METHYLATION, A MAJOR MECHANISM OF P16 CDKN2 GENE INACTIVATION IN HEADAND NECK SQUAMOUS CARCINOMA/

We studied 11 head and neck squamous carcinoma (HNSC) cell lines and 4 6 primary tumors for p16 gene status by protein, mRNA, and DNA genetic /epigenetic analyses to determine the incidence, the mechanism(s), and the potential biological significance of its inactivation, Of the 11 cell lines, only 1 showed intact p16 and 10 lacked its protein and mRN A: DNA analysis of these 10 cell lines showed 2 homozygous deletions, 6 methylations at exon 1 and 2, and 2 with no detectable abnormalities , In primary tumors, 16 (34.7%) of the 46 showed detectable p16 protei n and mRNA; of these, 12 had no DNA abnormalities and 4 had only exon 2 methylation. Loss of p16 expression was found in three tumors with c oncurrent mutation at exon 2 and methylation at exon 2 (two) and both 1 and 2 (one), Of the 30 tumors that lacked p16 protein, 27 also lacke d mRNA, 1 had detectable p16 mRNA, and 2 failed RT-PCR amplification, Twenty-two of the thirty tumors showed DNA alterations and eight manif ested no abnormalities; DNA alterations comprised 6 homozygous deletio ns, 2 concurrent mutations and methylation of exon 2, and 13 with meth ylation at exon 1 and exons 1 and 2 (12 with methylation only and 1 wi th mutation) at exon 1. Except for patients' gender (P = 0.02), no sig nificant correlation between p16 and clinicopathological factors was o bserved, We conclude that in HNSC 1) intragenic p16 alterations are in frequent events, 2) methylation of exon 1 constitutes a common mechani sm in silencing the p16 gene, 3) p16 inactivation may play an importan t role in the early development and progression of HNSC, and 4) no ass ociation between p16 alterations and conventional clinicopathological factors was noted in this cohort.