Preferential Loss of Expression 0f16INK4a Rather Than p1' in Breast Cancer1

The tumor suppressor p16�4" has been shown to be inactivated in numerous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which pJ6INK4as located in more than one-half of primary breast tumors. However, mutational analysis of these same tumors revealed mutation of pJ6�"�' to be infrequent. Other possible modes of inactivation, such as de novo meth- ylation and homozygous deletion, have since been shown to occur in numerous neoplasias. Furthering the complexity of this locus, a transcript overlapping the p16�"4" coding se- quence and encoding a novel peptide with growth-suppres- sive activity, �J�4RF, has been described. To clearly eluci- date 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 encodingp16�\'K4�� (exon la), mutational analysis of exon 1� by single-strand conformational polymorphism analysis of pJ9ARF transcripts, and expression of both a and ftran- scripts by reverse transcription PCR. Homozygous deletion of p16, as determined by interphase chromosomal fluores- cence in situ hybridization, was observed in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon la was observed in an additional 17% (4 of 23). Reduced expression of pJ6INK4a was observed in 11 tumors (48%), including all those in which homozygous deletion or com- plete methylation was observed. No mutations of exon 1� were detected, and expression of its transcript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression. These results further sup- port pJ6" as a target of inactivation in the 9p2l region for breast cancer.