Major Histocompatibility Class II (MHCII) and Germinal Center Associated Gene Expression Correlate with Overall Survival in Ritiximab and CHOP-Like Treated Diffuse Large B Cell Lymphoma (DLBCL) Patients Using Formalin Fixed Paraffin Embedded (FFPE) Tissues.
Authors: Rimsza Lisa M. , LeBlanc Michael L. , Roberts Robin A. , Unger Joseph M. , Miller Thomas P. , Sabalos Constantine M. , Martel Ralph R. , Campo Elias , Rosenwald Andreas , Connors Joseph M. , Sehn Laurie H. , Johnson Natalie , Gascoyne Randy D. 
Published: October 2019

Abstract
Large studies using snap frozen tissue for gene expression profiling (GEP) have identified a limited number of genes predicting outcome in DLBCL treated with CHOP alone (17 genes from Rosenwald et al NEJM 2002, 13 from Shipp et al, Nat Med 2002, 6 from Lossos et al, NEJM 2004, 2 from Tome et al, Blood 2005). Our objective was to test the prognostic value of these genes (38 total, 36 different genes) using FFPE tissues and compare results from patients treated with CHOP or CHOP-like regimen alone (C) (N=93) to those treated with CHOP + rituximab (R) (N=101). We used a multiplexed quantitative nuclease protection assay, the ArrayPlate (High Throughput Genomics), to measure mRNA levels reliably in FFPE samples as recently described. (Roberts et al, Lab Invest, ePub, 13 Aug 2007). Of 194 cases attempted, there was only 1 that did not result in adequately detectable signal. Univariate associations between gene levels and patient OS were performed using Cox proportional hazard regression models. For C alone cases, gene expression levels were significantly correlated with overall survival (OS) at p < 0.05 for 15/36 prognostic genes including the MHCII genes HLA-DR and HLA-DP, as well as C-MYC, BCL-6, BCL-2 and ten other genes. These genes represented all 4 Rosenwald prognostic signatures, 3/6 of Lossos genes, and 4/13 Shipp genes. In the R-treated cases, 8/36 genes correlated with OS including HLA-DR and HLA-DQ, the germinal center B-cell (GCB) genes BCL-6, and SERPINA9 as well as C-MYC, PLAU, PDCD4, and MnSOD. Again, all 4 Rosenwald signatures were represented. In our assay, genes that represent key gene expression signatures previously identified as the most prognostically important in large discovery-oriented GEP experiments were significantly associated with patient OS using FFPE tissues and retained significance in the R-treated patients, suggesting that previous findings remain relevant in the R-CHOP era. For most genes in both treatment groups, the estimated hazard ratios tended in the direction originally predicted, suggesting these genes would be significantly related to survival in a larger, more powered study. Previous studies have relied on unfixed, snap-frozen tissues which are available only in a minority of patients for GEP or quantitative RT-PCR measurements of mRNA expression. The ArrayPlate assay can assess prognostic genes in patients for which only FFPE material is available, is highly correlated with discovery microarray results, and should be easily adaptable as more prognostic genes are identified. Importantly, previously identified prognostically important genes such as MHCII and GCB genes remain correlated with survival in the R-CHOP treatment era.