Christine P. Hans

Addition of Rituximab to Standard Chemotherapy Improves the Survival of Both the Germinal Center B-Cell–Like and Non–Germinal Center B-Cell–Like Subtypes of Diffuse Large B-Cell Lymphoma

PURPOSE Diffuse large B-cell lymphoma (DLBCL) includes at least two prognostically important subtypes (ie, germinal center B-cell-like [GCB] and activated B-cell-like [ABC] DLBCL), which initially were characterized by gene expression profiling and subsequently were confirmed by immunostaining. However, with the addition of rituximab to standard chemotherapy, the prognostic significance of this subclassification of DLBCL is unclear. PATIENTS AND METHODS We studied 243 patient cases of de novo DLBCL, which included 131 patient cases treated with rituximab plus standard chemotherapy (rituximab group) and 112 patient cases treated with only standard chemotherapy (control group). The cases were assigned to GCB or non-GCB subgroups (the latter of which included both ABC DLBCL and unclassifiable DLBCL) on the basis of immunophenotype by using the Hans method. Clinical characteristics and survival outcomes of the two patient groups were compared. RESULTS The clinical characteristics of the patients in the rituximab and the control groups were similar. Compared with the control group, addition of rituximab improved the 3-year overall survival (OS; 42% v 77%; P < .001) of patients with DLBCL. Rituximab-treated patients in either the GCB or the non-GCB subgroups also had a significantly improved 3-year OS compared with their respective subgroups in the control group (P < .001). In the rituximab group, the GCB subgroup had a significantly better 3-year OS than the non-GCB subgroup (85% v 69%; P = .032). Multivariate analyses confirmed that rituximab treatment was predictive for survival in both the GCB and the non-GCB subgroups. CONCLUSION In this retrospective study, we have shown that the subclassification of DLBCL on the basis of the cell of origin continues to have prognostic importance in the rituximab era.

BCL2 Translocation Defines a Unique Tumor Subset within the Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup.

BCL2 Expression Is a Prognostic Marker for the Activated B-Cell–Like Type of Diffuse Large B-Cell Lymphoma

BACKGROUND The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, as well as primary mediastinal DLBCL. PATIENTS AND METHODS In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression. RESULTS There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup. CONCLUSION Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

LMO2 Protein Expression Predicts Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Anthracycline-Based Chemotherapy With and Without Rituximab

PURPOSE The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab. PATIENTS AND METHODS DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome. RESULTS In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis. CONCLUSION We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

Expression of PKC-beta or cyclin D2 predicts for inferior survival in diffuse large B-cell lymphoma

We sought to determine whether identification of poor-risk subgroups of diffuse large B-cell lymphoma (DLBCL) using immunohistochemical stains would have practical utility with regard to prognosis and therapeutic decisions. Tissue microarray blocks were created using replicate samples of formalin-fixed, paraffin-embedded tissue from 200 cases of de novo DLBCL. The sections were stained with antibodies to proteins that are expressed by activated or proliferating B cells including MUM1, FOXP1, bcl-2, survivin, protein kinase C-beta (PKC-β), cyclin D2, cyclin D3, and Ki-67. In univariate analysis, tumor expression of cyclin D2 (P=0.025) or PKC-β (P=0.015) was associated with a worse overall survival, whereas none of the other markers was predictive of overall survival. Patients with DLBCL that expressed either cyclin D2 or PKC-β had a 5-year overall survival of only 30% as compared to 52% for those who were negative for both markers (P=0.0019). In multivariate analysis, the expression of cyclin D2 or PKC-β was an independent predictor of poor overall survival (P=0.035). Cyclin D2 and PKC-β expression will be useful in designing a ‘biological prognostic index’ for patients with DLBCL.

Prognostic significance of VEGF, VEGF receptors, and microvessel density in diffuse large B cell lymphoma treated with anthracycline-based chemotherapy

Vascular endothelial growth factor-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of lymphoma cell proliferation and/or survival induced by autocrine vascular endothelial growth factor receptor-mediated signaling. We have recently shown that diffuse large B cell lymphomas expressing high levels of vascular endothelial growth factor protein also express high levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether tumor vascularity, or expression of vascular endothelial growth factor protein and its receptors, contribute to patient outcomes. Our results show that increased tumor vascularity is associated with poor overall survival (P=0.047), and is independent of the international prognostic index. High expression of vascular endothelial growth factor receptor-1 by lymphoma cells by contrast is associated with improved overall survival (P=0.044). The combination of high vascular endothelial growth factor and vascular endothelial growth factor receptor-1 protein expression by lymphoma cells identifies a subgroup of patients with improved overall (P=0.003) and progression-free (P=0.026) survival; these findings are also independent of the international prognostic index. The prognostic significance of overexpression of this ligand-receptor pair suggests that autocrine signaling via vascular endothelial growth factor receptor-1 may represent a survival or proliferation pathway in diffuse large B cell lymphoma. Dependence on autocrine vascular endothelial growth factor receptor-1-mediated signaling may render a subset of diffuse large B-cell lymphomas susceptible to anthracycline-based therapy.