WAF1 CIP1 GENE POLYMORPHISM AND EXPRESSION IN CARCINOMAS OF THE BREAST, OVARY, AND ENDOMETRIUM/

The p53 gene is altered in approximately 50% of human cancers and is c onsidered to be important in the pathogenesis of these malignancies. T he p53 protein product regulates the transition from G1 to S phase of the cell cycle and entry to the DNA damage repair pathway. As alterati ons in this pathway appear to be important in a variety of human cance rs, downstream effector proteins of p53 are potential sites for somati c alterations. WAF1/Cip1, also known as WAF1, Cip1, sdi1, or CAP20, co des for a 21-kd protein (p21(WAF1/Cip1)), which was recently described as a universal inhibitor of cyclins and is thus critical in cell cycl e control. Mutations in WAF1/Cip1 are potentially important in human m alignancies because they could affect the control of the cell cycle. T o understand whether mutations of WAF1/Cip1 occur in cancer, we screen ed 53 cases of invasive breast carcinoma, 35 cases of ductal carcinoma in situ (DCIS), 53 ovarian carcinomas, and 47 endometrial carcinomas in the second exon of WAF1/Cip1 (90% of the open reading frame). p21(W AF1/Cip1) expression was characterized with immunohistochemistry. Cell s from the blood of 21 normal individuals were also characterized usin g single-strand conformational polymorphism analysis, DNA sequencing a nd restriction analysis. Single-strand conformational polymorphism ana lysis demonstrated an altered mobility pattern for exon 2 in 12 invasi ve breast cancers (22.6%), 5 DCIS of the breast (14%), 8 invasive ovar ian carcinomas (15%), and 9 endometrial carcinomas (19%). In total, 20 9 samples were screened, and 38 cases (18.2%) had an altered codon 31. Each case with altered single-strand conformational polymorphism, ana lyzed by DNA sequencing and/or restriction analysis, showed the same a lteration of codon 31, a C to A transversion encoding a change in amin o acid sequence from serine to arginine (31Ser --> 31Arg). DNA from th e blood of 21 normal individuals showed the same alteration in WAF1/Ci p1 in 4 cases (19%). Furthermore, paired normal tissue was available f or 3 of 20 breast carcinomas with the Ser31Arg transversion. Normal DN A from all 3 cases showed the same 31Arg alteration as found in the tu mor tissue. These results indicate that codon 31 is a polymorphic site and that the serine to arginine shift is a polymorphism. p21(WAF1/Cip 1) expression, identified by immunohistochemistry, was found to vary i n a pattern that depended both on the tissue type and on the presence or absence of the codon 31 polymorphism. Using pair-wise comparisons i n breast DCIS, we found higher protein expression in tumor nuclei as c ompared with benign stromal cell nuclei (P = 0.002) or normal ductal e pithelium (P = 0.005). Invasive breast cancer specimens showed a trend in p21(WAF1/Cip1) immunostaining similar to DCIS but did not reach st atistical significance (P = 0.12). However, when cases with extensive desmoplastic reaction were excluded, a statistically significant assoc iation (P = 0.019) similar to that in DCIS was noted. In contrast to t he breast tumors, ovarian carcinomas exhibited significantly greater p 21(WAF1/Cip1) expression in the benign stromal (fibroblast) nuclei sur rounding the tumor than in the carcinoma cell nuclei (P = 0.016). Endo metrial carcinoma revealed no difference in staining when comparing be nign tissue with carcinoma (P = 0.99); however, unlike breast and ovar ian carcinomas in which there was no correlation between p21(WAF1/Cip1 ) expression and the presence or absence of the alteration at the 31st codon, endometrial carcinomas showed an increased (P = 0.056) with 31 Arg. These results demonstrate tissue-specific expression pattens of W AF1/Cip1 in different tumors which appears to be characteristic of the tumor type.