Supplementary Data from Expression of p21 Protein Predicts Clinical Outcome in DLBCL Patients Older than 60 Years Treated with R-CHOP but not CHOP: A Prospective ECOG and Southwest Oncology Group Correlative Study on E4494

Abstract Purpose: To prospectively investigate the prognostic significance of p21 and p53 expression in diffuse large B-cell lymphoma in the context of the U.S. Intergroup trial comparing conventional cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy to rituximab-CHOP (R-CHOP) induction, with or without maintenance rituximab. Experimental Design: Immunohistochemical staining of 197 paraffin-embedded biopsy specimens was scored by an independent panel of experts. Results: The cyclin-dependent kinase inhibitor, p21, was expressed in 55% of cases examined. In a multivariable analysis adjusting for International Prognostic Index score and BCL2 status, p21 expression was a significant, independent, favorable predictive factor for failure-free survival (relative risk, 0.3; P = 0.001) and overall survival (relative risk, 0.3; P = 0.003) for patients treated with R-CHOP. Expression of p21 was not predictive of outcome for CHOP-treated patients. Only p21-positive cases benefited from the addition of rituximab to CHOP. Among p21-positive patients, treatment with R-CHOP was associated with a higher failure-free survival rate at 5 years compared with CHOP (61% versus 24%; P = 0.01). In contrast, no significant differences were detected in failure-free survival according to treatment arm for p21-negative patients. Expression of p53, alone or in combination with p21, did not predict for outcome in univariable or multivariable analyses. Conclusions: In this study, p21 protein expression emerged as an important independent predictor of a favorable clinical outcome when rituximab was added to CHOP therapy. These data suggest that rituximab-related effects on lymphoma survival pathways may be functionally linked to p21 activity. Clin Cancer Res; 16(8); 2435–42. ©2010 AACR.

Introduction and methods: Gene expression profiling has been used to define three molecular subtypes of diffuse large B cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL).To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high resolution, genome-wide copy number analysis (aCGH) coupled with gene expression profiling.We defined minimal common regions (MCRs) that were recurrently altered in copy number and identified those that dysregulated the expression of their constituent genes.Statistical differences in MCR frequency between DLBCL subtypes were corrected for multiple hypothesis testing using a false discovery rate (FDR) calculation.Results: Of 258 MCRs that occurred in more than 5% of any DLBCL subtype, 31 were utilized differentially by the DLBCL subtypes (p<0.005,false discovery rate <0.05).Amplification of the oncogenic mir-17-92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent in GCB DLBCL but did not occur in ABC DLBCL.Conversely, deletion of the INK4a/ARF tumor suppressor locus, gain of chromosome arm 18q, and trisomy 3 occurred almost exclusively in ABC DLBCLs and were associated with inferior outcome within this subtype.BCL2 and NFATC1 are candidate oncogenes affected by the chromosome 18 aberrations.An amplicon on chromosome 19 was detected in 20% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs.The gene most upregulated by this amplicon was SPIB, which encodes an ETS family transcription factor.Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines, but not to GCB DLBCL, PMBL or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL.Conclusions: In summary, aCGH revealed novel copy number abnormalities in DLBCL that had significant different frequencies in the three DLBCL subtypes.These data provide genetic evidence that the molecular DLBCL subtypes are distinct diseases that utilize different oncogenic pathways.

Over the past two decades, the incidence of follicular lymphoma has increased. Contemporary treatments include combinations of chemotherapy and monoclonal antibodies, radioimmunotherapy, new targeted agents and stem-cell transplantation. Prognostic tools are becoming more important in helping clinicians and patients decide on the most appropriate therapeutic regimens. Gene expression profiling and biomarkers are promising additions to this armamentarium. When patients do require therapy, the addition of rituximab to chemotherapy seems to improve remission duration and may improve overall survival. Radioimmunotherapy capitalises on the capacity to target radiation directly to malignant cells, and is currently approved for the treatment of relapsed/refractory follicular lymphoma. Further investigation is needed to clarify the role of stem-cell transplantation in follicular lymphoma. Only well-designed clinical trials can provide answers to the many questions that remain regarding the optimal treatment and sequence of treatments for patients with follicular lymphoma.

Follicular lymphoma (FL) is characterized by its responsiveness to initial therapy, a pattern of repeated relapses, and a tendency for histologic progression to a process resembling diffuse, large B-cell lymphoma. Treatment decisions are complicated by the many effective options now available including combinations of conventional chemotherapy and monoclonal antibody, radioimmunotherapy, new targeted agents, and autologous and allogeneic stem cell transplantation. For selected patients, "watch and wait" or involved field irradiation may still be the most appropriate strategy. When therapy is required, a combination of rituximab and conventional chemotherapy results in improved outcomes compared to chemotherapy alone. Radioimmunotherapy alone or in combination with chemotherapy is an attractive strategy for patients with relapsed disease and may prove to be appropriate first line therapy. The role of stem cell transplant in FL requires further investigation. Novel agents with varied mechanisms of action continue to be developed. Enrollment of patients into clinical trials designed to address the many unanswered questions in FL is essential to improving clinical outcomes.

Posttranslational processing of Ras proteins has attracted considerable interest as a potential target for anticancer drug discovery. Rce1 encodes an endoprotease that facilitates membrane targeting of Ras and other prenylated proteins by releasing the carboxyl-terminal 3 amino acids (ie, the -AAX of the CAAX motif). Homozygous Rce1 mutant embryos (Rce1(-/-)) die late in gestation. To characterize the role of Rce1 in hematopoiesis, we performed adoptive transfers and investigated cells from the recipients. Rce1(-/-) fetal liver cells rescued lethally irradiated recipients and manifested normal long-term repopulating potential in competitive repopulation assays. The recipients of Rce1(-/-) cells developed modest elevations in mature myeloid cells (neutrophils + monocytes), but remained well. Bone marrow cells from mice that received transplants of Rce1(-/-) activated extracellular signal-related kinase (ERK) normally in response to granulocyte-macrophage colony-stimulating factor. These data suggest that pharmacologic inhibitors of Rce1 will have minimal effects on normal hematopoietic cells.