Negative Diffuse Large B-cell Lymphoma Research Articles

A Case of Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma Occurring in Thyroid Gland

Epstein-Barr virus positive diffuse large B-cell lymphoma is very rare malignant tumor in thyroid gland. Compared to Epstein-Barr virus negative diffuse large B-cell lymphoma, it tends to have more extranodal involvement, does not respond to chemoimmunotherapy well, and has worse prognosis. We have encountered a patient with Epstein-Barr virus positive diffuse large B-cell lymphoma of thyroid gland in 75-year-old female presenting with aggravating dyspnea and neck pain.

Development of Chimeric Antigen Receptor (CAR) T Cells Targeting MET in Lymphomas and Solid Tumors

Currently, multiple versions of CAR T cells targeting the cell surface protein CD19 are approved by the FDA to treat refractory B-cell malignancies. However, durable remissions range from 60% in B-cell leukemias to 40% in B-cell lymphomas. Major reasons for resistance to CD19-CAR treatment relate to heterogeneous or loss of CD19 expression. In addition, CD19-CAR leads to B-cell depletion which increases the risk for infection. These limiting factors necessitate research into alternative targets for CAR therapy against B-cell lymphomas. While HGF/MET signaling has been extensively studied in solid tumors, studies have also shown important roles of HGF/MET in hematologic malignancies. Approximately 30-70% of diffuse large B-cell lymphomas (DLBCL) express MET with moderate to strong intensity; as do other lymphomas such as classic Hodgkin lymphoma, Burkitt lymphoma, primary effusion lymphoma, and multiple myeloma. We hypothesize that these MET-positive B-cell lymphomas can potentially be targeted with chimeric antigen receptor (CAR) T-cells against MET receptor. We first looked at the DepMap database and found that a significant subset of aggressive and large B-cell lymphoma cell lines express MET such as Raji, Karpas-422, OCI-LY3, and OCI-LY132. Of note, some of them, like OCI-LY3 and OCI-LY132, lack detectable CD19. Flow cytometry using anti-MET antibody confirmed MET expression in these cell lines. Further analysis of RNAseq data from 500 CD19-CAR T-naïve primary patient samples (GSE125966) revealed MET expression in 62% (294/474) of CD19-positive and 61% (15/26) of CD19-low to negative DLBCL . We then constructed a second-generation anti-MET-CAR by fusing the scFv fragment of an anti-MET monoclonal antibody with the human CD28 hinge/transmembrane/cytoplasmic domain and CD3z cytoplasmic domain. Stable MET-CAR Jurkat T cells were incubated with various target lymphoma cells for 16-24 hours, and the percentage of Jurkat T cells expressing the activation marker CD69 was determined by flow cytometry. MET-CAR Jurkat showed increased CD69 expression in MET-positive lymphoma cell lines such as Karpas-422 and OCI-LY3. Karpas-422 demonstrated dose dependent activation of MET-CAR Jurkat cells, with higher CD69 expression in response to increasing amounts of MET antigen. OCI-LY3, a lymphoma cell line negative for CD19 but positive for MET, led to only basal level of CD69 expression in CD19-CAR Jurkat but elevated CD69 level in MET-CAR Jurkat. After showing MET-CAR Jurkat T cell activation by lymphoma cell lines, we tested cytotoxicity using primary human CD3+ T-cells against A549, a lung cancer cell line with known high level of MET expression, as a positive control for MET-CAR mediated cytotoxicity. Activated primary human T cells were electroporated with MET-CAR mRNA. Twelve hours post electroporation, the reprogrammed T cells were mixed with A549 cells at a 5:1 effector: target ratio (E:T). CellCyte proliferation analysis showed reduced A549 cell proliferation in the presence of MET-CAR T cells, in comparison to mock electroporated CD3+ T cells. We further demonstrated that the same mRNA MET-CAR T-cell was able to mediate substantial cytotoxicity against DLBCL cell lines such as Karpas-422 and OCI-LY3 ( Figure 1). At varying E:T ratio, MET-CAR T cell exhibited significant cytotoxicity against Karpas-422. Also, consistent with the CD69 expression data, while CD19-CAR T cell showed only low level of cytotoxicity against the CD19-/MET+ OCI-LY3 cell line, MET-CAR T cell was able to mediate substantial tumor cell lysis. In vivo testing against a xenograft model is currently underway. In summary, the preliminary data demonstrate that MET-CAR is capable of mediating cytotoxicity against MET-positive DLBCL. Some cell lines such as OCI-LY3 are negative for CD19 but positive for MET expression, and MET-CAR is capable of mediating cell lysis while CD19-CAR is ineffective. These results suggest that some large B-cell lymphomas negative for CD19, either intrinsic or secondary to loss of CD19 as a result of resistance mechanism, may show MET expression and will likely benefit from MET-targeted immunotherapy. Figure caption: Figure 1. Cytotoxicity of MET-CAR T cell against DLBCL cell lines. (A) MET-CAR against Karpas-422 at various E:T ratio. (B) Cytotoxicity of MET-CAR against OCI-LY3 which is positive for MET but negative for CD19.E:T ratio is 10:1.

Ophthalmoplegia and Tumor Lysis Syndrome: An Uncommon Presentation of Posttransplant Lymphoproliferative Disorder

A 55-year-old man presented with 1 month of fatigue, nausea, vomiting, and weight loss, and blurry vision in his left eye. He has a history of type 1 diabetes mellitus after allogenic kidney and pancreas transplantation 15 years prior. He was found to have tumor lysis syndrome (TLS), total left-sided ophthalmoplegia and ptosis, and left cavernous, abdomen, and pelvic masses. Biopsy revealed an Epstein-Barr virus–negative diffuse large B-cell lymphoma, a type of posttransplant lymphoproliferative disorder (PTLD). It is essential to consider PTLD in transplant patients who present with TLS and compressive neurologic deficits.

Case report of primary CD20 negative diffuse large B-cell lymphoma

ABSTRACTFew groups of aggressive non-Hodgkin’s lymphomas (NHL) that are refractory to standard chemotherapy are rarely reported. Primary CD20 negative diffuse large B cell lymphoma (DLBCL) without human immunodeficiency virus infection is an uncommon presentation and this case report is challenging in terms of diagnosis and treatment as well.

Mutation Analysis and Effect on Prognosis of Direct Antiglobulin Test Positivity in Diffuse Large B-Cell Lymphoma

IntroductionDirect Antiglobulin Test (DAT) positivity occurs at higher rates in patients with Non-Hodgkin Lymphoma and is often associated with poor prognosis. This study examined the genetic differences between DAT positive (DAT +) and DAT negative (DAT-) DLBCL cases, and the effect of DAT positivity on survival and revised international prognostic index (R-IPI) in Diffuse Large B-Cell Lymphoma (DLBCL).MethodOne hundred and thirteen cases that had undergone DAT testing at the time of diagnosis as part of a lymphoma screen were included. DAT was performed by the column agglutination method. Cell of origin was determined using the Hans criteria. DNA was extracted from 7 DAT+ and 8 DAT- patient samples and targeted next-generation sequencing (NGS) was performed. Non-synonymous mutations were then grouped into B-cell pathways in DAT+ and DAT- groups. Analysis of survival outcomes was assessed by the Kaplan-Meier method and Cox proportional hazards regression.ResultsThe median age of the cohort was 65.0 (Interquartile Range [IQR] 55.3-74.5) and the median follow up time was 2.1 years (IQR 0.7-5.0). Of the 113 cases, 23 (20.3%) were positive and 90 cases (79.7%) were negative. One of the DAT+ cases was diagnosed with clinical autoimmune hemolytic anaemia at the time of DLBCL diagnosis. The DAT+ group were significantly more likely to have a lactate dehydrogenase (LDH) level > upper limit of normal (ULN) at the time of diagnosis (91.3% vs 67.4%, p=0.034), and to be of non-Germinal Centre (non-GC) origin (63.2% vs 33.3%, p=0.033).An average of 10.07 mutations per DLBCL clone were found on NGS (DAT+ 10.14 vs DAT-10.00). Mutations were found in genes and B-cell pathways associated with oncogenesis and autoimmunity in both the DAT+ and DAT- groups. The most recurrently mutated genes were KMT2D (n=13), MYOM2 (n=9), EP300 (n=8), SPEN (n=7), and ADAMTSL3 (n=7), which were all mutated in both groups. The genes associated with the non-canonical NFκB pathway were found to be more frequently mutated in the DAT+ group, with 6 mutations in the DAT+ group compared with 1 mutation found in the DAT- group. Within the DAT+ group, three mutations were found in BIRC3, a gene involved in apoptosis regulation and previously linked to type 1 diabetes mellitus.There was a non-significant difference in overall survival between the DAT+ and DAT- groups (p=0.19), with a trend towards DAT+ patients being less likely to survive. DAT significantly influenced the effect of R-IPI on survival (p=0.017). Within the DAT- group, R-IPI maintained its strong predictive power for overall survival with three clear groups (p <0.0001). Within the DAT+ group, no patients fell into the “very good” R-IPI prognosis group, while the other two R-IPI groups were not significantly different in their overall survival (p=0.74).ConclusionBoth DAT positive and DAT negative patient groups showed high rates of non-synonymous mutations in genes that are frequently targeted in DLBCL. These findings suggest mutations in the non-canonical NFκB pathway, which is associated with lymphomagenesis and autoimmunity, may be more common in patients who are DAT positive. This study did not demonstrate a significant effect on survival of DAT positivity in patients with DLBCL, but a moderating influence on R-IPI's survival effect. This moderation may indicate that autoimmunity negates the survival benefits of a better patient risk profile. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinicopathological Study of Methotrexate-Associated Lymphoproliferative Disorders in Rheumatoid Arthritis Patients: Histological Classification and Predictive Factors for Disease Progression

Introduction: Rheumatoid arthritis (RA) patients treated with methotrexate (MTX) may develop lymphoproliferative disorders (LPD), and MTX-associated LPD (MTX-LPD) are recognized as iatrogenic immunodeficiency-associated LPDs. However, the clinicopathological features of MTX-LPD have not been well documented, and distinct diagnostic criteria have not been established. This study analyzed clinicopathological features of MTX-LPD to clarify its characteristics.Methods: Between 2004 and 2015, 193 patients with RA were diagnosed with MTX-LPD at Kurume University. Lesions were classified into the following 4 diagnostic categories: (1) reactive hyperplasia (RH) - lesions with lymphoid proliferation characterized by retention of architecture; (2) polymorphic LPD (Poly-LPD) - diffuse and destructive lesions composed of plasma cells, immunoblasts, and variably-sized lymphocytes, with lesions not fulfilling criteria for lymphoid neoplasms in immunocompetent hosts; (3) diffuse, large B-cell lymphoma (DLBCL) type - fulfilling criteria for immunocompetent hosts; (4) classic Hodgkin lymphoma (CHL) type - fulfilling morphologic and immunophenotypic criteria of CHL. Low grade B-cell lymphoma and peripheral T-cell lymphoma were excluded from this study.Results: All 193 patients received low-dose MTX; and the patients were classified as having RH (n=28), Poly-LPD (n=33), DLBCL type (n=107), and CHL type (n=25) lesions. The clinical features of patients with RH, Poly-LPD, DLBCL-type, and CHL-type lesions were as follows: median age, 62 (range, 26-81 years), 68 (42-83 years), 69 (39-88 years), and 66 (46-85 years); extra-nodal involvement, 11% (3/28), 36% (12/33), 70% (73/105), and 16% (4/25); multiple involved sites, 46% (12/26), 48% (16/33), 62% (64/104), and 48% (12/25), respectively. Of the patients who discontinued MTX without concurrent chemotherapy or surgery at the time of diagnosis, 100% (14/14), 73% (22/30), 49% (34/70), and 11% (2/19) of patients with RH, Poly-LPD, DLBCL-type, and CHL-type lesions had spontaneous regression. Among those who received chemotherapy, 78% (7/9), 72% (48/67), and 70% (14/20) of patients with Poly-LPD, DLBCL-type, and CHL-type lesions had complete remission. One, 17, and 2 patients with Poly-LPD, DLBCL-type, and CHL-type lesions had disease- or treatment-related death during the follow-up period (median, 25 months; range, 1-143 months).Respective histological features of RH, Poly-LPD, DLBCL-type, and CHL-type lesions were as follows: necrosis - 0% (0/27), 48% (16/33), 34% (36/107), 16% (4/25); angioinvasion - 0% (0/27), 9% (3/33), 14% (15/106), 4% (1/25); epithelioid clusters - 0% (0/27), 9% (3/32), 0% (0/105), 24% (6/25). Hodgkin/Reed-Sternberg-like cells were observed in 15% (4/27), 48% (14/29), and 25% (25/100) of patients with RH, Poly-LPD, and DLBCL-type lesions, respectively. Epstein-Barr virus-encoded RNA (EBER)-positive lymphocytes/neoplastic cells were observed in 64% (18/28), 75% (24/32), 54% (56/104), and 84% (21/25) of patients with RH, Poly-LPD, DLBCL-type, and CHL-type lesions, respectively. On immunohistochemical analysis, neoplastic cells, detected in patients with DLBCL-type and CHL-type lesions, were positive for each antibody as follows: CD20 - 98% (104/106), 0% (0/25); CD30 - 43% (40/93), 100% (25/25); CD15 - 6% (4/63), 48% (12/25); LMP-1 - 59% (43/73), 72% (18/25); EBNA-2 - 23% (15/65), 0% (0/22), respectively.Among patients who discontinued MTX without concurrent chemotherapy at diagnosis, RH and Poly-LPD patients had significant favorable chemotherapy-free survival (CFS) (p<0.0001, p=0.0120), while CHL patients had unfavorable CFS (p=0.0344) compared to DLBCL patients (Figure 1). Moreover, EBER-positive DLBCL patients had significantly favorable CFS compared to negative DLBCL patients (p=0.0046), although there were no significant differences in other categorized groups. Overall survival was not significantly different between any categorized groups; however, DLBCL patients with high risk according to the international prognostic index had unfavorable prognoses (p<0.0001).Conclusions: MTX-LPD patients of histological type RH and Poly-LPD lesions had favorable clinical outcomes, while CHL patients had significantly unfavorable clinical outcomes, compared to patients with DLBCL. Histological classification may help predict disease progression in patients with MTX-LPD. [Display omitted] DisclosuresTamaru:Takeda Pharmaceutical Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Nichirei Biosciences Inc.: Research Funding. Tokuhira:Ezai: Honoraria.

PET-CT Invisible Diffuse Large B-Cell Lymphoma Features a Silent B-Cell Receptor Signaling

Background:18F-fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET-CT) has been widely used in lymphoma patients for diagnosis, staging, assessment of response and surveillance. And the standardized uptake value (SUV), a commonly used indicator in PET-CT scan, can quantitatively reflect the glucose uptake and glycolysis of the tumor. Diffuse large B-cell lymphoma (DLBCL), one of the most common subtypes of aggressive lymphomas, is supposed to exhibit high standardized uptake value (SUV) in 18F-FDG PET-CT due to high rate of glucose (18F-FDG) uptake and high level of glycolysis. Nevertheless, PET-CT “invisible” DLBCL cases have also been described, which featuring a relatively low 18F-FDG uptake denoted by low SUV. Herein, we analyzed this subgroup of under-estimated disease.Methods: Nineteen cases each with PET-CT “invisible” (PET-CTi) and PET-CT “visible” (PET-CTv) de novo DLBCL were submitted for an immunohistochemical detection for the expression level of B-cell receptor (BCR) signaling-related molecules. Nine xenografts derived from BCR-dependent and BCR-independent DLBCL cell lines were further submitted for a small animal 18F-FDG PET-CT scanning and the following immunohistochemical detection for BCR signaling-related molecules.Results: The immunohistochemical expression level of pSYK (pY525/526), a key effector molecule for BCR signaling, was significantly lower in PET-CTi DLBCL tumor tissues compared with that in PET-CTv DLBCL cases (P <0.01). With regard to in vivo PET-CT experiment, the xenografts derived from BCR-independent DLBCL cell lines exhibited remarkable lower SUV level than those derived from BCR-dependent cell lines (P < 0.01). And BCR-independent DLBCL xenografts featured relatively lower expression level of pSYK compared with those BCR-dependent ones (P <0.01).Conclusions:18F-FDG PET-CT negative DLBCL features a silent BCR signaling and 18F-FDG PET-CT may be of use in evaluating BCR signaling status. DisclosuresNo relevant conflicts of interest to declare.

A Pilot Study of Brentuximab Vedotin, Rituximab and Dose Attenuated CHP in Patients 75 Years and Older with Diffuse Large B-Cell Lymphoma

Older patients with hematologic malignancies are underrepresented on prospective clinical trials relative to the incidence of disease in this group (Kanapuru et al, 2020). There are few studies in diffuse large B-cell lymphoma (DLBCL) focused specifically on older patients. Rituximab and dose attenuated cyclophosphamide, doxorubicin, vincristine, and prednisone (R-miniCHOP) has been studied on a prospective trial in fit patients aged 80 years and older. Seventy-two percent of patients on this study completed 6 cycles of R-miniCHOP. The overall response rate (ORR) was 73%, and the 2-year progression free survival (PFS) was 47% (Peyrade et al, 2011). Novel regimens are needed to improve upon the efficacy of therapy while preserving tolerability. Brentuximab vedotin (BV) has demonstrated activity in relapsed and refractory DLBCL (Jacobsen et al, 2015) as well as in combination with chemoimmunotherapy (Svoboda, 2020). This study evaluates the feasibility of BV with dose attenuated chemoimmunotherapy. Methods: Patients with both CD30 positive (cut off 1%) and CD30 negative DLBCL aged 75 years and older were enrolled on the study. Patients received six, 3-week cycles of BV 1.8 mg/kg, cyclophosphamide 400 mg/m2, doxorubicin 25 mg/m2, vincristine 1 mg and prednisone 40 mg/m2 days 1-5 (BV R-miniCHP). For the first cycle patients received BV and prednisone as a prephase starting one week prior to cycle 1. All patients received pegfilgrastim. All patients underwent geriatric assessments at screening, following prephase and at the end of treatment. The primary endpoint was feasibility of this regimen in older patients. The regimen was considered feasible if 71% of patients completed 6 cycles of treatment with a 90% confidence interval (CI)=(58.0, 90.6%). Secondary endpoints included toxicity, ORR and complete response (CR) evaluated by positron emission tomography, PFS and overall survival (OS). Response assessments used the Lugano Criteria (Cheson et al, 2014). PFS and OS were estimated using the Kaplan-Meier method. Results: Twenty-two patients were enrolled and started prephase with BV and prednisone. Their baseline characteristics are summarized in the table. Seventy-seven percent (17/22) of patients completed 6 cycles of BV R-miniCHP. Reasons for not completing treatment included progressive disease in 2 patients, myocardial infarction, fatigue, and an unrelated injury in 1 patient each. Twenty-one patients were evaluable for response. ORR was 86% (18/21) in all patients, with 67% (14/21) achieving CR. In CD30 positive patients the ORR was 80% (8/10) and the CR rate was 70% (7/10). In CD30 negative patients the ORR was 91% (10/11) and the CR rate was 64% (7/11). With a median follow up of 23 months, median OS and PFS (figure) were not reached. The 2-year PFS was 60.6%, 90% CI=(40.0%, 76.1%), and the 2-year OS was 73.9%, 90% CI=(52.4%, 86.8%). The most common adverse events (AEs) were fatigue (82%), anemia (50%), diarrhea (50%), dysgeusia (45%) and peripheral sensory neuropathy (45%). Grade ≥3 AEs seen in more than 2 patients included neutropenia (23%), fatigue (18%), pneumonia (18%), hypoxia (14%), thrombocytopenia (9%) and thromboembolism (9%). Grade ≥3 peripheral sensory neuropathy was seen in 9% of patients. There were two deaths in patients receiving study treatment. These included a myocardial infarction related to treatment, and a bowel obstruction secondary to disease progression. Three other patients have died in follow up with 2 secondary to disease progression and 1 due to an unrelated event. Conclusions: The study met its primary feasibility endpoint with 77% of patients completing 6 cycles of therapy. This regimen was delivered safely in this population and toxicities were consistent with those reported in larger prospective studies with R-miniCHOP or ofatumumab and miniCHOP (Peyrade et al, 2011; Peyrade et al 2017). Peripheral neuropathy is a key AE of interest given the inclusion of BV and while nearly half of patients experienced some peripheral neuropathy, it was severe in only 9% of pts. The ORR and CR rate, as well as the 2-year PFS compare favorably to other prospective studies in a population that included patients with high clinical risk, histologic transformation, and double hit lymphoma. BV and R-miniCHP may be a feasible regimen in older patients, and warrants further study based on these preliminary data demonstrating clinical activity and tolerability. Figure 1 Disclosures Reagan: Seattle Genetics: Research Funding; Curis: Consultancy; Kite, a Gilead Company: Consultancy. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Barr:Gilead: Consultancy; Verastem: Consultancy; Abbvie/Pharmacyclics: Consultancy, Research Funding; Genentech: Consultancy; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy; Morphosys: Consultancy; TG therapeutics: Consultancy, Research Funding; Merck: Consultancy. Friedberg:Acerta Pharma - A member of the AstraZeneca Group, Bayer HealthCare Pharmaceuticals.: Other; Roche: Other: Travel expenses; Kite Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Portola Pharmaceuticals: Consultancy; Astellas: Consultancy; Bayer: Consultancy.

PB1810 CLINICAL CHARACTERISTICS AND OUTCOMES OF DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL) LACKING THE IMMUNOGLOBULIN LIGHT CHAIN RESTRICTION (ILCR) BY FLOW CYTOMETRY

Background:The immunoglobulin light chain restriction (iLCR) detected by flow cytometric analysis is useful to prove the monoclonality of B‐cell lymphoma. However, we occasionally encounter cases of B‐cell lymphoma without iLCR. The lack of immunoglobulin expression on B‐cell lymphomas arises from abnormalities of gene transcription or translocation of fully assembled proteins on the cell surface. Few reports have evaluated patients of diffuse large B‐cell lymphoma (DLBCL) without iLCR, but the clinical characteristics and outcomes of DLBCL lacking iLCR has not been firmly elucidated.Aims:In the present study, we evaluated the clinical features and prognosis of DLBCL without iLCR.Methods:We conducted a retrospective analysis using a database of our hospital. From April 1, 2007 to March 31, 2017, patients who were performed flow cytometric analysis and diagnosed with DLBCL were eligible. The definition of iLCR was determined when κ‐positive cells were more than 3 times that of λ‐positive cells (κ‐DLBCL) or λ‐positive cells were more than 2 times that of κ‐positive cells (λ‐DLBCL) in dual‐color diagrams. We excluded cases with HIV infection, central nervous system involvement, intravascular lymphoma, primary mediastinal large B‐cell lymphoma, transformation of indolent lymphoma and cases in which flow cytometric analysis did not reflect the lymphoma cells. The endpoints of this study were overall survival (OS) and progression free survival (PFS) of iLCR‐positive/negative DLBCL and κ/λ‐DLBCL. The OS was defined as the time from diagnosis to the last follow‐up or death from any cause. The PFS was defined as the time from diagnosis to disease progression, relapse or death due to any cause.Results:70 patients were identified; 14 cases with iLCR‐negative (20%) and 56 cases with iLCR‐positive (80%), with a median age of 73 years (range 40–86), 80% &gt; 60 years, 44% male, 74% Stage III/IV, 63% IPI high–int/high, 41% were treated with CHOP‐based, 44% THP‐COP‐based (THP‐COP: pirarubicin, cyclophosphamide and vincristine on day 1 and predonisolone on days 1–5) regimen and 91% were combined with rituximab. Median observation time was 605 days (range 2–3223). There were no differences in clinical characteristics with regard to sex, age, serum lactate dehydrogenase (LDH), performance status (PS), stage, number of extranodal lesions, International Prognositic Index (IPI), B‐symptoms and bulky lesion between the two groups. Cell surface markers also showed no difference between iLCR‐positive and iLCR‐negative DLBCL; CD5 (21.4% vs 14.2%; p = 0.72), CD10 (44.6% vs 21.4%; p = 0.138), CD19 (92.8% vs 96.4%; p = 0.494), CD20 (100% vs 91.0%; p = 0.575), CD23 (21.4% vs 10.7%; p = 0.37), CD25 (28.5% vs 41.0%; p = 0.542), CD30 (14.2% vs 1.7%; p = 0.0997), sIgG(35.7% vs 30.4%; p = 0.758), sIgM (50.0% vs 67.9%; p = 0.229). 2‐year‐OS and PFS of iLCR‐positive and iLCR‐negative DLBCL were 61.2% vs 53.9% (p = 0.557) (Fig1) and 51.9% vs 55.0% (p = 0.924) respectively. There are 34 κ‐DLBCL and 22 λ‐DLBCL in 56 iLCR‐positive group. 2‐year OS and PFS of κ‐DLBCL and λ‐DLBCL were 64.3% vs 59.0% (p = 0.746) and 58.6% vs 47.8% (p = 0.255) respectively.Summary/Conclusion:In this study, we observed no difference in the clinical characteristics and outcomes between iLCR‐negative DLBCL and iLCR‐positive DLBCL. In iLCR‐positive DLBCL, the outcome of κ‐DLBCL is similar to that of λ‐DLBCL. Therefore, the presence or absence of iLCR and deviation of κ or λ light chain of DLBCL may not influence on the prognosis and selection of treatment.

Diagnostic and Prognostic Value of Nuclear Factor Kappa-B in Diffuse Large B Cell Lymphoma in Egyptian Patients with Hepatitis C Virus Genotype 4

Background: Members of the NFκB [p65] family have potential diagnostic and prognostic role in various inflammatory diseases and Lymphomas. Aim: We studied NFκB [p65] in paraffin blocks of hepatitis-C-virus [HCV] positive genotype-4 and HCV negative diffuse large B-cell lymphoma [DLBCL] patients, aiming at identification of its differential expression and prognosis in DLBCL and its subtypes; GCB and ABC. This is to establish its relation to HCV infection and its role in lymphogenesis. Besides assessing the role of new directly acting antiviral drugs [Sofusbuvir/Ledipasvir] concomitantly administered to [CHOP] combination in HCV positive DLBCL. Subjects and Methods: NFκB [p65] expression was assessed using Anti-NFκB [p65] antibody semi-quantitative technique in 30 newly diagnosed DLBCL patients [HCV positive [n = 15], HCV negative [n = 15]. Results: NFκB [p65] expression was higher in the HCV positive DLBCL patients than their HCV negative counterpart, with a positive correlation with the viral load [r = 0.536, p = 0.088]. NFκB [p65] expression was significantly more frequently detected in the ABC subtype than GCB subtype [p = 0.04]. Patients who expressed NFκB [p65] had higher incidence of extranodal involvement, advanced stages, higher LDH levels and IPI score. Besides, the expression of NFκB [P65] revealed an inferior overall response [OR] [p = 0.044]. Higher complete response rates to CHOP concomitantly with antiviral [ledipasvir/sofosbuvir] were encountered in the HCV positive group. In HCV positive group, NFκB [P65] displayed a positive relationship with the viral load and liver enzymes [p = 0.04], besides an inverse relation with serum albumin. This raises the possibility that NFκB [p65] expression is suggestive of the hepatic necro-inflammation in HCV patients. The ABC group presented more in advanced stages than GCB. Higher frequency of the ABC subgroup exhibited intermediate to high viral load, while it was less in the GCB. A statistically significant difference was found in the NFκB [p65] positive patients as regards MUM1 expression among the two groups [p ≤ 0.001]. Double positive [CD10+, MUM1+] and triple negative [CD10-, BCL6-, MUM1-] cases were encountered in the HCV positive group, and were characterized with a high NFκB [p65] expression. Conclusion: NFκB [p65] is expressed in patients with DLBCL, more frequently in ABC than in GCB subtypes. Expression of NFκB [p65] is associated with poor response to therapy in DLBCL. The NFκB [p65] disclosed an increased expression in HCV positive DLBCL compared to HCV negative group. The viral load displayed a positive correlation with the NFκB [p65] expression. Simultaneous administration of DAAs in combination with CHOP disclosed a better response and high tolerability.

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

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

Cd20 Expression and Effects on Outcome of Relapsed/ Refractory Diffuse Large B Cell Lymphoma after Treatment with Rituximab

Introduction:Down regulation of CD20 expression has been reported in diffuse large B cell lymphoma (DLBCL)). Therefore, it is important to determine whether chemotherapy with rituximab induces CD20 down regulation and effects survival.Objectives:To determine the incidence of down regulation of CD20 expression in relapsed DLBCL after treatment with rituximab and to compare outcomes and assess pattern of relapse between CD20 negative and CD20 positive cases.Methodology:We retrospectively reviewed patients with relapsed DLBCL who received rituximab in the first line setting at Aga Khan University Hospital between January 2007 and December 2014. Data were recorded on predesigned questionnaires, with variables including demographics, details regarding date of diagnosis and relapse, histology, staging, international prognostic index, treatment and outcomes at initial diagnosis and at relapse. The Chi square test was applied to determine statistical significance between categorical variables. Survival curves were generated by the Kaplan–Meier method.Results:A total of 54 patients with relapsed DLBCL were included in our study, 38 (70 %) males and 16(30%) females. Some 23 (43%) patients were at stage IV at the time of diagnosis and 34 (63%) had B symptoms. The most frequent R-IPI at diagnosis was II in 24 (44%) patients. Only 6 (11%) did not show CD20 expression on re-biopsy for relapsed/refractory disease, 2 with CD20 negative DLBCL responding to second line chemotherapy. A complete response after salvage chemotherapy was noted in 16 (29.6%) cases with relapsed/refractory DLBCL. Seven (13%) patients underwent an autologous bone marrow transplant as consolidation after second line treatment. Median overall survival was 18 months in CD20 positive vs. 13 months in CD20 negative patients.Conclusion:This study demonstrated that a small percentage of patients treated with rituximab lose their CD20 expression at the time of relapse. However, it is unclear whether this is associated with an inferior outcome.

Prevalence and Prognostic Significance of CD5 Immunohistochemistry in De-Novo DLBCL in a Western Population Cohort

Abstract Background: Various subsets of Diffuse Large B-cell lymphoma (DLBCL) are distinguished based on molecular and Immunohistochemical features. CD5 expressing DLBCL (CD5+ DLBCL) is increasingly recognized as a subtype of DLBCL with an aggressive disease course in small elderly Asian population cohorts comprising 5-10% of de-novo DLBCL. We attempt to describe its prevalence and prognostic implications in concert with IHC expression profiles of biomarkers Methods: A retrospective review of elderly patients (65yr-92yr) with de-novo Diffuse Large B-Cell lymphoma (DLBCL) uniformly treated with RCHOP was conducted between Jan 98 and Dec 2016. Patient demographics, clinical parameters and IHC markers were collected. Analysis using chi square, or Fisher exact where appropriate, was performed. Kaplan-Meier analysis and univariate and multivariate Cox regression analysis identified associations with overall survival. The protocol, and subsequent modifications, were reviewed and approved by the IRB at the Texas Tech University Health Sciences Center Results: One hundred fourteen de-novo DLBCL with known CD5 IHC status, Ki67, c-MYC, BCL2, BCL6, MUM1, CD10 marker status and Han's classifiers were evaluated. Median age of the CD5 negative cohort was 75yrs ±12.8 vs 80yrs±9.3 in the CD5+ subgroup (p=0.51). Median OS 6.7 years (SD 62.3mo; Range 1-256). CD5+ expression on IHC was seen in 23/110 (21%) patients. IHC marker status showed c-myc (12%), BCL2(42%) and BCL6(39%) respectively. Compared with matched group of patients with CD5 negative DLBCL, CD5+ DLBCL were more likely to be non-GCB type (14/23 vs 9/87; p 2 (14/23 vs 23/81; p=0.009) and Stage >III (p=0.004). Three patients with CNS involvement all had CD5 expression. Kaplan-Meier analysis showed that Han's non-GCB classification (p 70% (HR=0.33, P=0.017), BCL6 (HR=0.47, P=0.019) and CD10 (HR=0.2, P=0.003) were associated with worse outcomes. CD5+ expression was not significantly associated with OS (HR=0.76; -1.4 to 0.8; p=0.64) in multivariate model, however was significant in a univariate analysis (P Conclusion: CD5+ DLBCL subset is more common in this elderly western cohort than historically seen in the Asian population with a signal toward Hans non-GCB, higher R-IPI, higher Stage and perhaps higher risk of CNS involvement. More data is needed to evaluate if CD5+ de-novo DLBCL has any correlation with other antiapoptotic or proliferative markers like BCL2,6 and c-MYC and larger patient subset and prospective studies are needed to corroborate these findings Disclosures No relevant conflicts of interest to declare.

Clinical and Prognostic Significance of Epstein-Barr Virus in Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the largest subtype of lymphoma, but consists of heterogeneous groups with different prognosis. Many studies conducted to determine prognosis factors of DLBCL. Recently, several studies have been reported that the elderly Epstein-Barr virus (EBV)-positive DLBCL patients showed worse prognosis than the elderly EBV-negative DLBCL. (Oyama T et al. Clin Cancer Res 2007;13:5024-5032). On the other hand, other studies reported that EBV-positivity is not a prognosis factor in young-adult DLBCL patients (Hong JY et al. Annals of Oncology 2015;26:548-555, Nicolae A et al. BLOOD 2015;126:863-872). In the present study, retrospective analyses were carried out for patients with DLBCL diagnosed between 1990 and 2007 to analyze the clinical and prognostic significance of EBV.Patients: Those who met the following criteria were included in the study: (i) pathology confirmed de novo DLBCL, according to the WHO classification; (ii) adequate amount and quality of paraffin-embedded biopsy specimens or unstained slides for EBV-encoded RNA in situ hybridization.Statistical methods: Intergroup comparisons were carried out with Fisher's exact test for categorical variables and the Mann-Whitney test for age. For survival analysis, we carried out Kaplan-Meier and multivariate proportional hazard (Cox) analyses. Statistical analyses were carried out using the software Stata SE version 14.0.Results: A total of 456 patients were included. The median follow up duration of survivor was 100 months (range, 1.9-363). There were 236 male (51.75%) and 220 female patients (48.25%) with a median age of 67 (range, 22-94) years. 246 patients (54.0%) were in advanced (III/IV) stage, 134 (29.4%) presented with poor Eastern Cooperative Oncology Group Performance Status (ECOG-PS), 275 (60.3%) with elevated lactate dehydrogenase (LDH) level, 263 (57.7%) with extranodal involvement of 1 or more sites, and 64 (14.0%) with bulky mass. As a consequence, 211 patients (46.3%) were in high/high-intermediate International Prognostic Index (IPI) categories. 5 years overall survival (OS) was 66.2%, in total. EBV was detected in samples from 47 patients (10.3 %). The positivity was 10.6% (15 / 142) in patients of 60 years or younger, and 10.2 % (32 / 314) in those older than 60 years (p = 0.51). No differences in back ground date were found between EBV - positive and negative DLBCLs regarding age (median 66.5 vs. 64.8, P = 0.17), male sex (88.1% vs. 11.9%, P = 0.16), advanced stage (88.2% vs. 11.8%, P = 0.17), poor ECOG-PS (90.3% vs. 9.7%, P = 0.47), elevated LDH (89.5% vs. 10.6%, P = 0.48), extranodal sites of one or more (90.5% vs. 9.5%, P = 0.31), bulky mass (92.2% vs. 7.8%, P = 0.33), and higher IPI categories (88.6% vs. 11.4%, P = 0.29).The prognosis was also not different between EBV-positive and negative DLBCLs. The 5 years OS was 42.6% for EBV-positive DLBCLs, and 56.4% for EBV-negative DLBCLs (P = 0.11). The same results were found for the younger (53.3% vs. 66.9%, P = 0.30) and the elderly patients (42.9% vs. 51.8%, P = 0.18).Conclusion: In this practice based, uncontrolled study, EBV-positively wad not different in younger and elderly DLBCLs. Although EBV-positive DLBCL showed worse trend of prognosis, the difference was not statistically significant. The significance of EBV in DLBCL should be reconsidered in unbiased, large-scale patient date. [Display omitted] [Display omitted] [Display omitted] DisclosuresSuzuki:Chugai: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Hakko kirin: Honoraria. Suzumiya:Chugai: Research Funding, Speakers Bureau; Astellas: Research Funding; Toyama Chemical: Research Funding; Eizai: Research Funding; Takeda: Speakers Bureau; Kyowa Hakko Kirin: Research Funding.

The Adhesion Molecule ICAM-1 in Diffuse Large B-Cell Lymphoma Post-Rituximab Era: Relationship with Prognostic Importance and Rituximab Resistance

Background: Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor involving in cell-to-cell adhesion and as well as co-stimulatory molecule involved in T-cell lymphocyte infiltration and activation. In the pre-rituximab era, high level of ICAM-1 was reported to be a prognostic indicator of better clinical outcomes in diffuse large B-cell lymphoma (DLBCL). The value of ICAM-1 expression in the post-rituximab era remains unclear. To investigate the impact of ICAM-1 expression in DLBCL, we conducted a retrospective study in R+CHOP treated patients.Methods: Patients (N=102) with histologically proven DLBCL treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. Forty-three patients were treated with CHOP and 59 patients with R+CHOP. ICAM-1 expression was determined by immunohistochemistry (IHC). ICAM-1 expression was quantified by staining intensity or percentage of positive cells. Tumors were considered positive when at least 75% of tumor cells expressed ICAM-1. Progression free survival (PFS) and overall survival (OS) was plotted by Kaplan-Meier method and curves were compared with the long-rank test in both groups. Correlation between the ICAM-1 expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of <0.05 was considered to be statistically significant. Finally, ICAM-1 expression was determined in a panel of rituximab-sensitive (RSCL) and rituximab-resistant lymphoma cell lines (RRCL) by western blot. Correlation of ICAM-1 expression and rituximab anti-tumor activity was determined by standard complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) assays.Result: ICAM-1 expressionwas detected in 28 (27.5%) DLBCL patients. The rest of the patients tested negative for ICAM-1 (N=74; 72.5%). Differences in ICAM-1 expression were observed according to cell of origin DLBCL subtype. ICAM-1 expression was higher among germinal center B-cell (GCB) DLBCL (41.4%) vs. non-GCB subtype patients (19.6%, P=0.019). The response rates for R-CHOP and CHOP were, respectively, 89.4% and 77.7% (P=0.409) in ICAM-1 positive patients and 92.5% and 73.5% (p=0.027) in ICAM-1 negative patients. As previously described, ICAM-1 expression was associated with an improved PFS/OS (P=0.03/ P=0.05) in CHOP-treated DLBCL. On the other hand, no differences in PFS/OS were observed between ICAM-1 positive or negative DLBCL patients treated with R+CHOP. The addition of rituximab to CHOP chemotherapy resulted in an improved PFS in ICAM-1 negative DLBCL (P=0.018). On the other hand, the clinical outcome of ICAM-1 positive DLBCL patients treated with CHOP chemotherapy was similar than R+CHOP treated patients. Finally, loss of ICAM-1 expression level was found in our rituximab resistant cell lines, along with reduction of rituximab activity.Conclusion: Before therituximab era, ICAM-1 low expression correlated with worse PFS and OS. In our retrospective clinical analysis, we found that rituximab significantly improved the overall response rate and PFS in ICAM-1 negative subset patients. Our data also indicated that ICAM-1 expression level was higher in GCB than non-GCB subtypes, and this may contribute to better rituximab response in GCB cell types. Gradually exposed lymphoma cell to rituximab lost ICAM-1 expression and may contribute to the resistance to rituximab and chemotherapy agents. Further investigation of ICAM-1 expression needed to be study to enhance the rituximab therapy in lymphoma. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas.

Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.

Influence of Different Components of the Tumor Microenvironment on Human Patient-Derived Lymphoma Cell Engraftment in Immmunodeficient Mice

Patient-derived xenografts (PDX) have become reliable tools for preclinical research for many types of cancer. We recently reported the establishment of 4 human diffuse-large B-cell lymphoma (DLBCL) PDX in mice arising from patient prostate carcinoma tissue (Wetterauer,C. et al. 2015). All PDX were EBV associated and of ABC subtype as determined by the new algorithm of Choi et al. 2009. In contrast, none of the donor patients showed clinical or histological evidence of lymphoma disease. To address the question why lymphoma engraftment takes place in immunodeficient mice whereas patients do not show clinical evidence of disease, we compared growth behavior of the DLBCL PDX in 4 different mouse strains harboring different immunological deficiencies. Furthermore using 3 different implantation routes the dissemination behavior of these models into different immunological niches was evaluated. Results were compared to a similar study carried out with an EBV negative DLBCL PDX of a secondary, cerebral lymphoma (ABC-subtype).None of 4 EBV+ DLBCL PDX showed tumor growth after implantation in NMRI nu/nu (B- cells+, T cells-, NK cells+, n=5 mice/PDX) or 1147-F mice (B cells-, T cells+, NK cells+ n=5 mice/PDX). Subcutaneous implantation into NOD/SCID mice (B cells-, T cells-, NK cells+ n=5 mice/PDX) revealed tumor growth for 2 of 4 lymphomas displaying take rates of 80% (4 out of 5 mice) and 100% (5 out of 5 mice) within one model. Infiltration of murine NK cells in tumor tissue was determined by IHC (analyses ongoing). All 4 EBV+ lymphoma models showed stable growth in NOG mice (B cells-, T cells-, NK cells- n= 10 - 12 mice/PDX) with a take rate of 100% and an average passaging time of 20 days, ranging from 14 to 26 depending on the specific model. Taken together these results indicate that tumor growth of EBV+ lymphomas in mice depends on the NK cell activity as well as the B- and T cells status of the recipient mouse. These cell populations control tumor engraftment in mice, which might explain the absence of lymphoma disease in donor patients. In contrast the PDX of a clinically apparent EBV- DLBCL patient was able to circumvent NK cell block in NMRI nu/nu mice and showed similar take rates (100%, 5 out of 5 engrafted mice) but prolonged passaging times (35 days) as compared to NOG (26 days).Comparison of tumor cell engraftment after subcutaneous (s.c.), intravenous (i.v.) and intratibial (i.t.) injection of tumor cells in NOG mice revealed disseminated tumor growth for the EBV- DLBCL PDX exclusively when injected i.v. or i.t.. After subcutaneous implantation of lymphoma material, no tumor cells could be detected by flow cytometry or IHC in lymph nodes, blood, spleen, brain, liver, lung or bone marrow of recipient mice. However, lymphoma cells could be detected in liver and bone marrow of all (n= 8) i.v. and i.t. injected mice. Currently we are analyzing the dissemination behavior of the EBV+ lymphomas after injection into the different immunological niches as described above.In summary these data highlight the importance of the tumor microenvironment in lymphoma engraftment and dissemination in vivo. Accordingly we could show for our EBV associated lymphoma models that NK-, T- and B cell population control engraftment in vivo and are possibly involved in controlling EBV+ transformed lymphatic cells in patients. Furthermore, we showed that injection into the biologically relevant niche enables lymphoma cells to form multiple tumor nodules in distinct organs in vivo. These models contribute to a better understanding of the interplay between lymphoma cells and their microenvironment. They will thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies. DisclosuresTschuch:Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Haapaniemi:Biositehisto Oy: Employment. Schueler:Oncotest GmbH: Employment.

The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

A proportion of MYC translocation positive diffuse large B‐cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double‐hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double‐hit DLBCL, and whether there is a difference in clinical outcome between the double‐hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R‐CHOP (n = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double‐hits had the worst overall survival, followed by those with MYC/BCL2 double‐hits. In MYC translocation negative DLBCL treated by R‐CHOP (n = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double‐hit DLBCLs from those with an isolated MYC translocation.

MicroRNA 21 Expression Levels in HIV Negative and HIV Positive Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease with various morphological and molecular subtypes. It commonly occurs in the elderly as well as in people infected with HIV. According to the Hans' algorithm DLBCL can be classified prognostically into the favourable germinal centre (GC) subtype and the unfavourable non- GC subtype. The disease tends to be more aggressive in HIV infected individuals. Research on DLBCL has been largely conducted on HIV negative samples and it is still unclear how HIV affects the molecular mechanisms of the disease. We conducted a study to compare the expression levels of miR-21 in HIV negative and HIV positive DLBCL patients. Our results suggest that miR-21 expression levels are higher in HIV positive cases of DLBCL. We did not observe any statistically significant difference in miR-21 levels between DLBCL subtypes in both HIV negative and positive cases. Favourable prognosis in HIV negative patients was associated with high miR-21 expression and the reverse was true in HIV positive patients. In conclusion, our results indicate that HIV infection may affect the expression of miR-21 in DLBCL. This highlights the need for further studies on HIV positive DLBCL as the prognostic significance of miR-21 expression level may differ depending on the HIV status. Keywords: B-cell lymphoma; Micro RNA; Prognosis; HIV; miR-21; DLBCL Introduction Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) comprising of 30% of all NHL [1]. The disease shows marked heterogeneity, with morphological and molecular variants [2,3], making it difficult to treat. DLBCL is divided into two histological subtypes with prognostic significance according to the Hans' algorithm. The non-germinal centre (non-GC) subtype has a worse prognosis than the germinal centre B cell like (GC) subtype [4]. Recent data suggests that HIV gp120 induces B cells expressing mannose C type lectin receptors (MCLRs) to undergo immunoglobulin class switching by increasing the expression of activation-induced cytidine deaminase [5]. Chronic B-cell activation in HIV infection is driven by the production of B-cell stimulating cytokines which are secreted by gp120 bound monocytes [6]. These cytokines act by upregulating MCLRs on the surface of B cells, thus creating chronic B cell activation [5]. Therefore, people infected with HIV are at an increased risk of developing B cell NHL [7]. DLBCL is the most common NHL diagnosed in HIV infected individuals [8,9]. MicroRNAs are non-coding RNA molecules that silence the expression of target genes [10]. Gene silencing is achieved by imperfect or perfect pairing of microRNA with3’UTR of target mRNA thereby resulting in inhibition of translation or degradation, respectively [11,12]. They are transcribed as long transcripts

Clinical characteristics and treatment outcome in a Taiwanese population of patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Epstein-Barr virus-positive diffuse large B-cell lymphoma is a provisional entity in the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues. Reports on the characteristics and clinical outcome of this disease in different geographic regions showed great disparities. To define the clinical characteristics as well as the prognostic impact of Epstein-Barr virus infection on diffuse large B-cell lymphoma in Taiwan, we retrospectively investigated the Epstein-Barr virus status of 89 patients with newly diagnosed diffuse large B-cell lymphoma in our institute. Using a cutoff point of positive nuclear staining of Epstein-Barr virus-encoded RNA-1-in situ hybridization in ≥20% of the examined cells, we identified 15 cases (16.9%) of the entire study cohort as Epstein-Barr virus-positive diffuse large B-cell lymphoma. The clinical and laboratory features were not different between Epstein-Barr virus-positive and -negative diffuse large B-cell lymphoma patients. Univariate analysis showed patients with diffuse large B-cell lymphoma that were either Epstein-Barr virus-positive or had activated B-cell-like features had an inferior overall survival. Older age, advanced stage and lymphoma with activated B-cell-like features or Epstein-Barr virus-encoded RNA positivity were independent prognostic factors affecting overall survival on multivariate analysis. Patients with two or three of these adverse-risk factors were considered high risk and fared far worse than patients with no or only one adverse factor. Taken together, we demonstrated that a higher frequency of Epstein-Barr virus association was detected in a Taiwanese cohort of diffuse large B-cell lymphoma patients, and Epstein-Barr virus-encoded RNA positivity was shown to add important prognostic value in these patients.