RESUMO
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.
Assuntos
Canal Anal/patologia , Neoplasias do Ânus/patologia , Carcinoma de Células Escamosas/patologia , Metilação de DNA , Proteínas Adaptadoras de Transdução de Sinal , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Canal Anal/metabolismo , Neoplasias do Ânus/genética , Biópsia , Carcinoma de Células Escamosas/genética , Proteínas de Transporte , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Humanos , Imunoglobulinas/genética , Fatores de Transcrição Kruppel-Like , Proteínas de Membrana/genética , Proteína 1 Homóloga a MutL , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Serina-Treonina Quinases/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p14ARF/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Cervical carcinoma is a common malignancy among women worldwide, and its pathogenesis is related causally to human papillomavirus infection. The progression from precursor squamous intraepithelial lesions to cervical carcinoma requires additional genetic and epigenetic alterations that have not been characterized fully. The authors examined aberrant promoter methylation of multiple tumor suppressor genes in precursor squamous intraepithelial lesions. METHODS: A multiplex, nested, methylation-specific polymerase chain reaction approach was used to examine promoter methylation of 15 tumor suppressor genes in high-grade squamous intraepithelial lesions (HSIL, n = 11), low-grade squamous intraepithelial lesions (LSIL, n = 17), and negative tissues (n = 11) from liquid-based cytology samples. The area under the receiver-operating characteristic (ROC) curve was determined for individual methylated tumor suppressor genes and for gene combinations to evaluate test performance for the ability of methylation profiles to distinguish HSIL cytology samples from combined LSIL/negative cytology samples. RESULTS: Aberrant promoter methylation of DAPK1 and IGSF4 occurred at a high frequency in HSIL samples and was absent in LSIL and negative samples. There was a significant trend toward increased methylation with the increased severity of lesions, and the mean number of methylated genes was significantly higher in HSIL samples compared with LSIL and negative samples. Using the area under the ROC curve as a measure of test performance, the methylation of IGSF4 and DAPK1 had areas that were significantly greater than 0.5; thus, each had the ability to distinguish HSIL samples from combined LSIL/negative samples. The areas under the curve for the best two-gene combination (IGSF4/DAPK1) and the best three-gene combination (IGSF4/DAPK1/HIC1) were not statistically different from the best individual tumor suppressor gene (IGSF4) in distinguishing HSIL samples from combined LSIL/negative samples. CONCLUSIONS: Aberrant promoter methylation of tumor suppressor genes is an epigenetic alteration that occurs during neoplastic progression to cervical carcinoma. The methylation status of multiple tumor suppressor genes can be evaluated using ROC analysis to determine methylation profiles that can distinguish HSIL samples from combined LSIL/negative samples.