DETECTION OF P16 GENE DELETIONS IN GLIOMAS - A COMPARISON OF FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) VERSUS QUANTITATIVE PCR

The p16 protein plays a key role in cell cycle control by preventing C DK4 from inactivating the retinoblastoma protein (pRb). The correspond ing tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicate d in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 meth ods of detecting p16 deletion. Thirty diffuse gliomas were analyzed fo r p16 gene dosage. Dual color fluorescence in situ hybridization (FISH ) was performed on cytologic preparations using paired centromeric (CE N) and locus-specific probes for CEN9/p16, CEN8/RB, and CEN12/CDK4. Qu antitative PCR was performed using primers for p16, MTAP, and referenc e genes. Eleven cases were also studied using comparative genomic hybr idization (CGH). Abnormalities of the p16-CDK4-RB pathway were identif ied in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis furt her revealed tetraploidy/aneuploidy in 14 (47%), RE deletion in 11 (37 %), and CDK4 amplification in 1 (3.3%). There were 94% and 100% concor dance rates between CGH and FISH or PCR, respectively. Quantitative PC R was noninformative in 4 cases. Although FISH and quantitative PCR ar e both reliable techniques, each has limitations. PCR is likely to mis s p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysi s may miss small deletions. Replacement of the latter with a cosmid pr obe may improve the sensitivity of FISH in future experiments.