non-GCB Phenotype Research Articles

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.

The emerging role of lenalidomide in the management of lymphoid malignancies

Lenalidomide, a novel immunomodulatory drug (IMiD), is a promising therapeutic strategy for patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and B-cell lymphomas. Biologically, the mechanisms responsible for lenalidomide activity are yet to be clearly defined. Based on preclinical models and early correlative studies conducted in parallel to clinical trials, lenalidomide has been found to enhance natural killer (NK)- and T-cell activity against tumor cells, alter the balance of pro- and anti-inflammatory cytokines in the tumor bed, inhibit angiogenesis, and, to a lesser degree, induce cell cycle arrest and apoptosis in cancer cells. Together, all of these biological effects appear to play a role in the activity observed in CLL or lymphoma patients treated with lenalidomide. Given the effect in NK- and T-cell function, lenalidomide is an alternative strategy to enhance the antitumor activity of monoclonal antibodies (mAbs). Clinical responses have been observed in patients with relapsed/refractory CLL, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL) treated with lenalidomide single agent. The favorable toxicity profile and route of administration made the use of lenalidomide an attractive therapy for certain types of patients (i.e. elderly, chemotherapy unfit, etc.). The erratic but serious incidence of tumor lysis syndrome and/or tumor flare reactions provides challenges in the incorporation of lenalidomide in the management of previously untreated CLL or CLL/lymphoma patients with bulky adenopathy. Correlative studies and/or retrospective analysis of lenalidomide-treated patients had identified several biomarkers associated with clinical endpoints in CLL (i.e. changes in tumor necrosis factor alpha [TNF-α] or vascular endothelial growth factor [VEGF] levels) or DLBCL (non-GCB phenotype) patients, but need to be validated. Early studies evaluating the efficacy and toxicity of lenalidomide in combination with rituximab in previously untreated indolent lymphoma are promising and warrant further study. In addition, the evaluation of lenalidomide in the maintenance setting or in combination with other target-specific agents (i.e. proteasome inhibitors) in aggressive lymphomas is being addressed in ongoing clinical trials. In summary, lenalidomide is emerging as a biologically active and novel agent in the treatment of B-cell neoplasms. Future translational and clinical studies will further define the role of lenalidomide in the management of de novo or relapsed/refractory CLL or B-cell lymphomas and identify the subset of patients most likely to gain clinical benefit.

Blimp-1 protein and Hans classification on prognosis of diffuse large B-cell lymphoma and their interrelation

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL), is heterogeneous on molecular and clinical levels, therefore, its prognosis is difficult to predict. This study aimed to evaluate the value of Blimp-1 protein and Hans classification in predicting the prognosis of DLBCL and their interrelation. The clinical records of 136 patients with DLBCL were reviewed. The patients were followed up for 5-80 months (median, 39 months). Immunohistochemical staining for CD10, MUM1, Bcl-6, and Blimp-1 were performed on paraffin-embedded tumor tissues from the 136 patients. The correlations of Blimp-1 protein and Hans classification in prognosis of DLBCL and their interrelation were analyzed. Blimp-1 was detected in 38 (30.0%) patients, and was associated with a significantly shorter overall survival (OS) (P = 0.030). Using the Hans classification based upon the expression of CD10, Bcl-6, and MUM1, 54 patients had germinal center B-cell (GCB) phenotype and 82 had non-GCB phenotype. The 5-year OS rate was 75% in the GCB group and 52% in the non-GCB group (P = 0.020). The positive rate of Blimp-1 was 22.2% in the GCB group and 31.7% in the non-GCB group (P = 0.329). The Cox regression multivariate analysis showed that international prognosis index (IPI) and Hans classification had independent prognostic significance, whereas Blimp-1 was not an independent prognostic factor. The patients with GCB subtype of DLBCL had better prognosis than the non-GCB subtype. High level of Blimp-1 expression in the patients with DLBCL implies a shorter survival, but it is not associated with Hans classification.

Primary central nervous system lymphoma: Immunohistochemical profile and prognostic significance

Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma (NHL) with extranodal location affecting only the CNS, meninges and eye, without visceral or lymph node involvement. Its incidence has increased sharply over the past three decades, especially in immunocompetent subjects. Most PCNSL cases are diffuse large B-cell lymphomas (DLBCLs). However, it differs from nodal DLBCL in that it has a worse prognosis. DLBCLs are a heterogeneous entity and according to new genomic discoveries, classifications into prognostic subgroups have been embarked upon. Two prognostic algorithms were then prepared using a panel of immunohistochemical markers (CD10, Bcl6, MUM1/IRF-4, and Bcl2), thus categorizing DLBCL into two subgroups, GCB (germinal centre B-cell-like) or non-GCB, and into Group 1 or Group 2. Our goal is to apply both of these two sub-classifications to 39 PCNSLs, in order to assess their usefulness and prognostic relevance. 74.3% of our PCNSLs were of a non-GCB phenotype, corresponding to an activated postgerminal origin. They were evenly distributed across G1 and G2. Two- and 5-year overall survival rates were 34.8% and 19.6%, respectively. Younger age (<65) and a therapeutic combination of chemotherapy and radiotherapy significantly improved our patients' survival rates. The other clinical or biological markers tested had no prognostic impact. The two classifications did not reveal any significant survival difference. The recent discovery of a specific "transcriptional signature" of PCNSL, marking them out of DLBCL could account for the irrelevance of such prognostic classifications to PCNSL.

Lack of prognostic significance of the germinal-center phenotype in diffuse large B-cell lymphoma patients treated with CHOP-like chemotherapy with and without rituximab

The influence of the germinal-center B-cell (GCB) and the non-GCB phenotypes of diffuse large B-cell lymphoma (DLBCL) on the outcome of 92 patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like chemotherapy, with or without rituximab was determined in this study. The differentiation between the GCB and non-GCB types was arrived at by immunohistochemistry using previously published criteria. Thirty-nine patients had the GCB and 53 had the non-GCB type of DLBCL. Forty-nine patients were treated with rituximab and chemotherapy; 43 were treated with chemotherapy alone. The GCB and non-GCB group did not differ in their international prognostic index factors and score, presence of bulky disease, or frequency of rituximab treatment. Median follow-up of the surviving patients was carried out for 37 months. There was no difference between the GCB and non-GCB groups in both overall response rates (67 vs. 70%, respectively) and estimated rates of 3-year event-free (46 vs. 49%, respectively) and overall (54 vs. 56%, respectively) survival. In addition, no differences of the outcomes were observed between the subgroups treated with or without rituximab. The patients of this study with immunohistochemically determined GCB-type DLBCL did not have an improved prognosis, irrespective of whether they had received rituximab or not.

Prognostic impact of C-REL expression in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) with a germinal center B-cell (GCB) phenotype is believed to confer a better prognosis than DLBCL with an activated B-cell (ABC) phenotype. Previous studies have suggested that nuclear factor-κB (NF-κB) activation plays an important role in the ABC subtype of DLBCL, whereas c-REL amplification is associated with the GCB subtype. Using immunohistochemical techniques, we examined 68 newly diagnosed de novo DLBCL cases (median follow-up 44 months, range 1 to 142 months) for the expression of c-REL, BCL-6, CD10, and MUM1/IRF4. Forty-four (65%) cases demonstrated positive c-REL nuclear expression. In this cohort of patients, the GCB phenotype was associated with a better overall survival (OS) than the non-GCB phenotype (Kaplan–Meier survival (KMS) analysis, p = 0.016, Breslow–Gehan–Wilcoxon test). In general, c-REL nuclear expression did not correlate with GCB vs. non-GCB phenotype, International Prognostic Index score, or OS. However, cases with a GCB phenotype and negative nuclear c-REL demonstrated better OS than cases with a GCB phenotype and positive nuclear c-REL (KMS analysis, p = 0.045, Breslow–Gehan–Wilcoxon test), whereas in cases with non-GCB phenotype, the expression of c-REL did not significantly impact the prognosis. These results suggest that c-REL nuclear expression may be a prognostic factor in DLBCL and it may improve patient risk stratification in combination with GCB/non-GCB phenotyping.

Prognostic Significance of BCL6 Translocation in Diffuse Large B-Cell Lymphoma Patients Treated with R-CHOP

Translocation of the BCL6 oncogene is the most commonly-detected chromosomal abnormality in diffuse large B-cell lymphoma (DLBCL), but its prognostic significance remains uncertain. BCL6 translocation has been associated with variable outcomes in prior clinical series, and its clinical impact has not been reevaluated since the introduction of rituximab (R). The objective of this study was to determine the prognostic value of BCL6 translocation in DLBCL patients treated with both CHOP and R-CHOP immunochemotherapy.Methods We included 163 DLBCL patients treated with CHOP (n=72) or R-CHOP (n=73) at the British Columbia Cancer Agency between the years 1999–2002. Formalin-fixed paraffin-embedded diagnostic lymph node specimens were analyzed for BCL6 translocation on tissue microarray (TMA) by fluorescence in situ hybridization (FISH), using commercially available dual-color break-apart probes (Abbott Molecular, IL, USA). Immunohistochemical analysis was performed for BCL6, CD10, and MUM1 expression, and cases were categorized as germinal centre B-cell-like (GCB) or non-GCB using a standard algorithm (Hans, Blood 2004).Results BCL6 translocation status was evaluable in 145 cases, and was positive (BCL6+) in 26% (37/145). Median age (65 vs. 59 years, p=0.03) and overall IPI score (p=0.03) were higher in the BCL6+ than in the BCL6− subgroup. Presence of BCL6 translocation correlated with non-GCB phenotype by immunohistochemical analysis (p=0.03), and showed no significant association with BCL6 protein expression (p=0.32). Median follow-up duration was 4.8 years (range, 0.1–8.4). BCL6 translocation was associated with a trend toward decreased overall survival (OS) among patients treated with R-CHOP (5-year OS 46.7% vs. 68%, p=0.098), though not among CHOP-treated patients (5-year OS 36.4% vs.49.5%, p=0.393). Kaplan-Meier analysis limited to the BCL6+ subgroup showed no overall survival difference between patients treated with CHOP and R-CHOP (p=0.67).Conclusions BCL6 translocations are found in approximately 25% of patients with de novo DLBCL. Presence of BCL6 translocation appears to predict unfavorable outcome in DLBCL patients treated with R-CHOP. However, the adverse prognostic impact of BCL6 translocation is confounded by an association with high IPI score and with non-GCB phenotype. Prospective analysis within large clinical trials would help to determine the independent prognostic value of this marker in the rituximab era.

Gene Expression Profile Analysis According to Recurrent Gene Copy Number Abnormalities Defines a Diffuse Large B-Cell Lymphoma Subgroup Characterized by 9p21 Locus Deletion, Ribosome Machinery Deregulation and Poor Prognosis. A GELA Study

Genomic gains and losses play a crucial role in the development and progression of DLBCL. Some gains or losses are associated with particular morphologic or clinical manifestations and correlate with the “germinal center B-cell like” (GCB)/non-GCB phenotype, as defined by gene expression profiles (GEP). We previously developed a reliable and routinely single applicable PCR assay, which provided information regarding gain/loss of relevant genes, and prognosis in DLBCL, termed QMPSF (Multiplex PCR of Short Fluorescent Fragments). Here, we combined GEP and QMPSF approaches to delineate molecular pathways related to recurrent gene copy number abnormalities (GCNA) and assess their prognosis value in patients treated by R-CHOP. For this purpose a series of 69 newly diagnosed DLBCL, included in the 98–5 GELA trial with available tumor DNA was studied (median age = 69 years [59–79], IPI2–3: 64%; 4–5: 36%, 40 treated by R-CHOP and 29 by CHOP). A single QMPSF assay, validated by CGH array, to detect GCNA of 8 relevant genes including SIM1 (6q16), MYC (8q24), CDNK2A (9p21), RB1 (13q14), REL (2p13), BCL2 (18q21), TP53 (17p13), and CDKN1B (12p13) was performed. In addition a dedicated QMPSF assay that provides a “bar code” of the 9p21 locus containing CDKN2A (p16INK4a and p14ARF) and CDKN2B (p15INK4b) was designed. To delineate specific gene expression profile according to recurrent GCNA a subset of 52 patients were studied by both GEP (Affymetrix U133A) and QMPSF technologies. Gains of MYC, BCL2, and REL were observed in 13, 28 and 20 % respectively. DNA copy losses of TP53, CDNK2A, RB1 and SIM1 were observed in 9, 40, 6 and 17 % of cases respectively. Using supervised analysis, we delineated specific GEP according to the most frequent GCNA detected by QMPSF. Interestingly, a signature related to 9p21 locus (CDKN2A/CDKN2B) deletion was associated with an overexpression of several ribosome machinery coding genes and the involvement of distinct antiapoptotic molecular mechanisms. Subsequent genomic analysis with the dedicated assay indicated that in most of cases deletions were homozygous and abolished simultaneously p14arf and p16INK4a expression. With a median follow-up of 81 months, CDKN2A deletion, strongly correlates to a poor outcome in the entire cohort (5y OS=25% respectively vs.60% for patients in germline configuration, p=.003) and in the subgroup of patients treated by R-CHOP (5y OS=40% vs.70%, p=.04). Furthermore, prognosis impact of GCNA involving CDKN2A was validated in an independent set of 35 patients treated by R-CHOP. To conclude, combination of QMPSF and GEP may constitute a powerful approach to delineate new genomic pathways with prognosis impact in DLBCL. Notably, CDKN2A/CDKN2B loss, detected in more than one third of DLBCL patients constitutes a strong factor of chemoresistance that is not overcome by R combination. GEP indicates that this may be a consequence of an independent p14arf/p53 pathway, involving the well-established p14arf related ribosome regulation function.

Perforation predicts poor prognosis in patients with primary intestinal diffuse large B‐cell lymphoma

To elucidate the clinicopathological features and prognostic factors of primary intestinal diffuse large B-cell lymphoma (PI-DLBL). Archival tissues from 30 tumours were used for tissue microarray construction, immunohistochemistry and interphase fluorescence in situ hybridization for chromosomal translocation. The M:F ratio was 1.7:1, with a median age of 60 years. The ileum and ileocaecum were most frequently involved (40% each). Fourteen (47%) were at stage I(E) disease, 15 (50%) at stage II(E). Five (17%) tumours were perforated at presentation. The tumours expressed Bcl-6 (73%), MUM1 (70%), Bcl-2 (67%) and CD10 (23%). Nine (30%) were classified as germinal centre B-cell (GCB) phenotype and 21 non-GCB. Eight of 30 (27%), 7/30 (23%) and 2/29 (7%) cases were positive for rearrangements involving IGH, BCL6, and C-MYC loci, respectively, whereas all cases were negative for BCL2 and CCND1 translocation. Perforation was a poor prognostic indicator, with a hazard ratio of tumour-related death at 8.75 (P = 0.001). The differentiation antigens, GCB versus non-GCB phenotype, or lymphoma-associated translocations were of no prognostic significance. We found a higher rate of perforation and lower frequency of GCB phenotype in PI-DLBL in Taiwan compared with other geographical areas; perforation is a poor prognostic indicator.

Clinical relevance of CD10, BCL‐6 and multiple myeloma‐1 expression in diffuse large B‐cell lymphomas in Malaysia

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, and it is recognized to constitute a heterogenous group of neoplasms. It can be divided into germinal center B-cell-like (GCB) and non-GCB subgroups. The aim of the present study was to evaluate the utility of immunophenotype subgrouping of DLBCL in a cohort of multi-ethnic Asian patients. A total of 84 reconfirmed de novo DLBCL were immunostained for the expression of CD10, BCL-2, BCL-6 and multiple myeloma-1. Thirty-three (39.3%) had the GCB phenotype, and the remainder (60.7%), the non-GCB phenotype. The results concur with most reports using a similar method of stratification. Forty-five patients had complete demographic and phenotype studies and 42 patients did not have rituximab treatment and had sufficient data for survival rate analysis. Similar to other studies, patients with combined low and low-intermediate International Prognostic Index score had better overall survival (P = 0.006). But patients with GCB phenotype did not have better prognosis, and BCL-2 expression was not associated with better prognosis. The expression of BCL-6 was associated with lower overall survival rate (P = 0.038). No apparent difference in overall and disease-free survival was noted between patients with GCB and non-GCB disease. BCL-6 expression by tumor cells appears to be associated with poorer prognosis.

Low rather than high Ki-67 protein expression is an adverse prognostic factor in diffuse large B-cell lymphoma

The prognostic role of overexpression of Ki-67 protein in diffuse large B-cell lymphoma (DLBCL) is still unclear. Furthermore, immunohistochemical studies have suggested a correlation between markers of proliferation, B-cell differentiation and apoptosis, but the prognostic relevance of these findings has not been clarified. To investigate the prognostic impact of Ki-67, in the context of this correlation, a retrospective immunohistochemical study was performed on 199 DLBCL patients treated with curative intent. Patients with low Ki-67 expression (<49%) had significantly worse progression-free (PFS) and overall (OS) survival, independent of clinical risk factors. In addition, low Ki-67 correlated to bcl-2 expression but not to non-germinal centre B-cell-like (non-GCB) phenotype. Each of these factors had negative impact on PFS and OS, but low Ki-67 expression also remained as an adverse prognostic factor independent of non-GCB phenotype and bcl-2 expression. Together, these results suggest that low rather than high Ki-67 protein expression is of prognostic importance in DLBCL.

Biweekly CHOP therapy improves therapeutic effect in the non-GCB subtype of diffuse large B-cell lymphoma

Abstract CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) is accepted as the best available standard treatment for diffuse large B-cell lymphoma (DLBCL) patients; however, the therapeutic efficacy seems unsatisfactory. Additional rituximab will improve the cure rate, but it is not popular in China because of its increased medical cost. Germinal center B-cell (GCB) and non-GCB subtypes distinction have been described as independent prognostic factors, and provides likelihood for cure with chemotherapy. The aim of the study is to explore the association between Immunophenotype and treatment regimen. Between August 2003 and May 2006, 66 patients with DLBCL were enrolled, according to immunohistochemistry results (GCB and non-GCB phenotype), randomly assigned to receive either six to eight cycles of CHOP every 2 weeks or standard CHOP every 3 weeks. After a median follow-up duration of 32 months (range of 4 to 42 months), an estimated 3-year overall survival (OS) rate for the GCB patients were 68.2% and 55.6% for the biweekly CHOP regimen and standard CHOP regimen respectively, while the data were 62.8% and 37.9% respectively for the non-GCB cases. The biweekly CHOP therapy showed higher efficacy than standard treatment, and its superiority was more obvious with the non-GCB subgroup. All rights reserved.

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.

Diffuse large B‐cell lymphoma in the young in Japan: A study by the Osaka Lymphoma Study Group

Clinicopathological and immunophenotypical characteristics in 24 patients with diffuse large B-cell lymphoma (DLBCL) under 30 years of age in Osaka, Japan were examined, and the results compared to those of DLBCL patients aged over 40 years in Osaka and of young DLBCL patients in Western countries. The level of LDH and IPI score at initial diagnosis were significantly lower in young than older patients. The sex ratio (M:F) and age range (median) in the young and older groups were 1.18 and 11-30 (24.8) years and 1.59 and 42-87 (62.4) years, respectively. Extranodal presentation was higher in the young group (83.3% versus 60.0%, P < 0.05). Based on immunophenotyping with anti-CD10, bcl-6, and MUM1 antibodies, the cases were categorized as germinal center B-cell (GCB) (CD10+ or CD10-, bcl-6+, MUM1+) or non-GCB phenotype. The frequency of GCB type was significantly lower in the young group than older group (25% vs. 54%, P < 0.05), and much lower than that reported for young patients in Western countries. In situ hybridization revealed one of the young patients to be positive for Epstein-Barr virus (EBV). In the older group, none of 31 cases showed EBV positivity. Three year event-free and overall survival rates of young patients were better than those of the older patients, although not significantly different. DLBCL in the young in Japan is characterized by a much lower frequency of the GCB phenotype compared to that in Western countries.

Immunoblastic morphology in diffuse large B-cell lymphoma is associated with a nongerminal center immunophenotypic profile

Diffuse large B cell lymphomas (DLCBL) are a group of lymphomas whose biologic and prognostic diversity has been recently well characterized. There is also morphologic heterogeneity, but the relevance of subclassification remains uncertain. The World Health Organization Classification states that pathologists have the choice to use only the term diffuse large B-cell lymphoma or to use one of the specific morphologic variants. The aim of the present study was to evaluate if there is an association between immunoblastic morphology and the immunophenotypic profile in DLBCL. Two observers reviewed 117 DLBCL cases. Cases of immunoblastic lymphoma and cases of centroblastic polymorphic lymphoma with more than 50% immunoblasts were defined as having immunoblastic morphology. Immunohistochemistry was performed on tissue microarray slides to establish the immunophenotypic profile. Patients with immunoblastic morphology more frequently had a non-GCB phenotype (94%vs 6%). This finding suggests that the morphological subclassification of DLBCL does have biological meaning, in line with recent evidence indicating that the immunoblastic morphology should not be overlooked in lymphoma classification.

BCL6 Rearrangement Detected by FISH Analysis Is a Poor Prognostic Factor in the “Non-Germinal Center Phenotype” of Diffuse Large B-Cell Lymphoma.

Background: Diffuse large B-cell lymphoma (DLBCL), which is the most common type of adult non-Hodgkin lymphoma, is considered to be heterogeneous in cytogenetics, immunophenotype and clinical feature. As the results of gene expression profiling, DLBCL can be divided into prognostically significant 3 subgroups of germinal center B-like (GCB), activated B-like and type 3. Chromosomal translocations affecting the BCL6 locus at the 3q27 locus are common in DLBCL, however, the prognostic significance of BCL6 rearrangement is still controversial.Methods: Twenty-six cases of DLBCL were examined with interphase fluorescence in situ hybridization (FISH) on touch preparations of lymph nodes using LSI BCL6 dual color probes (Vysis) for the incidence of BCL6 rearrangement and immunohistochemistry on paraffin section using CD10, BCL6 and MUM1 for subclassfying “GCB phenotype” and “non-GCB phenotype”. The correlation of BCL6 rearrangement with survival was investigated in two subgroups of DLBCL.Results: Of the 26 DLBCL cases, 6 cases (23%) were considered GCB phenotype and 20 cases (77%) non-GCB phenotype. BCL6 rearrangements were detected in 2 of 6 cases (33%) with GCB phenotype and 9 of 20 (45%) with non-GCB phenotype (total 11/26, 42%). ALL 6 cases with the GCB phenotype achieved sustained complete remission after chemotherapy and are alive. On the other hand, complete remission rate was 22% for the cases with BCL6 rearrangement but 73% for the cases without BCL6 rearrangement in the non-GCB phenotype (p=0.069). BCL6 rearrangement had a significant adverse effect on progression free survival within the non-GCB phenotype (P=0.016), but there was no significant correlation between BCL6 rearrangement and overall survival.Conclusion: FISH-based technique of the BCL6 rearrangements using touch preparations of lymph nodes could be developed for the retrospective analysis on survival. BCL6 rearrangement showed a poor prognostic effect particular in the non-GCB subgroup of DLBCL. [Display omitted] [Display omitted]

Addition of Rituximab (R) to CHOP Improves Survival in the Non-GCB Subtype of Diffuse Large B Cell Lymphoma (DLBCL).

Background: The cell of origin (COO) distinction provides a prognostic and biologically relevant subclassification of DLBCL. Germinal center B cell (GCB) and non-GCB subtypes were originally characterized by gene expression studies and subsequently validated at the protein level by Hans et al., Blood 193: 275–82 (2004). The addition of R to CHOP chemotherapy has been shown to improve the outcome of patients with DLBCL. The underlying mechanism(s) responsible for this effect is largely unknown. However, it is known that R may preferentially prevent chemotherapy failure in DLBCLs that express Bcl-2 protein or fail to express Bcl-6 (Mounier et al., Blood 101: 4279–84 2003, Winter et al., Blood 107: 4207–13 2006). Bcl-2 over-expression and absence of Bcl-6 is more common in the non-GCB subtype. Thus, R may benefit mostly non-GCB lymphomas. To test this hypothesis we assessed the clinical impact of CHOP-R vs CHOP in DLBCL distinguished by COO subtypes.Method: We identified 163 patients with DLBCL treated with either CHOP or CHOP-R with available paraffin blocks and interpretable immuno-staining. All were de novo DLBCL cases diagnosed between 1999 and 2002 at the BCCA. The two treatment cohorts represent consecutive eras of therapy (Sehn et al., JCO 2005; 23: 5027–33), and thus the median follow-up of living patients was 5.1 and 4.0 y for CHOP and CHOP-R, respectively. HIV+ patients or those with active secondary malignancies were excluded. Tissue microarrays (TMA) were built using duplicate 0.6mm cores from paraffin embedded formalin fixed (FFPE) tissues and stained with antibodies against CD10, Bcl-6, MUM1, and Bcl-2. The COO distinction was determined using the method of Hans.Results: Patients were treated with either CHOP (81) or CHOP-R (82). Their clinical characteristics, including the IPI, were evenly matched. The median follow-up of living patients was 4.4 y. The IPI was predictive of overall survival (OS) (p<0.0001) for the entire study population. Six cases had uninterpretable immunostains resulting in 74 cases with a GCB phenotype and 83 with a non-GCB phenotype (n = 157). Overall, 71% and 75% of the cases over-expressed Bcl-2 and Bcl-6, respectively. Bcl-2 protein was expressed in 70% GCB cases and 73% non-GCB (p= 0.72). Bcl-6 was expressed in 96% GCB cases and 63% non-GCB cases (p<0.0001). In univariate analysis, the addition of R was associated with a better prognosis in the non-GCB cases (p=0.02), but not in the GCB cases (p=0.3). This survival difference was not solely explained by either Bcl-2 or Bcl-6 expression. The addition of R to CHOP chemotherapy and IPI were independent predictors of OS in non-GCB DLBCL (p=0.02; p=0.016, respectively). The addition of R was also of prognostic importance in the lymphomas over-expressing Bcl-2 (p=0.0081).Conclusion: Immuno-chemotherapy using CHOP-R is associated with better OS in DLBCL, due largely to its effect on the non-GCB subgroup. Although Bcl-2 expression does not contribute to the determination of COO distinctions, the OS of Bcl-2-positive DLBCL patients is significantly improved by the addition of R. These results provide insight into the possible mechanisms by which R exerts its beneficial therapeutic effect. [Display omitted]

A Quantitative Multiplex PCR Assay, Validated by a High Resolution Array Comparative Genomic Hybridization Analysis, Provides Information Regarding Genomic Imbalances and Prognosis in Diffuse Large B-Cell Lymphomas.

Genomic gains and losses may play a crucial role in the development and progression of diffuse large B-cell lymphomas (DLBCL). Some gains or losses are associated with particular morphologic or clinical manifestations and correlate with the “germinal center B-cell like” (GCB)/ non-GCB phenotype, as defined by gene expression profiles. To date, chromosomal-comparative genomic hybridization (CGH) and higher resolution array-CGH provide the more comprehensive view of these genomic imbalances. However, a CGH approach is expensive and not routinely applicable. By contrast, quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) is an inexpensive and sensitive method for the detection of genomic deletions or duplications based on the simultaneous amplification of short genomic fragments using dye-labelled primers under quantitative conditions. We designed and evaluated a QMPSF assay to determine simultaneously frequencies of gain/loss of several genes and their clinical relevance in DLBCL. A series of 68 newly diagnosed DLBCL with available conventional cytogenetics and treated with anthracycline-based frontline therapy was studied (median age = 59 years, IPI 0–1: 35%; 2–3: 41%; 4–5: 24%). 15 cases were selected for both CGH (using a high resolution, high performance 60-mer oligonucleotide-based microarray that contains ~ 43,000 probes, with an average spatial resolution of 35 kB) and QMPSF experiments. Considering CGH as the reference method, and using primers located on chromosome 3q27 (BCL6), 6q16 (SIM1), 6q22 (PTPRK), 8q24 (MYC), 9p24 (JAK2), 9p21 (CDNK2A), 12q13 (cdk2), 12q15 (mdm2), 12p13.1 (p27) and 17p13 (p53), the sensibility and specificity of our QMPSF assay to detect abnormal DNA copy numbers ranged between 80–100% and 90–100% respectively. A gain of DNA copy numbers of MYC, p27, and BCL6 was observed in 19, 30 and 19 % respectively. Losses of DNA copies of p53 or CDNK2A were observed in 20 and 34 % of cases. Some genes were almost exclusively amplified (MYC, p27, cdk2) or exclusively deleted (p53, CDNK2A, PTPRK), indicating genomic imbalances selection during oncogenesis. Interestingly, 6q16 (SIM1), 6q22 (PTPRK) and 9p21 (CDNK2A) deletions were predominantly observed in the non-GCB subtype, whereas MYC gains were mainly observed in the GCB subtype and especially in de novo t(14;18) positive DLBCL (p =.002). The prognostic value of a QMPSF assay including 5 genes (p53, MYC, CDNK2A, SIM1 and p27) was tested. With a median follow-up of 81 months, detection of more than 2 gene copy number abnormalities was related to a worse event-free and overall survival (OS = 10 months, n = 13) as compared to patients with 0–2 abnormalities (OS not reached, p = .003, n = 55). The prognosis value of this PCR remains regardless the GCB/non-GCB subtype or the IPI score. To conclude, we developed a reliable and routinely applicable PCR assay, which provided information regarding gain/loss of relevant genes, and prognosis in DLBCL.

Simian virus 40 tumor antigen expression and immunophenotypic profile of AIDS-related non-Hodgkin's lymphoma

Simian virus 40 (SV40) is associated with some systemic non-Hodgkin's lymphomas (NHL) among HIV-positive patients, based on assays for viral DNA sequences. To investigate the possible production of the viral transforming protein, we examined age-matched case–control specimens from patients with HIV/AIDS for the expression of SV40 large tumor antigen (T-ag). Masked specimens initially examined by polymerase chain reaction (PCR) for polyomavirus and herpesvirus DNA sequences were assessed for the expression of SV40 T-ag and phenotypic lymphocyte markers by immunohistochemistry (IHC). Fifty-five systemic NHL and 25 nonmalignant lymphoid and malignant nonlymphoid tissue control cases from two HIV community programs in Texas and New Jersey were scored for IHC positivity without knowledge of the PCR results. IHC showed expression of SV40 T-ag among B-cell lymphomas, whereas none of the control tissue samples were positive for T-ag (12/55, 22% vs. 0/25, 0%; P = 0.01). SV40 T-ag expression was detected only in B-cell lymphoma specimens that contained SV40 DNA sequences. Not all lymphoma cells in a positive specimen stained for T-ag, and the reaction was lower intensity than observed in SV40 hamster tumors. SV40 T-ag was demonstrated in both primary and recurrent tumors from one patient. A germinal center B-cell-like (GCB) profile was more frequently expressed by SV40-positive tumors than in Epstein–Barr virus (EBV)-related lymphomas (10/12, 83% vs. 6/13, 46%; P = 0.05), whereas a non-GCB phenotype was more frequent in EBV-positive than in SV40-positive lymphomas (7/13, 54% vs. 2/12, 17%; P = 0.05). This study shows that SV40 gene expression occurs in a fraction of cells in some B-cell lymphomas among patients with HIV/AIDS.

Diffuse Large B-Cell Lymphoma With Extra Bcl-2 Gene Signals Detected by FISH Analysis Is Associated With a “Non-Germinal Center Phenotype”

Amplification and translocation of the Bcl-2 gene has been detected in a certain subset of diffuse large B-cell lymphomas (DLBCL). The correlations among Bcl-2 protein expression, gene translocation or amplification, and the molecular signature determined by cDNA array are poorly understood. This study examined 25 cases with de novo nodal DLBCL. Interphase fluorescence in situ hybridization (FISH) analysis was performed to evaluate the Bcl-2 gene using IGH/BCL2 and CEP18 centromere probes (Vysis). When extra Bcl-2 gene signals were observed in each tumor cell and when these signals were in proportion to the extra CEP18 probe signals, we regarded the findings as indicating the presence of an additional chromosome 18; when extra Bcl-2 signals were observed but additional CEP18 signals were not, we regarded the findings as indicating the presence of gene amplification. A panel of 3 antigens (CD10, Bcl-6, and MUM-1) was applied to categorize each case as either a "germinal center B-cell (GCB) phenotype" or a "non-GCB phenotype." Of the 25 cases examined, 8 cases (32%) were classified as "GCB phenotype" and 17 cases (68%) were classified as "non-GCB phenotype." A FISH analysis revealed that t(14;18) was detected in 2 of the 8 cases (25%) with the "GCB phenotype" but in none of the 17 "non-GCB phenotype" cases. Extra Bcl-2 gene signals were detected in 7 of the 25 (28%) cases examined: n = 5 for an additional chromosome 18, n = 1 for gene amplification, and n = 1 for additional chromosome 18 + gene amplification. Extra Bcl-2 gene signals were exclusively detected in DLBCL with the "non-GCB phenotype"; these cases, with the exception of one, stained strongly positive for Bcl-2. The DLBCLs with Bcl-2 protein overexpression were classified into at least two heterogeneous molecular groups, based on the results of the FISH analysis.