DNA Methylation in Anal Intraepithelial Lesions and Anal Squamous Cell Carcinoma

Abstract

Purpose: Anal intraepithelial neoplasia is associated with human papillomavirus infection and may progress to invasive squamous cell carcinoma (SCC), which is increasing in immunocompromised patients. We hypothesize that anal intraepithelial neoplasia is associated with abnormal DNA methylation and that detection of these events may be used to improve screening programs. Experimental Design: Seventy-six patients were identified who underwent anal cytology screening and subsequent biopsy at our institution between 1999 and 2004. The specimens from these patients included 184 anal biopsies [normal, n = 57; low-grade squamous intraepithelial lesion (LSIL), n = 74; high-grade squamous intraepithelial lesion (HSIL), n = 41; and invasive SCC, n = 12] and 37 residual liquid-based anal cytology specimens (normal, n = 11; LSIL, n = 12; HSIL, n = 14). The methylation status of the following genes was determined for each biopsy and cytology sample using real-time methylation-specific PCR: HIC1, RASSF1, RARB, CDKN2A, p14, TP73, APC, MLH1, MGMT, DAPK1, and IGSF4. Results: Methylation-specific PCR analysis of biopsy samples revealed that DNA methylation was more common in SCC and HSIL than LSIL and normal mucosa. Specifically, methylation of IGSF4 and DAPK1 was prevalent in SCC (75% and 75% of cases, respectively) and HSIL (59% and 71%, respectively) but was absent in LSIL and normal biopsy samples. Methylation profiles of cytologic samples were similar to those found in the biopsy samples. Conclusions: Aberrant DNA methylation is a frequent event in anal HSIL and SCC. Methylation of IGSF4 and DAPK1 is specific for HSIL and SCC, and may serve as a useful molecular biomarker.

Screening for HIV-Associated Anal Cancer: Correlation of HPV Genotypes, p16, and E6 Transcripts with Anal Pathology

“…A few biomarkers have been investigated as adjunctive means to enhance the diagnostic accuracy of anal pathology specimens (15,(28)(29)(30)(31). The p16 protein (also known as p16 INK4a , MTS1, and CDKN2) is a common target of inactivation in human cancers but is overexpressed in high-risk HPV-related cervical cancers (32)(33)(34) and anal cancer (15,16).…”

“…Reactions were amplified for 40 cycles of 95jC for 30 s, 55jC for 1 min, and 72jC for 1 min; followed by a 5 min extension at 72jC. PCR products were detected with HPV genotyping strips (Roche) consisting of 29 probe lines detecting 27 individual HPV genotypes (6,11,16,18,26,31,33,35,39,40,42,45, 51 to 59, 66, 68, MM4, MM7, MM8, MM9) and two concentrations of the h-globin control probe. Briefly, PCR amplicons were denatured with 0.13 N NaOH.…”

Molecular Biomarkers Correlate with Disease-Free Survival in Patients with Anal Canal Carcinoma Treated with Chemoradiation

“…Methylation of several genes [59], human papilloma virus (HPV), p53, and AKT have been implicated in carcinogenesis of anal carcinoma [60]. Nilsson et al [61].…”

Epigenomic Characterization of Locally Advanced Anal Cancer

“…provided the first evidence of aberrant methylation in anal cancer among 11 candidate genes compared to normal tissue. 18 Subsequently using an array-based assay analyzing >1500 CpG sites, we observed differences in DNA methylation patterns in 20 genes in the progression from normal anal mucosa, carcinoma in situ, to invasive anal carcinoma in a small set of cases. 19 To date, a broad high-throughput characterization of methylation events in advanced anal cancer is clearly lacking.…”

“…18,19 Zhang et al . provided the first evidence of aberrant methylation in anal cancer among 11 candidate genes compared to normal tissue.…”