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 present study we investigated the alterations of this G1-phase protein pathway using immunohistochemical and molecular methods in a series of 55 breast carcinomas 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 there are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor suppressor 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 inverse relationship between p16 and pRb expression was found (p = 0.079). Analysis of the region that encodes for p16 by deletion mapping, a PCR-based methylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(INK4A) inactivation in breast carcinomas. The results of deletion mapping also 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/p16(INK4A) may play a role in breast carcinogenesis. In addition, microsatellite instability (MI), a marker of replication error phenotype (RER+), was observed with a frequency of 16% in the area examined and was inversely related with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcinomas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alterations. We speculate that there is an additional mechanism of CDKN2/p16(INK4A) inactivation. The relationship of p16 protein level pRb, status, the p16-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters revealed two significant correlations: one between normal pRb expression and lymph node involvement (p = 0.0263), and the other between microsatellite alterations (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 significant proportion of breast carcinomas, deregulation of the p16-pRb pathway in association with another, as-yet unidentified, TSG(s) of the 9p21-22 region may play a role in initiating or progressing the oncogenic procedure, while in other subgroups, alternative molecules may play this role.
Gorgoulis
V G
VG
Department of Histology and Embryology, School of Medicine, University of Athens, Greece.
Koutroumbi
E N
EN
Kotsinas
A
A
Zacharatos
P
P
Markopoulos
C
C
Giannikos
L
L
Kyriakou
V
V
Voulgaris
Z
Z
Gogas
I
I
Kittas
C
C
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002874:Chromosome Mapping; D002899:Chromosomes, Human, Pair 9; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry; D019656:Loss of Heterozygosity; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*