Immunoreactivity evaluation of mutant p53 gene product with DNA ploidy pattern in colorectal carcinoma

Abstract

Background/aims: Studying p53 protein expression in tumor cells is one of the effective methods for detecting p53 gene mutations. This study attempted simultaneous monitoring of p53 overexpression in colon cancer using immunohistochemical and immunoblotting techniques and also to compare abnormalities of p53 with DNA ploidy and clinicopathological variables.

Methodology: The occurrence of p53 protein expression was analyzed in forty-nine fresh colorectal cancer specimens by immunohistochemical and p53 protein expression also demonstrated by Western immunoblotting technique in 28 colorectal cancer specimens, using an anti-human p53 monoclonal antibody (Do-7), and 25 normal colon mucosa as a negative control. DNA ploidy in 36 specimens of colon cancer tissues was determined by Flow cytometry.

Results: Overexpression of p53 protein was detected immunohistochemically in 53.1% (26 of 49) of the tumor specimens. DNA ploidy was performed in 36 cases, 55.6% (20 of 36) of colon cancer specimens were DNA aneuploidy, p53 immunostaining was positive in 60% of cases with DNA aneuploidy compared to 31.3% in diploid tumors (p<0.001). There was no significant association between p53 immunostaining and clinicopathological variables. Overexpression of p53 protein was demonstrated in nuclear protein extract by immunoblotting in 75% (21 of 28) of colorectal carcinoma. Aneuploidy carcinomas were more frequently p53 positive by immunoblotting than DNA diploidy carcinomas; 76.5% (13 of 17) vs. 72.7% (8 of 11) (p<0.2). P53 expression by immunoblotting was more frequently found in good lymphocytic infiltration than moderate and poor lymphocytic infiltration (p<0.001). Also, p53 expression in right colon was significant with rectum (p<0.009). The incidence of p53 expression in Duke's stage B was significant if compared with Duke's stage C (p<0.005). Immuno-reactivity of p53 expression was detected by immunostaining and immunoblotting in 89.3% (25 of 28) of colorectal cancer. P53 immunoreactivity by immunostaining and immunoblotting were closely related to the clinicopathological variables such as pathological type (p<0.01), lymphocytic infiltration (p<0.0001), tumor grade, and tumor site (p<0.001). DNA aneuploidy was more frequently p53 positive than DNA diploid tumor by immunostaining and immunoblotting (p<0.001).

Conclusions: Immunohistochemistry confirmed by immunoblotting assay is a sensitive method for detecting the trace amount of p53 protein and provides valuable information for the understanding of colorectal cancer biology.