Germinal Center B-cell Subgroup Research Articles

Germinal center B-cell subgroups in the tumor microenvironment cannot be overlooked: their involvement in prognosis, immunotherapy response, and treatment resistance in head and neck squamous carcinoma

BackgroundMore than 60% of patients with head and neck squamous carcinoma (HNSCC) are diagnosed at advanced stages and miss radical treatment. This has prompted the need to find new biomarkers to achieve early diagnosis and predict early recurrence and metastasis of tumors. MethodsSingle-cell RNA sequencing (scRNA-seq) data from HNSCC tissues and peripheral blood samples were obtained through the Gene Expression Omnibus (GEO) database (GSE164690) to characterize the B-cell subgroups, differentiation trajectories, and intercellular communication networks in HNSCC and to construct a prognostic model of the associated risks. In addition, this study analyzed the differences in clinical features, immune cell infiltration, functional enrichment, tumor mutational burden (TMB), and drug sensitivity between the high- and low-risk groups. ResultsUsing scRNA-seq of HNSCC, we classified B and plasma cells into a total of four subgroups: naive B cells (NBs), germinal center B cells (GCBs), memory B cells (MBs), and plasma cells (PCs). Pseudotemporal trajectory analysis revealed that NBs and GCBs were at the early stage of B cell differentiation, while MBs and PCs were at the end. Cellular communication revealed that GCBs acted on tumor cells through the CD99 and SEMA4 signaling pathways. The independent prognostic value, immune cell infiltration, TMB and drug sensitivity assays were validated for the MEF2B+ GCB score groups. ConclusionsWe identified GCBs as B cell-specific prognostic biomarkers for the first time. The MEF2B+ GCB score fills the research gap in the genetic prognostic prediction model of HNSCC and is expected to provide a theoretical basis for finding new therapeutic targets for HNSCC.

BCL2 Expression Identifies a Population with Unmet Medical Need in Previously Untreated (1L) Patients with DLBCL

▪Introduction.High BCL2 protein expression has been associated with inferior outcome in multiple studies in diffuse large B-cell lymphoma (DLBCL) (Gascoyne et al. 1997; Salles et al. 2011; Horn et al. 2013; Tsuyama et al. 2017). Patients with high levels of BCL2 expression may benefit from treatment with venetoclax, a first-in-class, highly selective BCL2 inhibitor with demonstrated activity in cell lines and xenograft models (Souers et al. 2013, Punnoose et al. 2016). The evaluation of BCL2 as a prognostic marker has been complicated by the variability in recognized immunohistochemistry (IHC) thresholds (de Jong et al. 2007; Tsuyama et al. 2017) and the impact of cell-of-origin (COO) (Iqbal et al. 2011). Using a BCL2 IHC scoring algorithm that was developed to identify cell lines that are sensitive to BCL2 inhibition, and that could potentially identify patients likely to benefit from venetoclax, we evaluated the prognostic capability of BCL2 IHC in three independent cohorts of 1L DLBCL patients. Cohorts included two Phase 3 studies (MAIN [NCT00486759] and GOYA [NCT01287741]), and a representative cohort from the British Columbia Cancer Agency (BCCA) registry-based population, in which subjects received chemoimmunotherapy (either rituximab or obinutuzumab, combined with cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]).Patients and Methods. Pretreatment tumor samples were tested for BCL2 IHC using an algorithm that assesses both the percent of tumor cells stained and the intensity of staining, similar to the BCL2 algorithm proposed by Tsuyama et al. (Table 1).Whole-tissue slides from184 patients from MAIN were tested at Genentech , whole tissue slides from 366 patients from GOYA were tested at Hematogenex (Ventana assay) and tissue microarray slides from 310 patients from BCCA were tested at Ventana (Ventana assay). The NanoString Lymphoma Subtyping gene expression assay was used for classification of COO into germinal center B-cell (GCB), activated B-cell (ABC) or unclassified subtypes in MAIN (n=159), GOYA (n=353) and BCCA (n=302). Multivariate Cox regression analysis of progression-free survival (PFS) was performed in each study with the International Prognostic Index (IPI; The International NHL Prognostic Factors Project 1993) and/or COO as covariates. Given the similar treatment effect, patient data from both treatment arms in each study were merged for this analysis.Results. Baseline characteristics were similar for the BCL2 IHC evaluable and intent-to-treat (ITT) populations of these three cohorts. Prevalence of BCL2 positive patients was 48% in MAIN, 49% in GOYA and 58% in the BCCA cohort. The prevalence of BCL2 positive samples within the ABC subtype was 64%, 75% and 60% in the MAIN, GOYA and BCCA cohorts, respectively, and 40%, 38% and 56% within the GCB subtype, respectively. In univariate analysis, BCL2 positivity was associated with shorter PFS in all three cohorts (Figure 1, A-C). In the MAIN study, the prognostic effect of BCL2 appeared to be in the GCB subgroup, not ABC, as previously reported (Punnoose et al. ASH 2015). In the two largest studies of GOYA and BCCA cohorts, the prognostic effect of BCL2 was similar in ABC and GCB (not shown), and was independent of IPI (Table 2).Conclusions. Using a newly developed BCL2 IHC scoring algorithm, we demonstrate that DLBCL patients positive for BCL2 have a consistently inferior prognosis compared with patients negative for BCL2. The prognostic value of BCL2 IHC is independent of IPI and COO in GOYA and BCCA. The Ventana assay is currently being used in a Phase 2 study of venetoclax in combination with R-CHOP to evaluate the correlation (if any) of BCL2 expression with venetoclax + R-CHOP treatment (CAVALLI - NCT02055820). [Display omitted] DisclosuresSzafer-Glusman:Genentech: Employment. Peale:Genentech / Roche: Employment, Equity Ownership. Lei:Roche: Employment. Ray:Genentech/Roche: Employment. Farazi:Genentech: Employment. Kornacker:Roche: Equity Ownership; F. Hoffmann-La Roche: Employment. Oestergaard:F. Hoffmann-La Roche Ltd: Employment. Liu:F. Hoffmann-La Roche: Employment. Punnoose:Genentech: Employment.

Comparison of the algorithms classifying the ABC and GCB subtypes in diffuse large B-cell lymphoma.

Different immunohistochemical algorithms for the classification of the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of diffuse large B-cell lymphoma (DLBCL) are applied in different laboratories. In the present study, 127 patients with DLCBL were investigated, all treated with rituximab and cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP) or CHOP-like regimens between April 2004 and December 2010. Multi-tumor tissue microarrays were prepared and were tested according to 4 algorithms: Hans; modified Hans; Choi; and modified Choi. For 39 patients, the flow cytometric quantification of CD19 and CD20 antigen expression was performed and the level of expression presented as molecules of equivalent soluble fluorochrome units. The Choi algorithm was demonstrated to be prognostic for OS and classified patients into the GCB subgroup with an HR of 0.91. No difference in the expression of the CD19 antigen between the ABC and GCB groups was observed, but the ABC subtype exhibited a decreased expression of the CD20 antigen compared with the GCB subtype.

Low Expression and Somatic Mutations of the KLHL6 Gene Predict Poor Survival in Patients with Activated B-Cell like Diffuse Large B-Cell Lymphoma

IntroductionDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both clinically and biologically. Based on gene expression profiling, two major molecular subtypes, activated B-cell (ABC)- and germinal center B-cell (GCB)-like DLBCLs, have been recognized. More recently, next generation sequencing has also shed light on the mutational heterogeneity of these lymphomas. While several oncogenes are recurrently mutated and aberrantly expressed in DLBCL, little is known of their functional impact and association with survival.Design and MethodsTo identify mutated oncogenes in DLBCL, we performed whole-exome and RNA sequencing screen for tumor tissue and matched normal DNA from eight high-risk (aaIPI >2) DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with dose-dense chemoimmunotherapy followed by systemic central nervous system (CNS) prophylaxis.Initial screen was expanded to a Nordic discovery cohort consisting of 38 high-risk DLBCL patients treated in the same protocol. Gene expression was analyzed from the tumor tissue using Affymetrix Human Exon 1.0 ST arrays, quantified with MEAP (Multiple Exon Array Preprocessing, probe annotation version 70) algorithm, and expression of the mutated genes identified. Samples were classified into GCB, ABC and nonclassified subgroups using the Lymphochip gene predictor. At the time of the analysis, median follow-up time was 65 months and predicted 5-year OS 76%. Survival association of the identified mutations and gene expression was validated using RNAseq data from 92 (Cancer Genome Characterization Initiative (CGCI) repository), and oligonucleotide-based microarray expression data from 233 (Lymphoma/Leukemia Molecular Profiling Project, LLMPP) DLBCL patients, respectively. Patients were treated with chemoimmunotherapy in both validation cohorts.ResultsIn our initial screen 922 genes were affected by somatic mutations. The genes included both novel and previously identified lymphoma-related oncogenes, such as MYD88, CD79B and DTX1. We highlight mutations affecting GC marker Kelch-like family member 6 (KLHL6), which is a predicted oncogene and involved in B-cell receptor (BCR) signaling in DLBCL.In the CGCI validation cohort 16% of the patients carried non-silent KLHL6 mutations. According to Kaplan Meier analysis, the patients with KLHL6 mutations had significantly worse survival in comparison to the patients without mutations (5-year OS; 53% vs 84%; p = .010). In multivariate analysis, the prognostic impact of KLHL6 mutations on the survival was independent of IPI (OS; RR, 3.060; 95% CI, 1.233-7.598; p = .016). KLHL6 mutations were equally distributed between the molecular subtypes. However, when the molecular subtypes were analyzed separately, KLHL6 mutations remained predictive for poor survival only in the ABC-like DLBCL (OS; RR, 6.727; 95% CI 1.576-28.714; p= .010).KLHL6 expression was further analyzed in the Nordic discovery cohort. Consistent with its role as a GC marker, KLHL6 expression tended to be higher in the GCB- than the ABC-like DLBCLs. Overall, low KLHL6 expression was associated with poor survival (5-year OS; 93% vs 65%; p = .044). The difference in survival was restricted to the ABC subtype. The prognostic impact of KLHL6 expression in the patients with the ABC-like DLBCL was confirmed in independent LLMPP validation cohort.ConclusionsThe results show that although high KLHL6 expression is characteristic for the GCB DLBCL, mutations and low expression of KLHL6 are clinically more relevant in the ABC subtype pinpointing the patients with the most dismal prognosis. DisclosuresLeppä:CTI Life Sciences: Honoraria; Roche: Honoraria, Other: Travel Expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses; Bayer: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Amgen: Research Funding.

Association Between the Immunohistochemical GCB/non GCB Classification and the Outcome of the Patient With Diffuse Large B - Cell Lymphoma Treated With Rituximab - CHOP Regimen: Single Centre Experience

Diffuse large B-cell lymphoma (DLBCL) is sub-classified in to molecular subgroups that correspond to different stages of lymphocyte development- namely germinal center B-cell (GCB) and non- germinal center B-cell DLBCL (non-GCB). In fact, some studies suggest that the distinction between the GCB subgroup and non-GCB had important prognostic implications in the cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP) treatment era with better outcome for GCB DLBCL patients. However, recently the results from one large prospective study indicated that in Rituximab (R)-CHOP treatment settings other markers than immunohistochemical features predict the outcome of the DLBCL. In order to investigate the prediction value of the immunohistochemical GCB/non GCB classification of DLBCL of the outcome of our DLBCL patient treated with the R-CHOP regimen we conducted a retrospective study. Our study enrolled 132 DLBCL patients diagnosed and treated with R-CHOP regimen at the University Clinic of Hematology in the period between February 2002 and December 2007. The median follow-up of the patients was 36 months. The biopsy samples were immunostained and analyzed for markers of germinal center (BCL6), post-germinal center (MUM1) and apoptosis (BCL2). The patients were categorized as GCB subgroup (68; 51, 6%) or non-GCB subgroup (64; 48, 4%). The median overall survival time (OS) were 65, 25 months in GCB group and 61, 1 month in non-GCB group, and time to treatment (TT) were 60, 85 and 57,75 months respectively for the both groups. The groups were statistically comparable regarding the both parameters. They were also comparable regarding the age, gender distributions and all others already established prognostic parameters as performance status, advanced International Prognostic Index (IPI), albumin level except for the low IPI 0-2 which was statistically associated with the GCB group (p=.024). In this retrospective study the addition of rituximab to CHOP-based chemotherapy in patients with GCB DLBCL was not associated with statistical survival advantage and better outcome. Also the study indicates that immunohistochemical markers do not really reflect the molecular diversity of the tumor. In that line, results support the studies that suggest that Rituximab eliminates or modulates the significance of some already established prognostic markers for DLBCL. In context of the modern therapy, previously recognized markers should be re-evaluated and new prognostic indicators for DLBL has to be identified.

Cell Origin Subtypes Predict Outcomes in Localized-Stage Diffuse Large B-Cell Lymphoma Treated with Curative Radiotherapy

Diffuse large B-cell lymphoma (DLBCL) is regarded as a heterogeneous group of lymphomas. The aims of this study were to determine the clinical significance and prognostic value of different immunophenotypic profiles in localized-stage head and neck DLBCL treated with curative radiotherapy. We included 102 localized-stage head and neck DLBCL patients in this study. We classified DLBCL patients into germinal center B-cell (GCB) and non-GCB groups by immunohistochemical analysis. Statistical analysis was used to correlate the GCB and non-GCB subgroups, CD5 and Ki67 expression, B-ALPS (a modified International Prognostic Index for early stage lymphoma), chemotherapy regimen, and sex. Multivariate analysis was performed using the Cox proportional hazard regression model to compare cause-specific survival (CSS) and relapse-free rate (RFR) distributions. The cell of origin classification (GCB or non-GCB subtypes) was an independent predictor of CSS (p = 0.040) and RFR (p = 0.023). In the non-GCB group, chemotherapy with rituximab was an independent predictor of CSS. In conclusion, this study shows that the prognosis of the non-GCB group was significantly poorer than that of the GCB group, and that rituximab improved CSS in localized-stage head and neck DLBCL, especially in the non-GCB group.

CD99 expression and newly diagnosed diffuse large B-cell lymphoma treated with rituximab-CHOP immunochemotherapy

In order to evaluate prognostic value of CD99 expression in patients with diffuse large B-cell lymphoma (DLBCL) who underwent treatment with rituximab-CHOP immunochemotherapy, immunohistochemistry for CD99/CD10/BCL-2/BCL-6/MUM-1 was performed on nodal DLBCL specimens from 70 patients. Patients were classified as either germinal center B-cell (GCB) subtype or non-GCB subtype according to the Muris algorithm. A superior 2-year event-free survival (EFS) was observed in patients with the GCB subgroup, compared to those with the non-GCB subgroup (p = 0.034). The distribution of CD99 expression (29 patients; 41.4%) did not show deviation according to subtype and was not prognostic for survival in the entire patient population. Among patients with the GCB subgroup, better EFS and overall survival (OS) were observed in CD99+ patients, compared to CD99- patients. Conversely, among patients with the non-GCB subgroup, inferior EFS and OS were reported in CD99+ patients. Superior 2-year EFS (p = 0.004) and 2-year OS (p = 0.003) were observed in patients with GCB/CD99+ and non-GCB/CD99- compared to the others, and the combination classification was found to be an independent prognostic factor.

The t(14;18)(q32;q21) Characterizes a Subset of Patients with Diffuse Large-B Cell Lymphoma of Germinal Center Origin with Poor Outcome: Report From the International DLBCL Rituximab-CHOP Consortium Program Study

Abstract 949 Introduction:Diffuse large B cell lymphoma (DLBCL) has a highly variable outcome, and individual risk assessment is largely based on clinical features. Gene expression profiling (GEP) stratifies patients into those with germinal center B-cell (GCB) and activated B-cell subtype (ABC) subtype with different prognoses. These groups have been shown to predict prognosis in patients treated with CHOP or R-CHOP. Conversely, the role of other recognized prognostic markers, such as BCL2 gene abnormalities or Bcl2 expression has been questioned in the new therapeutic era.Materials and Methods: In 438 patients treated with R-CHOP for de novo DLBCL, we analyzed the tumors by immunohistochemistry for Bcl2 protein expression and by interphase fluorescence in situ hybridization (FISH) for BCL2 translocation and other abnormalities. All cases were successfully studied by GEP. The cutoff for Bcl2 protein expression, 60%, used as prognostic factor was determined using receiver operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were assessed. Results:The t(14;18)(q32;q21) was detected in 82 cases (18.7%) and BCL2 gains occurred in 63 cases (14.3%). Both t(14;18) and BCL2 gains strongly correlated with higher levels of Bcl2 protein expression (p<0.0001 for both). Presence of t(14;18) was associated with the GCB subtype (p<0.0001), whereas BCL2 gains were associated with the ABC subtype (p=0.004). BCL2 gains were not predictive of PFS in any patients’ subgroups. Conversely, within the GCB subtype, patients with the t(14;18) displayed a significantly worse outcome compared to GCB patients without t(14;18) with a 5-year PFS of 45% vs 68%, respectively (p<0.0001). Outcome of patients with DLBCL associated with t(14;18) was similar to patients with the ABC subtype (45% vs 48%, p=0.30, Figure 1). No impact of the t(14;18) and BCL2 gains was observed on patients with ABC-DLBCL. Using immunohistochemistry, patients with Bcl2 positive (>60%) tumors had significantly inferior PFS in the GCB subgroup (p=0.03), but not in the ABC subgroup (p=0.54). Multivariate analysis revealed that the presence of the t(14;18), but not Bcl2 protein expression, was independent of the International Prognostic Index in predicting outcome of our patients. Conclusions:Patients with the GCB subtype and t(14;18) exhibit a significantly worse prognosis than patients without t(14;18) when treated with R-CHOP. The assessment of t(14;18) by FISH approach not only functions as a valuable prognosticator for individual risk estimation in GCB-DLBCL patients in addition to the established parameters, but also provides valuable result for therapeutic intervention. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acquired somatic mutations in non-coding region of bcl-6 gene in germinal center-type diffuse large B-cell lymphoma

To investigate the mutation of 5' non-coding region of bcl-6 gene in germinal center B-cell (GCB) subtype of diffuse large B-cell lymphoma (DLBCL). t(14;18) detection and immunohistochemical staining (EnVision method) were performed in 60 cases of DLBCL, which were divided into GCB and non-GCB subtypes. Polymerase chain reaction (PCR), single-strand conformation polymorphism and direct DNA sequencing were used to identify mutations in the 5' non-coding region of the bcl-6 gene. Seven of 60 cases showed t(14;18) translocation in the major breakpoint region. Using minimally acceptable criteria, 18 of 60 cases were probably to be germinal centre derived. Bcl-6 mutations were detected in 12 of 60 cases (20.0%) of DLBCL, with a significantly higher frequency in the GCB subgroups (7/18) than in the non-GCB subgroups (11.9%, 5/42). Bcl-6 mutations occurred most frequently in +363 and +469 sites. An association of bcl-6 mutation and GCB subgroup was obtained. The 5' regulatory region of the bcl-6 gene underwent less frequent somatic hypermutation during lymphomagenesis than the results of previous reports. Bcl-6 mutation occurred mostly in the GCB subtype and detection of t(14;18) seems helpful in the classification of DLBCL.

Expression and prognostic significance of differentiation markers in primary CNS lymphoma (PCNSL)

e13028 Background: BCL-6 expression has been reported to be associated with a better prognosis in PCNSL. However, significant differences of B-cell lymphoma subtypes exist in different geographic regions. The objectives of this study were to characterize the immunophenotypes of PCNSL in Japan and to determine their utility in predicting clinical outcomes. Methods: Immunostaining for CD5, CD10, BCL-6, and MUM-1 was done to determine the profile of diffuse large B-cell lymphoma of PCNSL from 27 immunocompetent patients in Japan. Then, the authors evaluated whether clinical and immunophenotypic markers were associated with progression-free or overall survival. Results: Germinal center B-cell (GCB) marker CD10 was positive in 22% of PCNSL samples, while another GCB marker BCL-6 was positive in 48% of samples. Post-GCB marker MUM-1 was positive in 81% of samples. Poor prognostic marker of systemic B-cell lymphoma CD5 was expressed in 15% of samples. In univariate analyses, only BCL-6 expression had a significant effect on progression-free survival (median, 6.1 months in BCL-6-positive patients versus 15.6 months in BCL-6-negative patients; p = 0.037). Patients with BCL-6 expression had a trend towards longer overall survival but nonsignificant. GCB subgroup (CD10-positive or CD10-negative/BCL-6-positive/MUM-1-negative) was not significantly associated with favorable survival. Conclusions: BCL-6 expression is associated with a poor prognosis in patients with PCNSL. The discrepancy between the reports from other groups and our results is potentially due to the racial, geographic or immunophenotypic differences of PCNSL. No significant financial relationships to disclose.

Immunohistochemical Expression of Germinal Center and Non-Germinal Center Markers in Primary Gastric Diffuse Large B-Cell Lymphoma

BACKGROUND: Recent studies have shown a correlation between diffuse large B-cell lymphoma (DLCBL) prognosis and molecular features using genome profiles by cDNA microarrays. Later reports have shown that immunohistochemical tests using markers as CD10, bcl-6 and MUM-1 to identify germinal center B-cell (GCB) and non-GCB patterns can have a similar value and are gaining major importance in assessing prognosis in patients with DLBCL. Gastric DLCBL is the most frequent extranodal non-Hodgkin lymphoma.AIM: To evaluate the frequency of GCB and non-GCB subgroups in primary gastric DLCBL and the impact of these tests in prognosis.PATIENTS AND METHODS: Patients older than 18 years with a diagnosis of primary gastric DLBCL were identified in a single institution from January 2002 to December 2005 and included in this analysis. Immunohistochemical stains were performed using antibodies against CD20, CD10, bcl-6 and MUM1 in all cases. Survival curves were obtained using the Kaplan-Meier method.RESULTS: Twenty-nine patients were included in this study. The median age at diagnosis was 68 years (range 40 to 86 years). Thirteen male and 16 female patients were included. All cases received chemotherapy with CHOP regimen. Cases were subclassified in GCB and non-GCB using CD10, bcl-6, and MUM1 expression. Four cases (14%) were considered GCB and 25 cases (86%) non-GCB. There were no statistical differences between both groups in the following variables: age, ECOG performance status, serum LDH levels, clinical stage and International Prognostic Index (IPI) score. No statistical difference in survival was observed between the GCB and non-GCB groups. The 3-year overall survival for the entire group was 30%.CONCLUSION: Immunohistochemical expression of non-GCB pattern is very frequent in primary gastric DLBCL in Peru. However, it does not show an effect in survival, probably related to the small number of cases included in the present study.

Activated B-Cell (ABC) Profile Does Not Have An Inferior Outcome Compared to Germinal Center B-Cell (GCB) Profile Determined by Immunohistochemistry Tissue Microarray in Poor Prognosis Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Rituximab and Dose- Dense and High-Dose Chemotherapy (HDC) Plus Autologous Stem Cell Transplantation (ASCT).

Introduction: A specific molecular signature divides DLBCL into GCB and ABC profiles; in pre-Rituximab era, GCB group had a better prognosis. The addition of Rituximab (R) to chemotherapy improves the outcome in DLBCL. It remains uncertain the prognostic significance of GCB and ABC subgroups in Rituximab era.Patients and methods: from August 2000 to September 2006, 120 previously untreated patients <61 years affected by DLBCL, at aa-IPI intermediate/high or high risk, were enrolled into a phase II GIMURELL trial: 4 courses of bi-weekly R-MegaCEOP14 (375 mg/m2 R on day 1, 1200 mg/m2 CTX + 110 mg/m2 EPI + 1.4 mg/m2 VCR on day 3 and 40 mg/m2 PDN days 3 to 7), 2 R-MAD (8 mg/m2 Mitoxantrone + 2 g/m2/12h Cytarabine + 4 mg/ m2/12h Dexamethasone days 1 to 3 and 375 mg/m2 R on day 4 and before peripheral blood stem cell harvest as in vivo purging) followed by ASCT with BEAM as conditioning regimen. Thus far, tissue microarray (TMA) blocks were created from 92 cases and they were studied in immunohistochemistry; sections were stained with antibodies to CD20, CD3, CD10, CD138, Ki67, Bcl6, Bcl2, MUM1 and cyclinD1. Patients were classified as GCB or non-GCB (ABC) according to the Hans' algorithm. The samples were scored positive for CD10, Bcl-6 and MUM-1 if 30% or more of tumor cells were stained. Cases with the coexpression of CD10 and MUM-1 were classified as GC-activated according to Chang and grouped with GCB for analysis purpose. Crude Kaplan-Meier overall (OS) and event-free (EFS) survival curves were estimated both overall and stratified by TMA features. Differences between curves were tested using the 2-tailed log-rank test..Results: 92 samples were centrally reviewed and studied in immunoistochemistry. Overall 69 were evaluable; 23 were excluded due to inadequate pathological materials in 16 and incorrect inclusion criteria in 7. Thirty-seven patients were classified as GCB, 24 as ABC and 8 as GC-activated. Patient characteristics were well balanced between GCB and ABC subtypes. With a median follow-up of 53 months, OS in all 92 patients was 81% (95% CI: 71% - 88%); at the same timeframe, OS in GCB was 77% vs 86% for ABC (p=.5). With a median follow-up of 46 months, EFS in all 92 patients was 74% (95% CI: 64% - 82%); at that time, EFS in GCB was 65% vs 87% for ABC (p=.2).Conclusions: Rituximab as adjuvant to dose-dense and HDC with ASCT support is effective in high risk DLBCL in both GCB and ABC subgroups. In this R-HDC+ASCT study ABC profile as determined by TMA does not represent a poor prognostic feature. However 25% of patients still experienced progression. Further and more detailed biological analysis are needed to better identify these patients.

Effect of addition of rituximab to CHOP on survival of patients in both the GCB and non-GCB subgroups of diffuse large B-cell lymphoma

8040 Background: Diffuse large B-cell lymphoma (DLBCL) includes at least two prognostically important subgroups, i.e. germinal center B-cell (GCB) and activated B-cell (ABC) DLBCL, which were initially characterized by gene expression profiling and subsequently validated by immunostaining. Bcl-2 has also been identified as a prognostic indicator in the ABC subgroup. However, with the addition of rituximab (R) to standard chemotherapy, the prognostic significance of this subclassification of DLBCL is unclear. Methods: We studied 119 cases of de novo DLBCL including 70 cases treated with R-CHOP and 49 cases treated with CHOP. The cases were assigned to either the GCB or non-GCB subgroups using the methodology described by Hans et al (Blood 2004; 103:275). Characteristics of the patients were compared using the Chi-square test. Overall survival (OS) and event-free survival (EFS) were estimated using the Kaplan Meier method and compared with the log-rank test. Results: The median age of the 119 patients was 67 years, ranging from 20 to 90 years, and there were 62 males and 57 females. The clinical characteristics of patients treated with CHOP versus R-CHOP, including the IPI, were comparable. R-CHOP was more effective than CHOP with improved 5-year EFS (63% vs 41%, p=0.013) and OS (78% vs 47%, p&lt;0.001). In both patient groups treated with R-CHOP or CHOP, the GCB subgroup had a significantly better 5-year EFS and OS compared to the non-GCB subgroup (OS: 91% vs 64% for R-CHOP, p=0.0073; 67% vs 31% for CHOP, p=0.034, respectively). Additionally, both the GCB and non-GCB subgroups treated with R-CHOP had a significantly improved OS compared to their respective subgroups receiving CHOP alone (GCB, p=0.015; non-GCB, p=0.019). Bcl-2 expression was not a significant predictor in either the GCB or non-GCB subgroups treated with R-CHOP (OS, GCB: p=0.32; non-GCB: p=0.43). Conclusions: In this retrospective study, we demonstrate that subclassification based on the cell of origin continues to have prognostic significance in patients with DLBCL treated with R-CHOP. Addition of rituximab to CHOP improves the overall survival of patients with DLBCL in both the GCB and non-GCB subgroups. No significant financial relationships to disclose.

Comparison of the expression and prognostic significance of differentiation markers between diffuse large B-cell lymphoma of central nervous system origin and peripheral nodal origin.

Whether diffuse large B-cell lymphoma (DLBCL) of primary central nervous system origin (PCNSL) is biologically different from DLBCL of peripheral nodal origin (NL) remains unclear. The purpose of this study was to compare the expression frequencies and prognostic significance of a panel of cell differentiation markers between these two disease entities. This study included HIV-unrelated patients with PCNSL (n = 51) and NL (n = 72) treated at four hospitals in Taiwan for whom archival tumor tissue was available. Immunohistochemistry for CD10, BCL-6, MUM-1, vs38c, CD138, and BCL-2 was done. CD10, BCL-6, and MUM-1 expression results were used to classify all cases into the germinal center B-cell (GCB) or the non-GCB subgroup. The prognostic significances of clinical and immunophenotypic markers were evaluated. Nuclear MUM-1 expression was significantly higher in PCNSL than in NL (P < 0.001; 84% versus 53%). PCNSL tumors were more frequently classified into the non-GCB subgroup than NL tumors (P = 0.020; 78% versus 62%). For patients with PCNSL, univariate analysis showed that patients with BCL-6 expression had a trend towards longer survival (P = 0.073; median survival, 25.3 versus 7.3 months), and multivariate analysis showed BCL-6 was an independent prognostic factor (P = 0.026). For patients with NL, both of univariate (P = 0.003) and multivariate analyses (P = 0.002) showed that GCB was significantly associated with favorable survival. The higher frequency of non-GCB subclassification, which was mainly contributed by nuclear MUM-1 expression in PCNSL implies that it has a more differentiated cellular origin than NL. BCL-6 expression in patients with PCNSL and GCB subgroup in patients with NL were favorable prognostic factors.

Dose-Adjusted EPOCH-Rituximab Is Highly Effective in the GCB and ABC Subtypes of Untreated Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is subdivided by microarray into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. These subtypes have distinct pathways of lymphomagenesis and outcome with 5-year survival of 59% and 31%, respectively, using CHOP. A molecular predictor showed high tumor proliferation and the ABC subtype are associated with a poor outcome. We showed DA-EPOCH overcomes the adverse effects of high tumor proliferation (BLOOD 99:2685, 2002) and rituximab overcomes the adverse effects of BCL-2, which is associated with the ABC subtype (Proc Am Soc Heme 102:abstr 356, 2003). To assess DA-EPOCH-R with G-CSF in GCB and ABC subtypes, we employed a validated immunohistochemistry (IHC) algorithm (BLOOD: 103:275, 2004). GCB was defined as CD10+ or BCL-6+ and MUM1-. ABC was defined as CD10- and BCL-6- or CD10-, BCL-6+ and MUM1+. Eligibility includes de novo DLBCL, no prior chemotherapy, any PS, HIV- and stage I thymic DLBCL > 5 cm and all stage II-IV. Characteristics of 77 enrolled pts include median (range) age 48 (12-85) and PS 1 (0-3); stage III/IV 48 (62%); and LDH > nl 42 (55%); extranodal sites >1 27 (35%); and HI/H IPI 26 (34%). Including all 77 pts, response is CR/CRu 72 (94%). At the median (range) follow-up of 28 (4-60) mos, PFS is 82% and OS is 83% (Fig 1). DFS is 87% at 28 mos with no relapses beyond 18 mos. PFS at 28 mos according to IPI risk is 95% for 0-2 factors and 54% for 3-5 factors. Using IHC, 44 pts were subdivided in GCB (35) and ABC (9) subtypes. The proportion of the ABC subtype vs. GCS is low and likely due to incomplete IHC for the ABC subtype. Specifically, 16 cases that were CD10- did not have adequate BCL-6 and/or MUM1 IHC for categorization. PFS at 28 mos for the GCB and ABC subgroups were 84% and 62%, respectively (Fig 2). DA-EPOCH-R employs a pharmacodynamic design where doses of doxorubicin, etoposide and cyclophosphamide are adjusted (i.e. normalized) to achieve neutrophil nadirs < 500/mm3. PK analysis shows that clearance of doxorubicin and etoposide varies by up to 1 log and that dose-adjustment compensates for these differences. This approach increases dose intensity in pts with high drug clearance while limiting toxicity. Based on 451 cycles, the target nadir ANC < 500/mm3 was achieved on 277 (61%) cycles but with 67 (15%) episodes of fever/neutropenia. Non-hematological toxicity was low and there were 2 treatment related deaths. These results suggest DA-EPOCH-R is highly effective in all subtypes of de novo DLBCL. A phase III study comparing R-CHOP and DA-EPOCH-R with microarray analysis of tumor biopsies is under development. [Display omitted] [Display omitted]