PREFERENTIAL LOSS OF EXPRESSION OF P16(INK4A) RATHER THAN P19(ARF) INBREAST-CANCER

The tumor suppressor p16(INK4a) has been shown to be inactivated in nu merous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which p16(INK4a) is located in more th an one-half of primary breast tumors, However, mutational analysis of these same tumors revealed mutation of p16(INK4a) to be infrequent, Ot her possible modes of inactivation, such as de novo methylation and ho mozygous deletion, have since been shown to occur in numerous neoplasi as. Furthering the complexity of this locus, a transcript overlapping the p16(INK4a) coding sequence and encoding a novel peptide with growt h-suppressive activity, p19(ARF), has been described, To clearly eluci date the target of aberrations affecting this subchromosomal region an d approximate frequency in breast cancer, we performed a comprehensive study including pld deletion analysis by means of interphase chromoso mal fluorescence in situ hybridization, methylation analysis of the fi rst exon encoding p16(INK4a) (exon 1 alpha), mutational analysis of ex on 1 beta by single-strand conformational polymorphism analysis of p19 (ARF) transcripts, and expression of both alpha and beta transcripts b y reverse transcription PCR, Homozygous deletion of p16, as determined by interphase chromosomal fluorescence in situ hybridization, was obs erved in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon 1 alpha was observed in an additional 17% (4 of 23), Reduced exp ression of p16(INK4a) was observed in 11 tumors (48%), including all t hose in which homozygous deletion or complete methylation was observed , No mutations of exon 1 beta were detected, and expression of its tra nscript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression, These results further support p16(IN K4a) as a target of inactivation in the 9p21 region for breast cancer.