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

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 (methylationspecific 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 exon1h in 21of 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 1h in 11of 19 assessable cases (58%). Overall, LOH of 9p was identified in 15/41 (37%). Homozygous deletion of exons 2 and 1h 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 1of 18 (6%). Hypermethylation of the p14 ARF 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 exon1h 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.

Prognostic significance of copy number alterations in adolescent and adult patients with precursor B acute lymphoblastic leukemia enrolled in PETHEMA protocols

“…Iacobucci et al 13 reported that CDKN2A/B deletions had independent significance for lower disease-free survival in a series of 112 adult Ph-positive patients, but other authors did not show any prognostic impact. 27 No differences in outcomes were observed from a comparison of heterozygous and homozygous CDKN2A/B deletions, 12 and this suggests an important haplo-insufficiency effect 28 or concomitant inactivation of the remaining allele by epigenetic events. 29,30 Although our patients were treated within protocols, some limitations should be pointed out.…”

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

“…53 Although P16INK4A deletion has an undisputed role in transitional bladder carcinoma progression, P16INK4A DNA methylation is highly variable between individual bladder carcinoma patients and the contribution of epigenetic factors to bladder carcinoma risk is still controversial. 54,55 UTI-induced CDKN2A methylation could provide a mechanism for the observed correlation between recurrent UTI and bladder carcinoma risk. [56][57][58] After infection, bladder uroepithelial cells undergo apoptosis that results in shedding of infected cells and intracellular pathogens, thereby reducing the overall pathogen load.…”

Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

“…Traditional CGH and array-GCH have corroborated many of these findings but have also defined several recurrent high-level amplifications and deletions (3)(4)(5)(6)(7)(8). The key results of these investigations are losses of chromosome 9, or 9p and 9q, frequent amplification of 6p22 (9)(10)(11), and loss of RB1 (12). Apart from chromosomal changes, several genes are known to be mutated in bladder cancer including FGFR3, PIK3CA, KRAS, HRAS, NRAS, TP53, CDKN2A, and TSC1, of which activating mutations in FGFR3 and inactivating mutations in TP53 are the most frequent.…”