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 (PCa) with lethal potential. Here, we pre-analytically, analytically and clinically validated a robust immunohistochemistry (IHC) assay to detect subclonal and focal TP53 missense mutations in PCa. 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. 54 formalin fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE PCa 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 pre-analytic variables. Clinical validation was performed in two partially overlapping radical prostatectomy (RP) 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 (PPV) of p53 nuclear accumulation for underlying missense mutation was 84% (38/45), while the negative predictive value (NPV) was 97% (56/58). In a cohort of men who experienced biochemical recurrence after RP, the multivariable hazard ratio for metastasis among cases with p53 nuclear accumulation compared to those without was 2.55 (95% CI: 1.1–5.91). CONCLUSIONS IHC is widely available method to assess for the presence of deleterious and heterogeneous TP53 missense mutations in clinical PCa specimens.

If this is true, what does it imply? How end-user antibody validation facilitates insights into biology and disease

“…These findings show that even older antibodies can be re-examined using more modern technologies to validate their assay properties. Also, as is the case for PTEN, the p53 IHC assay was robust to pre-analytic fixation conditions and could be useful in identifying heterogeneous, subclonal TP53 alterations in the setting of prostate biopsies that may be difficult to detect without next generation sequencing approaches [89] . The ability to interrogate p53 status by a simple IHC assay in primary and metastatic prostate tumors could be highly useful for studies of disease pathogenesis and for developing predictive biomarkers.…”

“…Moreover, studies in prostate cancer showed that overexpression of p53 in tumor cells was predictive of poor outcome [88] , but there were very few studies analyzing in detail with automated clinical grade IHC assays whether positive IHC staining for p53 in prostate carcinoma correlated with TP53 mutation. Recently, using a series of cell lines with known TP53 status we worked up a previously existing IHC antibody that was being used in a clinical laboratory and found that it was highly specific for TP53 missense mutations in prostate cancer [89] . Using this assay on human prostate samples we reported that p53 overexpression was approximately 85% sensitive for detecting a TP53 missense mutation.…”

DNA damage repair alterations are frequent in prostatic adenocarcinomas with focal pleomorphic giant‐cell features

“…Briefly, PTEN immunohistochemistry was performed in a Clinical Laboratory Improvement Amendments (CLIA)‐accredited laboratory by use of a genetically validated protocol developed by our group for the Ventana autostaining system (Ventana/Roche Medical Systems, Tucson, AZ, USA), with a rabbit monoclonal antibody (D4.3; Cell Signaling Technologies, Danvers, MA, USA). p53 immunohistochemistry was performed in a CLIA‐accredited laboratory by use of a genetically validated protocol developed by our group, with a mouse monoclonal antibody (BP53‐11; Ventana/Roche Medical Systems). Scoring for both assays was performed with a dichotomous system as previously described …”

Pathomorphological Features of Primary Tumor and P53 Protein Expression as Predictors of Local Recurrence of Prostate Adenocarcinoma Under Hifu Treatment Conditions

“…The evaluation of staining intensity is based on the data suggesting that a high degree of p53 expression significantly correlates with the gene mutation, while a weak reaction can be associated with a physiological accumulation of the protein in the conditions of cellular stress or disturbance of some regulatory interactions [11]. The 10% cutoff was often used for the evaluation of positive expression in the studies of PC [8,12,13]. A lower fraction of positive nuclei (<10%) correlated stronger with the presence of a wild gene in the analysis of ovarian carcinoma [11].…”