Variakojis, Daina

8038 Background: P21 is a downstream effector protein of p53 that induces cell cycle arrest by inhibiting cyclin/cyclin dependent kinase (cdK) complexes. In this prospective correlative study, we investigated the prognostic significance of p21, p53, Bcl-6, and Bcl-2 in diffuse large B cell lymphoma (DLBCL) in the context of E4494, a randomized trial comparing conventional CHOP to rituximab(R)-CHOP induction, and maintenance R (MR) to observation for responding patients. Methods: Protein expression was quantified by immunohistochemical (IHC) staining of 198 DLBCL paraffin-embedded biopsy specimens and scored by an expert panel of hematopathologists. Results: No differences in the distribution of patient characteristics were detected between p21+ and p21- cases and between p53+ and p53- cases as well as across treatment arms. Among all study patients, there were no differences in clinical outcomes between p21+ and p21- cases. However, when analyzed by induction arm (removing the effect of MR), R-CHOP patients had marginally better FFS (p=0.06) if p21+; among CHOP-treated patients, p21 status had no effect on outcome. For R-CHOP patients but NOT CHOP patients, p21+ was an independent, favorable prognostic factor after adjusting for Bcl-6, IPI and Bcl-2 in multivariate analysis (FFS relative risk 0.3; p=0.003; OS relative risk 0.4; p=0.02). P21+ patients treated with R-CHOP had higher %FFS at 5 years compared to p21+ patients treated with CHOP (61 ± 7% vs. 24 ± 7%; p=0.007) but p21- R-CHOP and CHOP patients had similar FFS (37 ± 7% vs. 35 ± 7%; p=0.64). P53 staining (+ scored as either >20% or 50%) did not predict for FFS or OS in uni- or multivariate analyses. The p53+/p21- phenotype, a possible surrogate for mutated p53, was not prognostic. Further, no significant correlation was seen between p53 and p21 expression by IHC. Conclusions: These data suggest that p21 expression by IHC predicts for favorable outcome in older DLBCL patients treated with R-CHOP but not in those treated with CHOP. Furthermore, p21+ identifies patients who benefit from the addition of rituximab to CHOP. Complex interactions between p53, p21, and Bcl-6, as well as other unknown pathways, may account for this treatment-related effect and should be further investigated. [Table: see text]

Abstract Abstract 87 Background: The International Prognostic Index (IPI) remains the most powerful predictor of clinical outcome in DLBCL in the R-CHOP era, serving as a useful surrogate for the biology we are just beginning to understand. With a follow-up of more than 9 years, its uniformly staged and treated patients and its prospectively collected unstained slides for correlative studies, the clinical data set from E4494, the US Intergroup trial comparing CHOP and RCHOP in patients >60 years with DLBCL, stands as a valuable resource for investigating prognosis in DLBCL. Based on immunohistochemistry, we previously showed that rituximab modulates the prognostic significance of some biomarkers in DLBCL. With these same specimens, the quantitative nuclease protection assay (qNPA), a methodology for measuring mRNA levels in FFPET, was used to develop separate prognostic signatures for the CHOP and RCHOP arms that can be simply applied using stored unstained slides. Methods: Five micron unstained FFPET sections from 183 eligible and evaluable cases enrolled on E4494 and submitted for prospective immunohistochemical correlative studies more than ten years ago were used for this analysis. Tissue was scraped from slides and a multiplexed qNPA was performed in triplicate using a customized Array Plate assay (HTG, Inc) for 43 genes of interest. TBP served as a housekeeping gene. Association between standardized log gene expression and patient failure-free survival (FFS) and overall survival (OS) was obtained using the Cox proportional hazards model. A weighted analysis was used to eliminate the confounding effect of maintenance rituximab. Genes that showed at least marginal significance in the univariate analysis were used to perform LASSO (penalized method to select best subset) to select a final list of genes in the multivariate analysis. Using the predictive model for either CHOP or R-CHOP induction, individual risk scores were calculated based on the multivariable model, and cases were dichotomized into low and high risk groups based on the median risk score. The model was then validated using the Lenz dataset (NEJM, 2008). Results: In six cases, tissue from slides prepared >10 years ago was compared to freshly cut sections from corresponding blocks, and showed excellent concordance (corr=0.86). On-study characteristics for the 176 cases with analyzable data were representative of the greater E4494 patient population. Six gene predictors were developed for each arm of the trial: RCHOP: FN1, LMO2, AKT1, HIF1a, AKT3, BCL2; and CHOP: PDCD4, HLADRB1, COL3A1, LMO2, ROBO4, TP53. Both signatures proved powerful predictors of FFS and OS among CHOP (FFS: p=0.0031; OS: p=0.0013) and RCHOP (FFS: p=0.001; OS=p=0.0015) treated patients. When adjusted for the clinically-based IPI, the gene-risk score retained its significance (CHOP: FFS p=0.0007; OS p=0.0011; RCHOP: FFS p=0.003; OS, p=0.001) while the IPI became only marginally significant (CHOP: FFS: p=0.06, OS p=0.06; R-CHOP: FFS p=0.09, OS p=0.02), suggesting that the gene predictor accounted for much of the predictive power of the IPI. The predictive model was then validated using the Lenz dataset for OS. The predictor models for CHOP and R-CHOP-treated patients effectively dichotomized patients into prognostic subgroups (CHOP: p<0.0001; RCHOP: p=0.0014; see figure below) and this difference was maintained when the subset over age 60 was analyzed (CHOP: p=0.0008; RCHOP: p=0.017). When adjusted for the IPI, the molecular predictor developed for CHOP treated patients remained robust (OS: P=.0001; HR 2.39), while the molecular predictor for RCHOP predicted OS marginally (p=.06; HR 1.82), with shorter followup than the CHOP cohort. Conclusions: Unstained slides from FFPET stored for many years may be used to investigate gene expression in lymphoma biopsy specimens for which there is mature followup. Gene risk scores based on the expression of a limited number of genes are powerful predictors of clinical outcome. Disclosures: Pollock: HTG, Inc.: Employment, Equity Ownership. Botros:HTG, Inc.: Employment, Equity Ownership. Horning:Genentech: Employment, Equity Ownership.