Primary Nodal Diffuse Large B-cell Lymphoma Research Articles

Clinicopathological differences in MYC and BCL2 protein expression between primary extranodal and nodal diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) exhibits clinical, genetic, and immunohistochemical heterogeneity. However, the differences between primary extranodal or nodal DLBCL and double-expressor lymphoma (DEL), which is characterized by high MYC and BCL2 expression, remain unclear. This study aimed to elucidate the clinicopathological features, response to therapy, and clinical outcomes of primary extranodal (n=61) and nodal (n=128) DLBCL. Patients with primary nodal DLBCL had higher BCL2 expression than those with extranodal DLBCL (p=0.048), with high MYC expression and DEL as poor prognostic factors. Conversely, in patients with primary extranodal DLBCL, high BCL2 expression, low BCL6 expression, non-germinal center B-cell-like type, and DEL indicated poor prognosis. DEL was significantly associated with progression free survival and overall survival in patients with primary extranodal DLBCL (p=0.014 and p=0.021, respectively) but not in patients with primary nodal DLBCL (p=0.37 and p=0.084, respectively). Our findings highlight primary extranodal DEL as a strong adverse prognostic factor in DLBCL.

CXCR4 and CCR7 Expression in Primary Nodal Diffuse Large B-Cell Lymphoma—A Clinical and Immunohistochemical Study

BackgroundA few studies have evaluated the expression of chemokine receptors CXCR4 and CCR7 in diffuse large B-cell lymphoma (DLBCL); however, the association between CXCR4 and CCR7 with bone marrow (BM) involvement and their synergistic effect on prognosis is still unclear. Our study investigated this aspect. MethodsSpecimens were obtained from 61 primary nodal DLBCL patients and 100 reactive proliferative lymphadenitis patients. CXCR4 and CCR7 expression levels were examined by immunohistochemical staining; the relationship between these levels and clinical parameters and the differences in overall survival were analyzed. ResultsCXCR4 and CCR7 overexpression was observed in the malignant lymph node tissues from most DLBCL patients. CCR7 expression was significantly higher in the non-GCB than the GCB subtype; CXCR4 positivity rates showed no significant difference between the 2 subtypes. In DLBCL patients with BM involvement, CXCR4 was overexpressed in almost all BM samples, but CCR7 expression was low in BM. CXCR4 overexpression was associated with advanced Ann Arbor stages, MYC overexpression, and increased extranodal infiltration; CCR7 was associated with advanced Ann Arbor stages and elevated LDH. Like the case for CCR7, the survival rate of CXCR4-positive DLBCL patients was significantly lower than that of the CXCR4-negative patients. CXCR4+CCR7+ patients had the lowest survival rate. ConclusionsThere is a positive correlation between CXCR4 overexpression and BM involvement. CXCR4 and CCR7 overexpression is associated with poorer overall survival, especially in CXCR4 and CCR7 copositive patients. CXCR4, CCR7, Ki-67 index, and MYC were independent prognostic factors for DLBCL. Blocking CXCR4 and/or CCR7 can be a novel therapeutic strategy for DLBCL.

Serum Albumin and Peripheral Blood Neutrophils at Diagnosis May Provide Additional Prognostic Information in Primary Nodal Diffuse Large-B-Cell Lymphoma Patients Treated with R-CHOP

Background and Aim: Diffuse large B-cell lymphoma (DLBCL) can present both as a primary nodal or extranodal neoplasm. Some studies claimed a separate origin for nodal and extranodal lymphomas and it has been even suggested that these could be regarded as separate nosological entities. However, the standard of care in DLBCL patients (pts) is rituximab-CHOP immunochemotherapy (R-CHOP), and the prognostic stratification is performed by the Enhanced Revised International Prognostic Index (NCCN-IPI), identifying 4 distinct [low (L), low-intermediate (LI), high-intermediate (HI) and high (H)] risk groups (RGs). A lot of new prognostic markers such as serum albumin (SA), serum β2-microglobulin (B2M), hemoglobin level (Hb), absolute neutrophil (ANC), lymphocyte (ALC), monocyte (AMC) and platelet counts etc. have been introduced into the clinical practice to perform better pts' stratification. However, data on the importance of these factors particularly in primary nodal (PN) DLBCL pts are still limited. Therefore, we aimed to access the prognostic impact of these markers regarding overallsurvival (OS) across the different NCCN-IPI RG of R-CHOP treated PN-DLBCL pts.Patients and Methods: We retrospectively reviewed the clinical outcome of 174 R-CHOP treated PN-DLBCL pts at a median age 58.4 years. Pts were stratified using NCCN-IPI into L (24.1%), LI (43.1%), HI (24.7%) and H (8.1%) RGs. Laboratory levels of SA, B2M, Hb, ANC, ALC, AMC and PC were recorded, and LMR and NLR - calculated. A receiver operating characteristic (ROC) curve analysis was used to illustrate in our data set the best cut off values of SA, B2M, Hb, ANC, ALC, AMC, PC, LMR and NLR to predict OS by Kaplan-Meier method. Univariate analysis to evaluate differences between variables was performed by the log rank. A multivariate analysis was performed by Cox proportional-hazards models.Results: The estimated 5-year OS was 79.4%, 51.5%, 20.1% and 16.2% for NCCN-IPI L, LI, HI and H-risk pts, respectively (p<0.001). Univariate analysis showed that inferior OS was associated significantly with decreased SA (≤39.4 g/L), elevated B2M (>3.2 mg/L), elevated ANC (>5.19 x 109), reduced ALC (≤1.38 x 109), elevated AMC (>0.515 x 109), decreased LMR (≤1.77), increased NLR (>2.97), lower Hb level (≤134 g/L), presented as dichotomized variables. Multivariate analysis confirmed the independent prognostic impact only for SA (p<0.001) and ANC (p=0.011).Based on the dichotomized SA and ANC values a SA/ANC prognostic index (PI) was created stratifying pts into 3 RG: favorable (F) [SA >39.4 g/L and ANC ≤5.19 x 109], intermediate (I) [SA ≤39.4 g/L or ANC >5.19 x 109] and poor (P) - risk [SA ≤39.4 g/L and ANC ≤5.19 x 109] populations. The estimated 5-year OS differed significantly in SA/ANC PI RG, as follows: 92.8% in F-RG, 48.4% in I-RG, and 0% in P-RG (p<0.001). Median OS for I- and P- SA/ANC PI RG was 2.54 and 1.13 years, respectively and not reached for the F-risk pts.We sought to determine whether the SA/ANC PI may provide additional prognostic information within the NCCN-IPI RG. No statistics could be calculated within the L-RG due to the low number of deaths - 9.5% (4/42), and in the H-RG due to the low number of patients (n=14), respectively. However, within the LI-RG the SA/ANC PI allowed us to discriminate 3 subgroups, characterized by significant differences in the OS (p<0.001): no patient within the P-RG was alive at 5years and the median OS was only 1.13 years; while 5-years OS was 77% and 87.7% in the I-RG and F-RG, respectively, and the median was not reached in both RG. Similarly, within the NCCN-IPI HI-RG the application of SA/ANC PI allowed us to discriminate 3 subgroups, characterized by significant differences in OS: no patient within the P-RG was alive at 5 years and the median OS was 1.29 years; while 5-years OS was 30.6% (median OS - 1.66 yrs) in the I-RG and 100% (median OS not reached) in the F-RG. The introduction of the SA/ANC PI allowed for defining favorable subgroups within the IPI LI- and HI RGs with 5-yrs OS comparable to IPI L-RG.Conclusion: The present study provided evidence for the independent prognostic significance ofSA and ANC in regard to survival in patients with PN-DLBCL. Adding these variables to prognostic models such as the NCCN-IPI score might improve the predictive ability, particularly within the NCCN-IPI LI and HI risk groups, where the introduction of SA/LMR PI allowed for identifying favorable subgroups comparable to the NCCN-IPI L-RG in terms of OS. DisclosuresNo relevant conflicts of interest to declare.

Clinical Characteristics and Outcomes of Limited-Stage Primary Extranodal Versus Nodal Diffuse Large B-Cell Lymphoma (DLBCL) in Thailand: A Nationwide Multi-Institutional Registry of 920 Cases in Thailand

IntroductionThe difference in origin between nodal and extranodal DLBCL may contribute to the distinct clinical outcomes and prognoses. Previous studies including patients with both limited and advanced diseases showed various clinical results. The purpose is to study the clinical characteristics, and outcomes of patients with limited stage DLBCL according to the primary site of lymphoma.MethodsFrom the 4,371 patients in a multi-institutional registry of newly diagnosed lymphoma in Thailand between 2007-2014, there were 920 patients with limited stage DLBCL, excluding those with primary central nervous system lymphoma. The baseline patient characteristics and clinical outcomes were analyzed according to the primary site of diseases.ResultsMajority of patients had extranodal diseases (n= 535, 58.2%) while the other 385 cases (41.8%) had nodal DLBCL. The five most common primary sites of extranodal disease were Waldeyer ring (12.2%), stomach (7.3%), intestine (6.8%), sinonasal cavity (6.7%) and breast (2.8%) (Figure 1). Baseline characteristics, treatment and responses were well balanced between the two groups except for a slightly higher proportion of stage I disease in extranodal DLBCL (36.8% vs 28.8%, P =0.01) (Table 1). Two-third of patients received CHOP chemotherapy while the others were treated with R-CHOP. Radiotherapy (RT) was delivered to one-fifth of the patients in each group. Overall response rates were comparable between patients with nodal and extranodal diseases (68.6% vs 66.7%). With a median follow up of 52 months, there were no significant differences of progression free (PFS) (57.6 vs. 60.8%, P =0.46) and overall survival (OS) (63.1% vs. 65.6%, P =0.20) between patients with extranodal and nodal diseases. Among patients with extranodal manifestations, those with primary lymphoma at pancreas & gall bladder, pleura & lung, adrenal gland & kidney and intestine had the worst 4-year OS (33.3%, 39.3%, 53.3%, and 53.7%, respectively) (Figure 2). RT improved PFS (71.7% vs. 54.5%, P =0.03) and OS (74.7% vs. 62.9%, P =0.02) only in primary nodal DLBCL. The two prognostic factors for survivals were being treated with R-CHOP (HR for PFS 0.84, 95%CI: 0.74-0.96; HR for OS 0.70, 95%CI: 0.55-0.91) and the IPI scores (HR for PFS 1.49, 95%CI: 1.24-1.80; HR for OS 1.86, 95%CI: 1.37-2.51).ConclusionAmong limited stage DLBCL in Thailand, primary extranodal disease was more common than the nodal manifestations. Primary sites of origins did not affect baseline characteristics as well as the outcomes. Patients with unusual site lymphomas, however, had an inferior survivals warranted a further investigational therapy.Table 1The clinical characteristics of limited stage DLBCL according to disease of originClinical characteristicsNodal DLBCL(n = 385)Extranodal DLBCL(n = 535)p-valueMale205 (53.2%)294 (55%)0.60Median age (years)54.956.20.23Stage Stage I Stage II111 (28.8%) 274 (71.2%)197 (36.8%) 338 (63.2%)0.01HIV seropositivity11 (3.4%)19 (4.3%)0.53Age ≥ 60164 (42.6%)230 (43%)0.90High serum LDH183 (47.5%)230 (43%)0.17ECOG ≥ 246 (11.9%)60 (11.2%)0.73IPI Low (IPI = 0-1) Low-intermediate (IPI =2) High-intermediate (IPI = 3)285 (74.0%) 81 (21.0%) 19 (4.9%)406 (75.9%) 108 (20.2%) 21 (3.9%)0.82Chemotherapy regimens R-CHOP CHOP98 (29.0%) 227 (67.2%)160 (33.7%) 286 (60.2%)0.25Radiotherapy94 (24.9%)120 (22.8%)0.46Response Overall response Complete response261 (68.6%) 227 (59.7%)352 (66.7%) 322 (61%)0.24*IPI: International prognostic index [Display omitted] [Display omitted] DisclosuresKhuhapinant:Roche: Honoraria.

Low expression of microRNA-146b-5p and microRNA-320d predicts poor outcome of large B-cell lymphoma treated with cyclophosphamide, doxorubicin, vincristine, and prednisone.

Although diffuse large B-cell lymphoma (DLBCL) encompasses a biologically and clinically diverse set of diseases, increasing evidence has pointed to an important role of microRNAs (miRs) in the pathogenesis of DLBCL. We report here that low expression of miR-146b-5p and miR-320d is associated with poor prognosis of DLBCL patients treated with the standard cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen and that this is related to the inhibitory effect of these miRs on DLBCL cell proliferation. Analysis of a retrospective cohort of 106 primary nodal DLBCL samples from patients who were treated with CHOP showed that, when the median survival period (40.8 months) was used as the cutoff point, miR-146b-5p and miR-320d were expressed at lower levels in DLBCLs with poor prognosis. Indeed, whereas low expression of miR-146b-5p was correlated with reduced progression-free survival, low expression of miR-320d was associated with decreases in both progression-free survival and overall survival. Moreover, miR-146b-5p and miR-320d were expressed at significantly lower levels in DLBCLs with the MYC t(8;14) translocation. Functional studies demonstrated that overexpression of miR-146b-5p or miR-320d inhibited DLBCL cell proliferation, wheareas knockdown of miR-146b-5p or miR-320d promoted proliferation of DLBCL cells. Taken together, these results suggest that low expression of miR-146b-5p and miR-320d may be predictive of compromised responses of a subset of DLBCL patients to treatment with the CHOP regimen and that restoration of these miRs may be useful to improve the therapeutic efficacy of CHOP.

Matched‐pair analysis comparing the outcomes of primary breast and nodal diffuse large B‐cell lymphoma in patients treated with rituximab plus chemotherapy

Primary breast diffuse large B-cell lymphoma (DLBCL) is an extremely rare presentation of non-Hodgkin's lymphoma that has been associated with poorer clinical outcomes compared with nodal DLBCL in the pre-rituximab era. The aim of this study was to investigate the impact of rituximab on clinical outcomes in patients with primary breast DLBCL. Data from 25 female patients with primary breast DLBCL receiving rituximab plus chemotherapy were matched to 75 female patients (1:3) with nodal DLBCL by following five established prognostic factors (age, Ann Arbor stage, Eastern Cooperative Oncology Group performance status, serum lactate dehydrogenase level and B symptoms). Overall survival (OS) was similar between primary breast and nodal DLBCL groups (3-year OS rate, 82.2% vs. 90.7%, respectively; p = 0.345). In the analysis of immunohistochemically defined prognostic subgroups, 19 of 20 available cases in the primary breast DLBCL group displayed a non-germinal center (GC) phenotype. Compared with patterns of recurrence, extranodal progression in the breast or central nervous system (CNS) was significantly higher in the primary breast DLBCL group than in the nodal DLBCL group (p < 0.001). Additionally, the stage-modified International Prognostic Index was the only independent prognostic factor for OS in this population. This suggests that clinical outcomes of primary breast DLBCL might no longer be inferior to those of nodal DLBCL in the rituximab era, which might be associated with the intrinsic biologic characteristics of the non-GC phenotype. However, despite including rituximab, extranodal progression in the breast or CNS was problematic. This study was registered at www.clinicaltrials.gov as no. NCT01266668.

Genotypic and Phenotypic Differences between Nodal and Extranodal Diffuse Large B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases that have diverse clinical, pathological, and biological features. Here, it is shown that primary nodal and extranodal DLBCLs differ genomically and phenotypically. Using conventional comparative genomic hybridization (CGH), the authors assessed the chromosomal aberrations in 18 nodal, 13 extranodal, and 5 mixed DLBCLs. The results demonstrate significantly distinct chromosomal aberrations exemplified by gains of chromosomal arms 1p, 7p, 12q24.21-12q24.31, and 22q and chromosome X and loss of chromosome 4, 6q, and 18q22.3-23 in extranodal compared with nodal DLBCLs. Nodal DLBCLs showed an increased tendency for 18q amplification and BCL2 protein overexpression compared with extranodal and mixed tumors. Using a panel of five antibodies against GCET1, MUM1, CD10, BCL6, and FOXP1 proteins to subclassify DLBCLs according to the recent Choi algorithm, the authors showed that the genomic profiles observed between the nodal and extranodal DLBCLs were not due to the different proportions of GCB vs ABC in the two groups. Further delineation of these genomic differences was illuminated by the use of high-resolution 21K BAC array CGH performed on 12 independent new cases of extranodal DLBCL. The authors demonstrated for the first time a novel genome and proteome-based signatures that may differentiate the two lymphoma types.

Biological Characterization of Nodal versus Extranodal Presentation of Diffuse Large B-Cell Lymphoma using Immunohistochemistry

Diffuse large B cell lymphoma (DLBCL) of primary nodal (PN) or primary extranodal (PEN) origin may differ immunophenotypically, in that PEN lymphoma cells may originate from activated rather than germinal center B (GCB) cells. We evaluated the relationship between DLBCL clinicopathological features, including expression of B-cell differentiation markers, and primary tumor site. Expression of CD10, Bcl-6, Bcl-2, and MUM1 was determined in paraffin-embedded tissues from 123 patients with DLBCL. Of the 123 patients with DLBCL, 40 (32.5%) had the GCB and 83 (67.5%) had the non-GCB phenotype. Fifty-one patients (42%) showed disease involvement at PEN sites, including 29 with disease in the gastrointestinal (GI) tract (14 in the stomach, 15 in the intestine). Of these 51 patients, 16 (31.4%) were classified with the GCB and 35 (68.5%) with the non-GCB subtype. There were no differences in the frequencies of GCB and non-GCB subtypes among primary sites. Of the 72 patients with PN DLBCL, 22 (31%) had the GCB and 50 (69%) had the non-GCB subtype. There were no differences in the frequencies of GCB and non-GCB subtypes between patients with PN and PEN DLBCL. Although lactate dehydrogenase (LDH) concentration > normal, stage >II, and rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) treatment were independent predictors of overall survival (OS), GCB subtype, and presence of PEN disease failed to predict survival upon multivariate analysis. There was no difference in GCB and non-GCB phenotypes between patients with PN and PEN DLBCLs. Additional studies are needed to further assess molecular differences between the two groups.

Matched Pair Analysis Comparing the Outcomes of Primary Breast and Nodal Diffuse Large B Cell Lymphoma In Patients Treated with R-Chop; Consortium for Improving Survival of Lymphoma (CISL) Study.

AbstractAbstract 1790 IntroductionThe addition of rituximab to standard chemotherapy has substantially improved the survival in patients with diffuse large B cell lymphoma (DLBCL). Previous studies in the pre-rituximab era have identified the worse outcomes in primary extranodal DLBCL compared with nodal DLBCL. However, there have been reported conflicting datas about outcomes of primary extranodal DLBCL compared with nodal DLBCL in the rituximab era. Primary breast DLBCL is one of the extremely rare extranodal lymphoma. As in other primary extranodal lymphoma, few clinical studies have been reported for investigating the efficacy of rituximab in patients with primary breast DLBCL. For clarifying this, a large randomized trial comparing survival in patients with primary breast DLBCL is required. However, the rarity of primary breast DLBCL makes large trial virtually difficult in single center or study group. Additionally, retrospective studies for evaluating the role of rituximab in primary breast DLBCL had bias according to the difference of treatment period between CHOP and R-CHOP era. Thus, to investigate the impact of rituximab in primary breast DLBCL, we performed a matched pair analysis following strict matching criteria in patients with primary breast and nodal DLBCL treated with R-CHOP. Materials and methodsPrimary breast DLBCL patients treated with R-CHOP was identified from 11 hospitals in Korea between May 2004 and August 2009. The eligibility criteria included: (1) histologically confirmed DLBCL, (2) Ann Arbor stage I or II of primary breast DLBCL, defined as isolated breast involvement with or without nodal disease, (3) received front-line treatment with R-CHOP. Each primary breast DLBCL patient was matched to three nodal DLBCL patients treated with R-CHOP during the same period from the data registry of Korean Lymphoma Working Party. The patients were matched for 5 known prognostic factors: age (<60 vs. ≥60), Ann Arbor stage (I vs. II), Eastern Cooperative Oncology Group (ECOG) performance status (PS) (0-1 vs. 2–3), serum LDH level (normal vs. elevated), and B symptom (absent vs. present). All factors should be matched between the four matched patients. ResultsTwenty-five patients with primary breast DLBCL were identified. The median age at diagnosis was 56 (range, 21–79) years and all patients were female. The Ann Arbor stage was I in 13 patients (52%) and II in 12 patients (48%). ECOG PS was 0 or 1 in 23 patients (92%), B symptom was present in 1 patient (4%), and serum LDH level was elevated in 9 patients (36%). Thus, stage-modified international prognostic index (IPI) was 0 or 1 in 20 patients (80%). Eight patients (32%) were received 3 or 4 cycles of R-CHOP followed by involved field radiotherapy and 17 patients (68%) were treated with 6 to 8 cycles of R-CHOP. After matching process, stage-modified IPI, treatment strategy, radiation dose, and follow-up duration as well as 5 matching factors were not significantly different between primary breast and nodal DLBCL groups. With a median follow-up of 34.3 (range, 4.4–76.2) months, 3-year progression-free survival (PFS; 70.0% [59.9-80.1] vs. 85.2% [79.9-90.5], p=0.145) and overall survival (OS; 82.2% [72.6-92.8] vs. 90.0% [86.0-94.0], p=0.528) was not statistically different between primary breast and nodal DLBCL groups. In multivariate analysis, 2 or 3 risk factors of stage-modified IPI were independent prognostic factor for worse PFS (hazard ratio [HR], 3.18; 95% CI, 1.22–8.30) and OS (HR, 4.88; 95% CI, 1.55–15.33). Comparing 3-year cumulative incidence of progression between primary breast and nodal DLBCL, extranodal progression in the breast or central nervous system (CNS) was significantly higher in the primary breast DLBCL than nodal DLBCL (23.6 ± 9.3% vs. 1.4 ± 1.3%, p<0.001, respectively). ConclusionsIn the post rituximab era, the survival outcomes of primary breast DLBCL were not significantly inferior to those of nodal DLBCL. These results suggest adding rituximab improve survival in primary breast DLBCL as in nodal DLBCL, so that the results provide evidence to add rituximab in this rare extranodal DLBCL. However, even including rituximab, extranodal progression in the breast or CNS was observed still high. Thus, further larger studies of international collaboration to confirm these results are warranted. Disclosures:No relevant conflicts of interest to declare.

MGMT methylation in diffuse large B-cell lymphoma: validation of quantitative methylation-specific PCR and comparison with MGMT protein expression

Aims:(1) To validate a quantitative real time methylation specific PCR assay (MethyLight) for the detection of O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status (MS) in diffuse large B-cell lymphoma (DLBCL). (2)...

Identification of genes putatively involved in the pathogenesis of diffuse large B-cell lymphomas by integrative genomics

Diffuse large B-cell lymphomas (DLBCL) are highly heterogeneous with regard to clinical presentation and outcome. DLBCL copy number aberrations have been identified previously, of which the deletion at 6q21-24 was significantly associated with a highly favorable clinical response to chemotherapy. In this study, we aimed to identify genes implicated in this and other genomic regions with recurrent losses and/or gains. To identify implicated genes, we superimposed array comparative genomic hybridization (aCGH) data onto a microarray expression dataset of 42 clinically well-characterized primary nodal DLBCL biopsies. We confirmed that loss of 6q21-24 is significantly associated with a highly favorable clinical response to chemotherapy. Our approach identified 316 significant genes restricted to 32 chromosomal regions, including 24 genes identified at 6q21-24. In an independent dataset, 18% of overexpressed genes in gained regions and 55% of down-regulated genes in deleted regions were validated. In summary, using integrative genomics novel onco and tumor suppressor genes were identified in DLBCL that were not recognized by expression profiling alone.

No Benefit from Rituximab Containing Regimens in Patients with Primary Extranodal Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous category of lymphoid tumors that comprises different clinical forms not fully recognized in the WHO classification. In this regard, extranodal (EN) DLBCLs have particular clinicobiological features and outcome, sometimes related to the specific site where the lymphoma arises. Nowadays, rituximab plus chemotherapy (CT) is the gold-standard in the treatment of DLBCL. However, the superiority of rituximab-CT (R-CT) over CT alone has not been addressed for all the clinical subsets of the disease and, in fact, the clinical role of the new therapies might be different for primary nodal or EN DLBCLs. The aim of this study was to assess the impact of rituximab in patients suffering from nodal or EN DLBCL. Two-hundred and thirty non-immunocompromised patients (112M/118F; median age, 61 years) diagnosed with CD20+DLBCL in a single institution between 1997 and 2006 (five years before and after establishing R-CT as the standard treatment in DLBCL) and treated with adriamycin-containing regimens were the subject of the present study. The series included 148 primary nodal and 82 EN DLBCL. Patients with primary CNS lymphoma were excluded and lymphomas arising at Waldeyer ring were considered as nodal DLBCL. The main EN sites were GI (n=26), bone (n=13), soft tissue (n=13), lung/pleura (n=9), liver (n=9), and other (n=12). Main clinico-biological and evolutive variables were analyzed. One hundred nineteen patients received only CT and 111 R-CT. Eighty-seven cases with available information were assigned to germinal center B-cell-like (GCB) (41%) or non-GCB (59%) groups according to the Hans method (Blood 2004; 103:275–82) based on CD10, BCL6 and MUM1 expression. Main initial features, including the primary nodal or EN origin, international prognostic index (IPI), and GCB/non-GCB categories were similar for CT and R-CT groups. No correlation was observed between the GCB/non-GCB groups and the primary site of the tumor, although nodal lymphomas more frequently expressed MUM1 than EN (69% vs. 31%, respectively; p=0.01). CR rate and 5-year overall survival (OS) according to the treatment arm (CT vs. R-CT) is detailed for the whole series and for the nodal and EN groups in the table and OS curves depicted in the figure. In the whole series, variables predicting poor OS in the multivariate analysis were high-risk IPI (RR 2.5; p<0.001), primary nodal involvement (RR 1.6; p=0.04) and no R-CT treatment (RR 1.9; p=0.002). In the nodal group, IPI and no R-CT maintained the prognostic value, whereas in the primary EN only IPI predicted OS. Moreover, no difference in OS was observed according to the nodal or EN origin in those patients receiving R-CT. Biological subtypes GCB vs. non-GCB did not add predictive information neither in the whole series nor in the nodal or EN groups. In conclusion, patients with primary EN DLBCL seem to have little benefit from the use of R-CT. Nevertheless, this intriguing observation should be confirmed in further prospective studies.Complete response CR (%)5-years OS (%)CTR-CTCTR-CTAll cases (n=230)5979*4670*Primary nodal (n=148)5478*3471*Primary extranodal (n=82)68787069*p<0.002 R-CT vs. CT [Display omitted]

Prognostic significance of primary extranodal diffuse large B-cell lymphoma (DLBCL) in patients treated with R-CHOP

8583 Background: Previous studies in the pre-rituximab era have identified important clinical differences between nodal and primary extranodal DLBCL. We have examined the prognostic significance of primary extranodal DLBCL in the post-rituximab era. Methods: Using the Lymphoid Cancer Database of the British Columbia Cancer Agency, all patients ≥18 years of age diagnosed with DLBCL between January 1999 and May 2006 and treated with an R-CHOP regimen were included for analysis. Patients were excluded if they were HIV positive, presented with disease in the testicular or central nervous system, or had known coincident indolent lymphoma. Primary extranodal DLBCL was defined as disease confined to one or more localized extranodal sites, with no or minimal nearby nodal involvement. Results: 513 patients were identified with the following characteristics: median age 62 y (range 19–93), male 59.1%, stage III/IV 53.8%, elevated LDH 49.5%, and performance status ≥2 38.2%. While 350 (68.2%) had at least some degree of extranodal involvement, only 133 (25.9%) had primary extranodal DLBCL. Among patients with primary extranodal disease, 70 (52.6%), 37 (27.8%) and 26 (19.6%) had 1, 2 and ≥3 extranodal sites, respectively. The most commonly involved extranodal sites included bone (33.1%), soft tissue (28.6%), stomach (15.0%), intestine (12.8%), and sinus (10.5%). Thirty-two percent, 20%, 0% and 48% of patients with primary extranodal DLBCL had stage I, II, III, and IV, respectively. This is in contrast to 11%, 34%, 26% and 29% for the nodal DLBCL group. Primary extranodal DLBCL was less commonly associated with an elevated LDH (37.6% vs. 53.7%, p=0.001). The distribution of International Prognostic Index scores was similar between the two groups. With a median follow-up of 2.8 years, no significant difference in overall survival was detected between primary extranodal DLBCL and nodal DLBCL analyzed either as a group or by individual stages. Furthermore, we did not identify any specific primary extranodal sites that confer a worse prognosis. Conclusion: In the post-rituximab era, the previously identified survival difference in primary extranodal DLBCL is no longer observed in this large cohort. No significant financial relationships to disclose.

Expression of TNF‐receptor associated factor 2 correlates with poor progression‐free survival time in ABC‐like primary nodal diffuse large B‐cell lymphomas

Tumour necrosis factor (TNF)-receptor associated factor 2 (TRAF2) is an adaptor molecule involved in nuclear factor (NF)-kappaB activation, which is characteristic of in vitro activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCL) and may result in expression of anti-apoptotic genes and poor response to chemotherapy. TRAF2 also has direct anti-apoptotic properties via interference with the apoptosis signalling cascade. The aim was to determine whether TRAF2 is preferentially expressed in ABC-like DLBCL, and whether expression correlates with clinical outcome. TRAF2 was expressed in nine of 20 tested ABC-like DLBCLs and in only one of 13 tested germinal centre B-lymphocyte (GCB)-like DLBCLs. High TRAF2 expression was correlated with high International Prognostic Index at time of presentation, high chance of relapse and short progression-free survival time in 44 tested DLBCLs. Furthermore, when analysis was restricted to ABC-like DLBCL only, TRAF2 expression was significantly associated with poor progression-free survival time. TRAF2 might be involved in activation of NF-kappaB in a subset of ABC-like DLBCL, and its expression is associated with a particularly poor outcome in primary nodal DLBCL patients. Because of its possible effect on to chemotherapy resistance, resistance, TRAF2 might be an attractive candidate as a molecular target for TRAF2+ DLBCL.

Bcl-6 protein expression, and not the germinal centre immunophenotype, predicts favourable prognosis in a series of primary nodal diffuse large B-cell lymphomas: A single centre experience

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. The immunohistochemistry-based algorithms for the determination of the cell of origin of DLBCL have been proposed as a practical method to validate and surrogate results obtained by gene expression profiling. We studied 71 patients with primary nodal DLBCL at diagnosis, who received anthracycline-based therapy with or without rituximab. Immunohistochemistry was performed using anti-CD10, Bcl-6, MUM1 and Bcl-2 antibodies in order to assess the ontogenic profile of neoplastic cells and to verify its relation with clinical outcome. Survival data were analysed using an explorative Cox model. The immunohistochemistry-based algorithms for the determination of the cell of origin of DLBCL were not associated with prognosis. By contrast, Bcl-6 expression was associated with a longer lymphoma-free survival while immunoreactivities for MUM1 or Bcl-2 were not significantly related to patient outcome. Bcl-6 expression alone proved to be a prognostic marker in primary nodal DLBCL and seemed to be more reliable to predict clinical outcome in these disorders than the immunohistochemical algorithms for the detection of the germinal centre/non-germinal centre immunophenotype.

Diffuse Large B-Cell Lymphomas of the Waldeyer's Ring Frequently Have a Germinal Center-Like Phenotype: A Clinico-Pathological Study of 209 Patients from the Groupe d'Etude des Lymphomes de L'Adulte (GELA).

Diffuse large B-cell lymphomas (DLBCLs) are markedly heterogeneous, and there is increasing evidence that the biological features of these tumors vary according to the primary site of disease (lymph node or various extranodal organs). The Waldeyer's ring (WR), a circular band of mucosa-associated lymphoid tissue composed of the palatine, lingual and pharyngeal tonsils, is the second most common site of extranodal invovlement by DLBCL. The presence of focal follicular features in tonsillar DLBCLs has been previously reported. In this study we included 209 adult patients with de novo DLBCL presenting in the WR consecutively included in the GELA protocols during a 10-year period (1993–2004). All patients (M/F ratio: 1,77/1, mean age 59 years) comprising 81% with stages I–II and 19% with stages III–IV disease, received intent-to-cure anthracyclin-based polychemotherapy. Morphology, pattern of growth, immunophenotype and differentiation profile were analyzed and correlated to the clinical features. Survival and outcome were analyzed in comparison to a matched cohort of patients with primary nodal DLBCL. By morphology, 55% of tonsillar cases were classified as centroblastic, 39% as centroblastic-polymorphous, 3% as immunoblastic, and 3% were unclassifiable. Among large biopsy specimens (n=79), 18% had a prominent nodular pattern consistent with transformed follicular lymphomas, 35% had a minor nodular component (<50%) interpreted as follicular colonization and 47% were purely diffuse. The nodular pattern correlated with centroblastic morphology (p=0,0007). The prevalence of antigen expression was: bcl-2: 105/189 (60%); CD10: 75/178 (42%); bcl-6 :40/76 (53%); mum-1:40/109 (37%). Global immunophenotypic profile assessed on 136 cases was GC-like in 60% and non-GC-like in 40%. The GC-like profile correlated with absence of bcl-2 expression (p<0,0001) and with centroblastic morphology (p=0,04). In multivariate analysis, the GC-like phenotype correlated with better OS (p=0.014), and CD10 expression correlated with better EFS (p=0.0061). For 144 paired tonsillar/nodal cases, the complete remission rate was significantly better for tonsillar patients (p=0.01) but the 5-y OS and EFS rates - respectively, 79,7% and 70,9% for tonsillar patients, and 76,7% and 66,7% for nodal patients - did not significantly differ. When restriciting the comparison to the cases with no factor of the age-adjusted IPI (109 paired patients), the primary tonsillar localization was associated with a higher 5-y EFS (78,5% vs. 71,2%; p=0,029) and 0S (84,7% vs. 79,8%; p=0,047) rates. In conclusion, WR DLBCLs frequently have a partially follicular pattern of growth, and a GC-like phenotype. In DLBCL patients with no factor of the age-adjusted IPI, the tonsillar localization appears to confer a better outcome than primary nodal involvement.

6026 POSTER Primary follicular lymphoma of the gastrointestinal tract; initial sites and the promise of the rituximab plus CHOP chemotherapy

Diffuse large B cell lymphoma (DLBCL) of primary nodal (PN) or primary extranodal (PEN) origin may differ immunophenotypically, in that PEN lymphoma cells may originate from activated rather than germinal center B (GCB) cells. We evaluated the relationship between DLBCL clinicopathological features, including expression of B-cell differentiation markers, and primary tumor site.Expression of CD10, Bcl-6, Bcl-2, and MUM1 was determined in paraffin-embedded tissues from 123 patients with DLBCL.Of the 123 patients with DLBCL, 40 (32.5%) had the GCB and 83 (67.5%) had the non-GCB phenotype. Fifty-one patients (42%) showed disease involvement at PEN sites, including 29 with disease in the gastrointestinal (GI) tract (14 in the stomach, 15 in the intestine). Of these 51 patients, 16 (31.4%) were classified with the GCB and 35 (68.5%) with the non-GCB subtype. There were no differences in the frequencies of GCB and non-GCB subtypes among primary sites. Of the 72 patients with PN DLBCL, 22 (31%) had the GCB and 50 (69%) had the non-GCB subtype. There were no differences in the frequencies of GCB and non-GCB subtypes between patients with PN and PEN DLBCL. Although lactate dehydrogenase (LDH) concentration > normal, stage >II, and rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) treatment were independent predictors of overall survival (OS), GCB subtype, and presence of PEN disease failed to predict survival upon multivariate analysis.There was no difference in GCB and non-GCB phenotypes between patients with PN and PEN DLBCLs. Additional studies are needed to further assess molecular differences between the two groups.

Mutations within the 5′ region of FAS/CD95 gene in nodal diffuse large B-cell lymphoma

CD95 is a cell-surface receptor that mediates apoptosis. A possible association between CD95 mutations and extranodal diffuse large B-cell lymphomas (DLBCL) has been reported. To further elucidate this question, a mutation analysis within the 5′ region and exon 9 of CD95 was performed in a series of 66 DLBCL patients, by polymerase chain reaction, single-strand conformational polymorphism, and sequencing in all cases. Four mutations, all within the 5′ region, were detected in three cases of primary nodal DLBCL (6.3% of primary DLBCL), probably originated as by-products of the somatic hypermutation process. No CD95 mutations in the two analyzed regions were detected in primary extranodal DLBCL, mediastinal large B-cell lymphoma (MLBCL), and DLBCL arising from indolent low-grade lymphomas. Because of our results, a review of published data with respect to the site of mutations was performed, which suggested a different distribution of mutations in nodal and extranodal DLBCL.

Profiling of apoptosis genes allows for clinical stratification of primary nodal diffuse large B‐cell lymphomas

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B-lymphocytes versus activated B-cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro-and anti-apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis-inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

Diffuse large B-cell lymphoma with expression of anaplastic lymphoma kinase protein: clinicopathologic and immunohistochemical study of 5 cases

To study the clinicopathologic features of diffuse large B-cell lymphoma (DLBCL) with expression of anaplastic lymphoma kinase (ALK) protein. Nine hundred and forty-five (945) cases of DLBCL (including 177 consultation cases) diagnosed according to the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues were enrolled into the study. Immunohistochemical study for anti-ALK-11 was performed using LSAB technique. The ALK-positive cases were further confirmed by immunohistochemical study using EnVision technique. Only ALK-positive cases by EnVision technique were further analyzed by immunostaining for antigens including CD20, CD3, CD30, EMA, granzyme-B, TIA-1 and PC. Immunoglobulin heavy chain gene rearrangement study was also performed and follow-up data collected. There were altogether 5 (4 males and 1 female) cases of DLBCL showing expression of ALK protein. The age of the patients ranged from 34 to 72 years. All were primary nodal DLBCL. One case belonged to clinical stage I, 2 in stage II and 2 in stage III. The duration of follow up ranged from 4 to 32 months. Three patients subsequently died and the longest survival was 32 months. Morphologic subtypes included centroblastic 2, anaplastic 1, immunoblastic with plasmacytoid differentiation 1 and plasmablastic 1. Immunohistochemically, 4 cases were CD20 positive (including 2 centroblastic, 1 anaplastic and 1 immunoblastic cases). The plasmablastic case expressed kappa light chain and was negative for CD20. Rearrangement of immunoglobulin heavy chain gene was demonstrated in all 5 cases studied. As for ALK protein staining, a mixed membranous and cytoplasmic (1 immunoblastic case), granular cytoplasmic (2 centroblastic and 1 anaplastic cases) and mixed nuclear and cytoplasmic (1 plasmablastic case) patterns were observed. Expression of ALK protein is a rare phenomenon in DLBCL and can be seen in centroblastic, anaplastic, immunoblastic and plasmablastic subtypes. It is often associated with aggressive clinical behavior and worse prognosis. A new pattern of ALK protein expression, mixed membranous and cytoplasmic, is reported.