INK4a Gene Expression and Methylation in Primary Breast Cancer: Overexpression of p16INK4a Messenger RNA Is a Marker of Poor Prognosis

Frequent deletions or mutations of the INK4 gene, which encodes the cyclin-dependent kinase 4 inhibitor p16 INK4a , have been documented in various human cancers, but little is known about the role of this tumor suppressor gene in primary breast cancer. We examined p16 INK4a mRNA expression and its relationship with cyclin D1 and estrogen receptor (ER) expression in 314 primary breast cancers using Northern blots probed with a p16 exon 1α-specific cDNA. Tumor samples overexpressing p16 INK4a were predominantly ER negative with low levels of cyclin D1. Cyclin D1 and ER mRNA levels in the high p16 INK4a expressers were significantly lower than those in the remainder of the population ( P = 0.0001). Furthermore, the mean p16 INK4a mRNA level in the ER-negative tumors was significantly higher than that in the ER-positive group ( P = 0.0001). Because the INK4 gene is frequently inactivated by de novo methylation, we investigated the frequency of INK4a exon 1α methylation in a subset of 120 primary breast cancers using methylation-specific PCR; 24 of these were methylated. These findings indicate that high expression of p16 INK4a and reduced expression due to de novo INK4a methylation are frequent events in primary breast cancer. In a subset of 217 patients for whom detailed clinical data were available, high p16 INK4a mRNA expression was associated with high tumor grade ( P = 0.006), ≥ 4 axillary lymph node involvement ( P = 0.004), ER negativity ( P = 0.0001), and increased risk of relapse ( P = 0.006). The significant negative correlation between p16 INK4a and ER gene expression raises issues regarding their functional interrelationships and whether high p16 INK4a expression may be associated with a lack of hormone responsiveness in breast cancer.