Preferential loss of expression of p16^INK4a rather than p19^ARF in breast cancer

The tumor suppressor p16^INK4a has been shown to be inactivated in numerous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which p16^INK4a is located in more than one-half of primary breast tumors. However, mutational analysis of these same tumors revealed mutation of p16^INK4a to be infrequent. Other possible modes of inactivation, such as de novo methylation and homozygous deletion, have since been shown to occur in numerous neoplasias. Furthering the complexity of this locus, a transcript overlapping the p16^INK4a coding sequence and encoding a novel peptide with growth-suppressive activity, p19^ARF, has been described. To clearly elucidate the target of aberrations affecting this subchromosomal region and approximate frequency in breast cancer, we performed a comprehensive study including p16 deletion analysis by means of interphase chromosomal fluorescence in situ hybridization, methylation analysis of the first exon encoding p16^INK4a (exon 1α), mutational analysis of exon 1β by single-strand conformational polymorphism analysis of p19^ARF transcripts, and expression of both a and β transcripts by reverse transcription PCR. Homozygous deletion of p16, as determined by interphase chromosomal fluorescence in situ hybridization, was observed in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon Iα was observed in an additional 17% (4 of 23). Reduced expression of p16^INK4a was observed in 11 tumors (48%), including all those in which homozygous deletion or complete methylation was observed. No mutations of exon 1β were detected, and expression of its transcript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression. These results further support p16^INK4a as a target of inactivation in the 9p21 region for breast cancer.