Purpose: To date, the significance of p16INK4A tumor suppressor gene inactivation in sporadic endometrial cancer (EC) has only rarely been described. In this study, we examined the alteration type and frequency of gene alterations [point mutations, aberrant promoter methylation and loss of heterozygosity (LOH)] in 50 sporadic ECs, and correlated the genetic findings with the immunohistochemical expression of the p16INK4A protein and the classical clinicopathological features.
Methods: Gene mutations were detected by PCR-SSCP-sequencing analysis, promoter hypermethylation by methylation-specific PCR (MSP), and LOH by PCR of the STS-marker c5.1.
Results: In total, p16INK4A alterations were found in 14 of 50 (28%) sporadic ECs. In six (12%) cases, two alterations occurred simultaneously. Partial p16INK4A deletions were found in four of 50 (8%) samples. There was one missense mutation (codon 70; CCC-->GCC) and one frameshift mutation (1-bp deletion in exon 2). Only 2 of 47 (4.2%) tumors exhibited aberrant promoter methylation. An allelic loss was detected in 12 of 50 (24%) carcinomas with a higher incidence in advanced endometrial carcinomas than in early-stage uterine tumors. p16INK4A alterations were generally accompanied by gene silencing, confirmed by aberrant protein immunostaining ( r=-0.442; P=0.001). There was a significant difference in the frequency of p16INK4A alterations between early (stage I; 18%) and advanced (stages II-IV; 58%) ECs ( P=0.022). One case showed complete protein loss, but absence of genetic alterations.
Conclusions: Our data indicate that p16INK4A inactivation plays a role in the tumorigenesis of the subset of sporadic ECs, particularly in cases exhibiting an aggressive clinical behavior. We demonstrate that p16INK4A methylation can act efficiently and similarly to other genetic alterations as one of the two necessary hits according to the Knudson two-hit hypothesis of tumor suppressor gene inactivation.