utImmunohistochemical detection of the alternate INK4a-encoded tumor suppressor protein p14ARF in archival human cancers and cell lines using commercial antibodies: Correlation with p16INK4a expression

Abstract

The INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF. Although the former is one of the most common targets for inactivation in human neoplasia, the frequency of p14ARF abrogation is not established. We have developed an immunohistochemical assay that allows the evaluation of p14ARF expression in formalin-fixed, paraffin-embedded tissues, using commercially available antibodies, p14ARF positive cells showed nuclear/nucleolar staining, which was absent in all cell lines and tumors with homozygous deletions of the INK4a gene. The assay was applied to 34 paraffin-embedded cell buttons, 30 non-small cell lung cancers and 28 pancreatic carcinomas, and the staining results were correlated with p16INK4a expression. Loss of p14ARF expression was common but less frequent than down-regulation of p16INK4a (53% versus 76% of all specimens). The p14ARF and p16INK4a expression pattern was concordant in 65 of 92 cases (71%). Significantly, 24 cases were p16INK4a-/p14ARF+, while the opposite staining pattern was observed in three cases, consistent with the notion that the two proteins have nonredundant functions. The immunohistochemical assay described here may facilitate studies on the prevalence and significance of aberrant p14ARF expression in human tumors.