Detection of p53 mutations by a panel of monoclonal antibodies and their clinical implications in colorectal adenocarcinoma
Authors: Courtney B. Herring, Chakrapani Chatla, Venkat R. Katkoori, Nirag C. Jhala, Denise Oelschlager, William E. Grizzle, Upender Manne
Journal: Cancer Research (2005)

1262  Purpose: Technical variations have remarkable influence in determining the clinical usefulness of molecular markers of colorectal cancer (CRC). E.g., we have reported that nuclear accumulation of p53 (p53nac), detected by immunohistochemistry (IHC) without using antigen retrieval (AR) methods, is a prognostic marker of poor survival only for non-Hispanic Caucasian patients with CRCs (Cancer 1998;83:2456-2467). In this study, we evaluated the usefulness of IHC using multiple antibody (Ab) clones of p53 in detecting p53nac in CRCs with and without p53 genetic alterations to find highest concordance between IHC and DNA sequencing methods. Methods: We evaluated 45 randomly selected CRC specimens from non-Hispanic Caucasian patients for p53nac using multiple Ab clones of p53 (Clones, CM1, DO-1, DO-7, PAb1801, BP53.12.1 & combination of PAb1801 and BP53.12.1) with and without citrate buffer-microwave AR method. All these cases were previously assessed for p53 status by direct DNA sequencing method. We selected >0%, ≥10%, ≥20% and ≥50% immunopositive cells in a tissue section as cut-off values to categorize tumors as positive for p53nac and correlated tumor positivity with underlying genetic mutations. We calculated sensitivity (Sn), specificity (Sp), positive predictive values (PPV) and negative predictive values (NPV) for all clones in detecting tumors with mutations or wild-type. We analyzed prognostic value of IHC data generated by different clones using Kaplan-Meier method. Results: In molecular analysis, 17 cases exhibited missense mutations and 8 cases were with complex mutations. The range of proportion of tumors positive for p53nac, using >0%, ≥10%, ≥20% and ≥50% as cut-off values, was 33 to 84 without AR and 60 to 89 with AR. All point mutations were detected by all Ab clones of p53 with and without AR when >0% and ≥10% cut-offs were used; however, there was a significant difference in Sn, Sp, PPV and NPV values between the methods and among different Ab clones. At 10% cut-off value in non-AR condition, PAb1801 and DO-1 clones exhibited better concordance, based on Sn, Sp, PPV and NPV values, between IHC and molecular sequence analyses methods. However, the non-AR p53nac data generated by BP53.12.1 (long-rank P=0.0354) and DO-1 (long-rank P=0.0399) clones had better correlation with patient survival at 10 years post-surgery when compared to all other Ab clones of p53. Conclusions: These studies demonstrate that the citrate buffer-microwave antigen enhancement method should not be used for the detection of p53nac because it increases the sensitivity of the assay and results in detecting false positives. Also, these findings suggest that detection of p53nac by routine IHC, using either BP53.12.1 or DO-1 clones, can be used as a prognostic marker of colorectal cancer specifically for Caucasian patients. This study is supported by a NCI/NIH grant CA098932-01.