Detailed gene expression analysis but not microsatellite marker analysis of 9p21 reveals differential defects in the INK4a gene locus in the majority of head and neck cancers

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Abstract
The INK4a gene locus on chromosome 9p21 encodes two proteins, p16INK4a and p14ARF, which influence cell cycle control regulated by pRb and p53. The objective of this study was to use different methods for the analysis of the incidence of changes at the INK4a locus in head and neck cancer (HNSCC). Primary tumours were analysed for allelic imbalances (AI) with microsatellite markers for chromosome 9, by immunohistochemistry (IHC) and IHC with enhanced sensitivity by tyramide signal amplification (TSA-IHC), and by RT-PCR. No homozygous deletions at 9p21 were detected. AI at 9p21, which was found in approximately 60% of the tumours, completely failed to indicate the functional inactivation of the two INK4a gene products. Immunostaining of normal squamous epithelia revealed very low levels of p16INK4a, whereas p14ARF was readily detectable. In 160 tumours, IHC suggested a loss of p16INK4a expression in 90%. However, by TSA-IHC, only 53.7% showed loss of p16INK4a expression, and this was consistent with the RT-PCR analyses. In 100 tumours analysed for both proteins, selective loss of p16INK4a occurred in 37%; loss of p14ARF was found in only 15%, and selective loss in only 4%; 11% of the tumours had lost both proteins. We conclude that only IHC with high sensitivity and the combined expression analysis of mRNAs and proteins is suitable for studying the role of INK4a in HNSCC. The INK4a gene expression defects are frequent but not universal and primarily affect p16INK4a. Their clinical impact is still not clear. Copyright © 2001 John Wiley & Sons, Ltd.