Analytic, Preanalytic, and Clinical Validation of p53 IHC for Detection of TP53 Missense Mutation in Prostate Cancer

Abstract

Purpose:TP53 missense mutations may help to identify prostate cancer with lethal potential. Here, we preanalytically, analytically, and clinically validated a robust IHC assay to detect subclonal and focal TP53 missense mutations in prostate cancer.Experimental Design: The p53 IHC assay was performed in a CLIA-accredited laboratory on the Ventana Benchmark immunostaining system. p53 protein nuclear accumulation was defined as any p53 nuclear labeling in >10% of tumor cells. Fifty-four formalin-fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE prostate cancer tissues (88 primary adenocarcinomas, 15 metastases) with known TP53 mutation status were studied. DU145 and VCaP xenografts were subjected to varying fixation conditions to investigate the effects of preanalytic variables. Clinical validation was performed in two partially overlapping radical prostatectomy cohorts.Results: p53 nuclear accumulation by IHC was 100% sensitive for detection of TP53 missense mutations in the NCI-60 panel (25/25 missense mutations correctly identified). Lack of p53 nuclear accumulation was 86% (25/29) specific for absence of TP53 missense mutation. In FFPE prostate tumors, the positive predictive value of p53 nuclear accumulation for underlying missense mutation was 84% (38/45), whereas the negative predictive value was 97% (56/58). In a cohort of men who experienced biochemical recurrence after RP, the multivariable HR for metastasis among cases with p53 nuclear accumulation compared with those without was 2.55 (95% confidence interval, 1.1-5.91).Conclusions: IHC is widely available method to assess for the presence of deleterious and heterogeneous TP53 missense mutations in clinical prostate cancer specimens. Clin Cancer Res; 23(16); 4693-703. ©2017 AACR.

Figure 1. Representative p53 immunostaining in formalin fixed paraffin embedded prostate cell lines and primary and castrate resistant prostate cancer (CRPC) prostate tumors

(A) RWPE-1 cells, which are TP53 wild-type (WT) show rare positive nuclei comprising less than 10% of the tumor on p53 immunostaining. In contrast, DU145 cells (with two mutations, p.V274 and p.P223L) and VCaP cells (p.R248W) with missense mutations show robust nuclear accumulation of p53. PC3 cells, which have a loss-of-function frameshift mutation (p.A138fs) and loss of heterozygosity (LOH) are entirely negative for p53 expression. All photomicrographs are reduced from 200×. (B) Only rare, weakly positive nuclear immunostaining for p53 is seen in benign prostate tissues and primary and CRPC tumors that are TP53 wild-type (WT), though some cytoplasmic staining is seen specifically in these tumors of unknown significance. The lack of nuclear positivity in benign prostate tissue and TP53 WT tumors meant that the IHC assay cannot distinguish tumors that are TP53 WT from those with loss-of-function alterations (homozygous deletion, frameshift, splice site or nonsense mutations). In contrast, tumors with missense mutations of TP53 (p.R175H, p.R273C, p.V157A, p.131N) are readily distinguishable from WT and show robust nuclear accumulation of p53 protein in the majority of cells. All photomicrographs are reduced from 200×.

Figure 2. Heterogeneous p53 immunostaining on standard histologic sections was common in primary tumors with discordant TP53 sequencing and p53 immunostaining results

(A) A tumor that was TP53 WT by sequencing shows focal p53 nuclear accumulation on standard section (arrow, upper left) with an adjacent area lacking nuclear accumulation (arrow, lower right). (B) A tumor that was TP53 WT by sequencing because a p.C135Y mutation was detected in <5% of cells, shows focal p53 nuclear accumulation on standard section (arrow, upper right) with an adjacent area lacking nuclear accumulation (arrow, lower left). (C and D) Gleason score 9 biopsies with clear focal nuclear accumulation of p53 in some, but not all tumor cells (arrows). All photomicrographs are reduced from 200×.

Figure 3. Effect of pre-analytic tissue fixation conditions (cold ischemia time and time in fixation) on p53 immunostaining in two xenograft models with known TP53 missense mutations

(A) Effect of variation of fixation duration (4, 12 and 24 hours in 10% neutral buffered formalin with immediate fixation) on p53 immunostaining in DU145 and VCaP xenografts. Minimal variation in p53 nuclear accumulation is seen in DU145 cells (left panels), while VCaP cells show a slight decrease in intensity of staining with longer fixation conditions (right panels), though number of cells staining is not markedly affected. (B) Effect of variation of cold ischemia duration (4, 12, 24 hours without fixation at room temperature with 24 hour fixation in 10% neutral buffered formalin) on p53 immunostaining in DU145 and VCaP xenografts. Minimal variation in intensity of p53 nuclear accumulation is seen in DU145 cells (left panels) or VCaP cells (right panels) with variation of cold ischemia time, though tissue quality clearly decreases with increased autolysis and background staining compared to 0 hours old ischemia time (compare with last row of both panels in A).

Figure 4. Cumulative incidence of metastasis in patients from intermediate and high risk prostate cancer case-cohort study

(A) or post-biochemical recurrence case-cohort study (B), stratified by p53 immunostaining status. Note that the case-cohort design makes a random sampling of the cohort and then is enriched with all the metastatic patients (not selected randomly). Thus, to get the full cohort, the controls are re-weighted by the inverse of sampling fraction (32, 33).