From these data we conclude that the occurrence of CDKN2 (p16/MTS1) mutation in primary breast cancer is a rare event and is not likely to be involved in human breast tumour carcinogenesis and progression.
Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas.
To further analyze its role, we transfected hormone-responsive human breast cancer MCF-7 cells with the mts1 gene under the control of a strong constitutive promoter.
CDKN2 is not deleted with high frequency in primary breast carcinomas, and the p16 gene does not play a role in breast carcinogenesis via this mechanism.
These data suggest that methylation and lack of expression of p16INK4a is not a central mechanism in the development of breast carcinoma, but rather that the gene is functioning and expressed in breast carcinoma more frequently than in normal breast tissue.
These results indicate that cytoplasmic accumulation of the p16 protein identifies a subset of highly malignant breast carcinomas with accelerated tumour proliferation and other unfavourable parameters in breast cancer.
Loss of heterozygosity analysis of primary breast cancer tumors has revealed a high frequency of deletion of DNA from 9p21-22 encompassing the MTSI (P16/CDKN2A) gene.
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.
Our data show that de novo methylation of exon 1 of p16INK4a can be demonstrated in plasma DNA of breast cancer patients, a fact that provides additional evidence of the tumour-related origin of free plasma DNA in cancer patients.
Alterations in polymorphic markers selected because they had been found to show a high rate of alterations in breast cancer in previous studies (D17S855, D17S654, D16S421, TH2, D10S197, and D9S161), as well as mutations in the p53 gene and aberrant methylation at the first exon of p16INK4a, were used to identify and characterize tumor and plasma DNA.
The significant negative correlation between p16INK4a and ER gene expression raises issues regarding their functional interrelationships and whether high p16INK4a expression may be associated with a lack of hormone responsiveness in breast cancer.
They also show that families with the CDKN2A 113insArg mutation have an increased risk not only of multiple melanomas and pancreatic carcinoma but also of breast cancer.
Mutations in the above genes are mutually exclusive and are infrequent in fhit, p16INK4a/p19ARF and H-ras genes suggesting that these genes may not play a major role in Indian breast carcinomas.
In a group with low p16(Ink4a) expression determined by Western blotting, four randomly selected tumors contained several identical clones with methylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blotting or MSP, increasing the potential significance of p16(Ink4a) methylation in breast cancer.