Comprehensive Analysis of CDKN2A Status in Microdissected Urothelial Cell Carcinoma Reveals Potential Haploinsufficiency, a High Frequency of Homozygous Co-deletion and Associations with Clinical Phenotype

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Abstract
Purpose: There are significant differences in reported frequencies, modes of inactivation, and clinical significance of CDKN2A in urothelial cell carcinoma (UCC). We aimed to address these issues by investigating all possible modes of inactivation and clinicopathologic variables in a single tumor panel. Experimental Design: Fifty microdissected UCCs were examined. CDKN2A gene dosage (quantitative real-time PCR), allelic status (microsatellite analysis), hypermethylation (methylation-specific PCR), mutation status (denaturing high-performance liquid chromatography and sequencing), protein expression (immunohistochemistry), and clinicopathologic variables (stage, grade, and disease recurrence during follow-up) were assessed. Results: Exon 2 was underrepresented in 20 of 46 (43%) and exon 1β in 21 of 46 (46%) of cases. Underrepresentation of exon 2 was accompanied by loss of heterozygosity (LOH) of 9p in 6 of 18 (30%) and of exon 1β in 11 of 19 assessable cases (58%). Overall, LOH of 9p was identified in 15/41 (37%). Homozygous deletion of exons 2 and 1β was detected in 16 of 46 (35%) and 10 of 46 tumors (22%), respectively. Co-deletion was most common, but exon 2–specific homozygous deletion was also detected. In tumors without homozygous deletion, p16 promoter hypermethylation was detected in 1 of 18 (6%). Hypermethylation of the p14ARF promoter or mutations in CDKN2A were not observed. Homozygous deletion of exon 2 or LOH on 9p were associated with invasion. Homozygous deletion of exon 2 or exon 1β was associated with recurrent disease. Conclusions: These results confirm CDKN2A as a clinically relevant target for inactivation in UCC and show that the true frequency of alteration is only revealed by comprehensive analysis. Our results suggest that CDKN2A may be haploinsufficient in human cancer.