABC type DLBL was higher as compared to GCB. This observation needs to be validated in a larger cohort. Recent literature makes it very clear that ABC type DLBL has a poor outcome as compared to GCB type. PDL1 may offer an attractive target for treatment evaluation in ABC type DLBL. Figure Disclosures Alam: Pfizer:Honoraria;Roche:Honoraria.McCarthy:Karyopharm:Consultancy, Honoraria;Magenta:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Janssen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Takeda:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Juno Therapeutics, a Bristol-Myers Squibb Company:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board , Research Funding is to Roswell Park, Research Funding;AbbVie:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Genentech:Consultancy, Honoraria;Starton:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Abstract IA08: The role of gene expression in the classification of aggressive B-cell lymphoma

Abstract Aggressive B-cell lymphomas collectively make up half of all lymphoma diagnoses. The current classification system, the 2017 revision of the WHO 4th edition, assigns these tumors into groups based on morphology, immunophenotype, site of disease, and the presence of recurrent chromosomal rearrangements. Accurate and reproducible diagnosis is required for selection of optimal treatment, prognostication, and ongoing basic research and clinical trials aimed at improving outcomes. Ideally, the taxonomy would continue to evolve towards further defining homogeneous groups of tumors sharing targetable biology. Gene expression (GE) profiling of tumors supports the biologic validity of the entities in the current classification and, along with genomic sequencing, is driving the identification of new lymphoma subtypes. In the mid-2000s GE profiling studies identified specific signatures that distinguish aggressive B-cell entities from each other—namely, primary mediastinal large B-cell lymphoma (PMBL) from diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) from DLBCL. The increasingly divergent treatment of these entities makes reliable diagnosis important. Prior to those studies, GE profiling identified 2 distinct subtypes of DLBCL. This binary division of DLBCL into the cell-of-origin groups of germinal center B-cell-like (GCB) and activated B-cell-like (ABC) has been foundational to our understanding of the pathology of DLBCL. However, recent failure of clinical trials to improve outcomes by adding targeted agents to R-CHOP in upfront treatment of ABC-DLBCL highlights that this binary division may not be sufficiently granular to support precision medicine. These signatures have been translated onto tractable technology platforms, including nuclease protection assay, RT-MLPA, and NanoString, allowing potential integration into diagnostic workflows. More recently, GE signatures have been described that identify distinct molecular subtypes within GCB-DLBCL. Working from the standpoint of tumors that have GE profiles sitting between BL and DLBCL (the “molecular high grade” signature [MHG]) or the GE signature of tumors with rearrangement of MYC and BCL2 (the “double hit” signature [DHITsig]), a sizeable group of GCB-DLBCL can be identified with poor prognosis. Finally, a group of DLBCL without mediastinal involvement have been shown to display the PMBL GE signature. These tumors share perturbation of the hallmark pathways of PMBL but arrive at this biology through different genetic mechanisms. These subtypes map onto, and complement, the newly minted genetics-based classifications of DLBCL. In order to arrive at a tractable unified molecular classification for aggressive B-cell lymphoma, ongoing efforts are needed to integrate GE and genetic aberrations across the disease spectrum. Such a classification framework holds the promise of improved diagnostic accuracy and reliability while providing the foundation for improving patient outcomes through precision medicine. Citation Format: David W. Scott. The role of gene expression in the classification of aggressive B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA08.

Mechanism of MiR-224 Affecting DLBCL Cell Proliferation and Invasion by Targeted Inhibition of PIK3CD

To investigate the potential mechanisms of miR-224 affecting the proliferation and invasion of diffuse large B-cell lymphoma (DLBCL) cells. The blood samples of 76 DLBCL patients(DLBCL group) diagnosed at the First Affiliated Hospital of Kunming Medical University from June 2011 to December 2017, and 41 healthy persons of physical examination (normal control group) as well as human lymphatic endothelial cells (HELC) and DLBCL cell lines HBL1, OCI-LY10, OCI-LY8, OCI-LY19 were collected. The expression of miR-224 and PIK3CD mRNA was measured by RT-qPCR. The proliferation of HBL1 cells was detected by CCK-8 method and colony formation test. The invasion ability of HBL1 cells was detected by Transwell test. Dual-luciferase reporter gene assay was used to verify the relationship between miR-224 and PIK3CD. The expression of PIK3CD protein was measured by Western blot. The expression of miR-224 in blood of DLBCL patients was very significantly lower than that in normal control group (P<0.01). The overexpression of miR-224 significantly decreased proliferation and invasion of HBL1 cells (all P<0.01). PIK3CD was a target gene of miR-224. Knockdown of PIK3CD significantly suppressed the proliferation and invasion of HBL1 cells (all P<0.01). MiR-224 plays a key role in the progression of DLBCL, and the proliferation and invasion of HBL1 cells can be suppressed by targeted inhibition of PIK3CD.

DLBCL with amplification of JAK2/PD-L2 exhibits PMBCL-like CNA pattern and worse clinical outcome resembling those with MYD88 L265P mutation

BackgroundRecently, copy number alteration (CNA) of 9p24.1 were demonstrated in 10% of diffuse large b-cell lymphoma (DLBCL), with gene expression and mutation profiles that were similar to those of primary mediastinal large B-cell lymphoma (PMBCL). However, their CNA-based profile and clinical impact still remain unclear.MethodsMultiplex ligation-dependent probe amplification were employed to investigate the prevalence of JAK2/PD-L2 amplification in DLBCL and their CNA-based pattern of driver genes. The clinical outcome and characteristics were also analyzed.ResultsUsing unsupervised hierarchical clustering, a small group of DLBCL (10.5%, 8/76) was clustered together with PMBCL as Cluster_2, demonstrating amplification of JAK2 (100%,8/8) and PD-L2 (75.0%,6/8). This subgroups of DLBCL demonstrated significant higher expression of PD-L1 than those with MYD88 L265P mutation(p = 0.024). And they exhibited dismal OS and PFS as compared with DLBCL_others(p = 0.003 and 0.001, respectively), which is similar to DLBCL with MYD88 L265P mutation.ConclusionsDLBCL with amplification of JAK2/PD-L2 exhibits CNA pattern that is similar to PMBCL, and demonstrates unfavorable clinical outcome that resembles those with MYD88 L265P mutation. It is essential to identify this subgroup of DLBCL who may acquire more benefits from the JAK2 and PD-L1 signaling inhibition.

Aberrant peripheral blood CD4+ CD25+ FOXP3+ regulatory T cells/T helper-17 number is associated with the outcome of patients with lymphoma.

Little is known about the clinical significance of the peripheral blood CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) and T helper-17 (Th17) cells in lymphoma patients. In this study, the prognostic and clinical significance of peripheral blood Tregs and Th17 cells were evaluated in lymphoma patients during different phases. The frequency of Tregs and Th17 lymphocytes was measured by flow cytometry method in 47 classical Hodgkin's lymphoma (cHL) and 48 diffuse large B cell lymphoma (DLBCL) patients. Our results showed that the frequency of Tregs and absolute Treg count was significantly reduced in relapsed patients compared to patients at the remission phase, as well as with newly diagnosed untreated patients in both groups. Patients who reached complete remission had elevated frequency of CD4+ FOXP3+ lymphocytes, Tregs, absolute Treg count, Treg/CD4 and Treg/Th17 ratio in the cHL group and CD4+ CD25+ cells in DLBCL group. The frequency of Tregs, absolute Treg count and Treg/Th17 ratio in cHL patients and CD4+ FOXP3+ and CD4+ CD25+ cells in DLBCL patients positively associated with survival rate. Moreover, the percentage of Tregs and absolute Treg count positively correlated with white blood cell, platelet count and ESR level in cHL patients and with white blood cell count in DLBCL patients. The initial number of Tregs/Th17 cells and also the Treg/Th17 ratio was not associated with changes in disease-free survival (DFS) in both groups. Therefore, higher frequency of peripheral blood Tregs and Treg/Th17 ratio might be associated with a favorable outcome in lymphoma patients, better response to chemotherapy and lower rate of relapse.

Comparing CAR T-cell toxicity grading systems: application of the ASTCT grading system and implications for management

AbstractVarious grading systems are currently used for chimeric antigen receptor (CAR) T-cell–related toxicity, cytokine release syndrome (CRS), and immune effector cell–associated neurotoxicity syndrome (ICANS). We compared the recently proposed American Society for Transplantation and Cellular Therapy (ASTCT) grading system to other grading scores in 2 populations of adults: patients (n = 53) with B-cell acute lymphoblastic leukemia (B-ALL) treated with 1928z CAR T-cells (clinicaltrials.gov #NCT01044069), and patients (n = 49) with diffuse large B-cell lymphoma (DLBCL) treated with axicabtagene-ciloleucel (axi-cel) or tisagenlecleucel after US Food and Drug Administration approval. According to ASTCT grading, 82% of patients had CRS, 87% in the B-ALL and 77% in the DLBCL groups (axi-cel: 86%, tisagenlecleucel: 54%), whereas 50% of patients experienced ICANS, 55% in the B-ALL and 45% in the DLBCL groups (axi-cel: 55%, tisagenlecleucel: 15%). All grading systems agreed on CRS and ICANS diagnosis in 99% and 91% of cases, respectively. However, when analyzed grade by grade, only 25% and 54% of patients had the same grade in each system for CRS and ICANS, respectively, as different systems score symptoms differently (upgrading or downgrading their severity), leading to inconsistent final grades. Investigation of possible management implications in DLBCL patients showed that different recommendations on tocilizumab and steroids across current guidelines potentially result in either overtreating or delaying treatment. Moreover, because these guidelines are based on single products and different grading systems, they cannot be universally applied. To avoid discrepancies in assessing and managing toxicities of different products, we propose that unified grading be used across clinical trials and in practice and that paired management guidelines with product-specific indications be developed.

Easix and Modified-Easix Are Early Predictors of Severe Cytokine Release Syndrome and Neurotoxicity in Patients Treated with Chimeric Antigen Receptor T Cells

Easix and Modified-Easix Are Early Predictors of Severe Cytokine Release Syndrome and Neurotoxicity in Patients Treated with Chimeric Antigen Receptor T Cells

Comparing Gradings of Immune Effector Cells Toxicities: Application of Astct Consensus Grading System and Implications for Clinical Management

Comparing Gradings of Immune Effector Cells Toxicities: Application of Astct Consensus Grading System and Implications for Clinical Management