Alterations of p16-pRb pathway and chromosome locus 9p21-22 in sporadic invasive breast carcinomas

The p16-pRb pathway represents a vital cell-cycle checkpoint. In the presen t study we investigated the alterations of this G1-phase protein pathway us ing immunohistochemical and molecular methods in a series of 55 breast carc inomas and correlated the findings with clinicopathological features of the patients. Furthermore, we examined its relationship with the status of the chromosomal region 9p21-22 performing a deletion map analysis because ther e are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor supp ressor genes (TSGs), this area harbors other TSG(s). Aberrant expression (Ab) of p16 and pRb was observed in 26 (47%) and 16 (29 %) of the carcinomas, respectively. A statistical trend pointing out an inv erse relationship between p16 and pRb expression was found (p = 0.079). Ana lysis of the region that encodes for p16 by deletion mapping, a PCR-based m ethylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(I NK4A) inactivation in breast carcinomas. The results of deletion mapping al so suggest that another TSG(s) may reside at the 9p21-22 area particularly at the D9S162 loci and that co-deletion of this putative gene with CDKN2/p1 6(INK4A) may play a role in breast carcinogenesis. Ln addition, microsatell ite instability (MI), a marker of replication error phenotype (RER+), was o bserved with a frequency of 16% in the area examined and was inversely rela ted with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcin omas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alter ations. We speculate that there is an additional mechanism of CDKN2/p16(INK 4A) inactivation. The relationship of p16 protein level pRb, status, the p1 6-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters reve aled two significant correlations: one between normal pRb expression and ly mph node involvement (p = 0.0263), and the other between microsatellite alt erations (LOH and or MI) and tumor size (p = 9.2 x 10(-3)). In view of the heterogenous nature of breast cancer, we suggest that in a s ignificant proportion of breast carcinomas, deregulation of the p16-pRb pat hway in association with another, as-yet unidentified, TSG(s) of the 9p21-2 2 region may play a role in initiating or progressing the oncogenic procedu re, while in other subgroups, alternative molecules may play this role.