Gene expression predicts overall survival in paraffin-embedded tissues of diffuse large B-cell lymphoma treated with R-CHOP

Abstract

Gene expression profiling (GEP) on frozen tissues has identified genes predicting outcome in patients with diffuse large B-cell lymphoma (DLBCL). Confirmation of results in current patients is limited by availability of frozen samples and addition of monoclonal antibodies to treatment regimens. We used a quantitative nuclease protection assay (qNPA) to analyze formalin-fixed, paraffin-embedded tissue blocks for 36 previously identified genes (N = 209, 93 chemotherapy; 116 rituximab + chemotherapy). By qNPA, 208 cases were successfully analyzed (99.5%). In addition, 15 of 36 and 11 of 36 genes, representing each functional group previously identified by GEP, were associated with survival (P < .05) in the 2 treatment groups, respectively. In addition, 30 of 36 hazard ratios of death trended in the same direction versus the original studies. Multivariate and variable cut-off point analysis identified low levels of HLA-DRB (< 20%) and high levels of MYC (> 80%) as independent indicators of survival, together distinguishing cases with the worst prognosis. Our results solve a clinical research problem by demonstrating that prognostic genes can be meaningfully quantified using qNPA technology on formalin-fixed, paraffin-embedded tissues; previous GEP findings in DLBCL are relevant with current treatments; and 2 genes, representing immune escape and proliferation, are the common features of the most aggressive DLBCL.

Figure 1

Overall survival in years for 3 representative genes showing patients treated with CHOP versus R-CHOP according to gene expression levels. HLA-DRB is cut above and below 25%; BCL6 and MYC are cut at median. (A) CHOP-treated cases, all IPI scores: (Ai) HLA-DRB; (Aii) BCL6; (Aiii) MYC (N = 93). (B) R-CHOP cases, all IPI scores, HLA-DR, BCL6, and MYC (N = 116).

Figure 2

Overall survival in years for patients treated with R-CHOP according to IPI score and expression levels of HLA-DRB and/or MYC. Cutoff point levels are above and below the median for both genes. Adverse gene level for HLA-DR is for expression below the median, whereas adverse gene level for MYC is for expression above the median (A) All IPI groups (N = 116). (B) Low IPI group (scores 0-2, N = 72). (C) High IPI group (scores 3-5, N = 36). The combined number of cases in panels B and C are fewer than in panel A because of several cases with missing IPI information.

Figure 3

Variable cutoff point analysis for HLA-DRB and MYC genes. Gene expression level on x-axis; log rank score on y-axis; permutation P value indicated. (A) HLA-DRB. (B) MYC. The peaks in the log rank scores indicate the most significant cut-off points in the data yielding the largest differences in overall survival.