Observational StudyScreening Strategies Among High-risk Populations for Anal Cancer

At each study visit, participants will receive a digital anorectal examination followed by HRA, with directed biopsies of suspicious lesions. An HRA-certified physician will collect anal swabs and HRA-directed biopsies for histological confirmation of disease and biomarker assays.

At each study visit, participants will receive a digital anorectal examination followed by HRA, with directed biopsies of suspicious lesions. An HRA-certified physician will collect anal swabs and HRA-directed biopsies for histological confirmation of disease and biomarker assays.

At each study visit, participants will receive a digital anorectal examination followed by HRA, with directed biopsies of suspicious lesions. An HRA-certified physician will collect anal swabs and HRA-directed biopsies for histological confirmation of disease and biomarker assays.

At each study visit, participants will receive a digital anorectal examination followed by HRA, with directed biopsies of suspicious lesions. An HRA-certified physician will collect anal swabs and HRA-directed biopsies for histological confirmation of disease and biomarker assays.

High-Resolution Anoscopy, or HRA, is a standard of care procedure for screening and detection of premalignant lesions of the anus. Using a small, thin round tube called an anoscope, the anal canal is examined with a high-resolution magnifying instrument called a colposcope. The application of a mildly acidic liquid on the anal canal facilitates the evaluation of abnormal tissue such as anal dysplasia. Biopsies for histological confirmation of disease will be taken. Biopsies' reporting will follow the terminology, criteria, and recommendations of the Lower Anogenital Squamous Terminology (LAST) project. Histology results will be reported as benign, condyloma acuminatum, AIN grades 1-3, or cancer. For the proposed project, aHSIL+ will be defined as AIN2 (p16 block-positive), AIN3, and cancer at the HRA encounter. All other cases will be classified as <aHSIL, including benign, condyloma acuminatum, and AIN1

Anal cytology collection (swab samples) will be performed as part of the standard of care during this study. The collected cells, suspended in the SurePath liquid-based medium, will be placed on a slide and microscopically examined by a board-certified pathologist. The sample will then be interpreted using the Bethesda System: negative for intraepithelial lesion or malignancy (NILM), ASCUS, LSIL, ASC-H, or HSIL. Pathologists interpreting the anal cytology will be blinded from hrHPV testing and biopsy histology.

Samples of exfoliated cells from the anal canal will be collected with Dacron swabs. To identify the infecting HPV genotype, DNA will be extracted from swab samples collected with the MagNA Pure LC DNA Isolation Kit III and an LC MagNA Pure system (Roche Diagnostics GmbH, Mannheim, Germany), followed by HPV typing using the TypeSeq assay, a laboratory prepared high-throughput next-generation sequencing assay developed by NCI will be performed as described. The assay uses three sequential PCR steps resulting in barcoded L1 amplicons sequenced and submitted to a bioinformatics pipeline for typing. This TypeSeq assay qualitatively detects and identifies 51 HPV genotypes (HPV types 3, 6, 11, 13, 16, 18, 26, 28, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 97, and 114), including 14 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68)

Samples of exfoliated cells from the anal canal will be collected with Dacron swabs. Dual immunostaining with p16/Ki-67 will be performed on the residual cytologic specimen by Roche MTM Laboratories (Heidelberg, Germany), using the CINtec®PLUS Kit according to the manufacturer's instructions. The anal cytology material will be stained with a mouse monoclonal antibody directed against human p16INKa (p16) protein (clone E6H4) and recombinant rabbit monoclonal antibody directed against human Ki-67 protein (clone 274-11AC3V1), using the BenchMark ULTRA instrument (Ventana Roche). Samples with insufficient cellularity will be excluded from the evaluation. A trained cytotechnologist will review all cases for cells staining positively with both markers. A slide will be considered positive if 1 or more squamous epithelial cell(s) stained positive for both p16 and Ki-67 and dual stain-positive cells will be semi-quantitatively assessed (0, 1, 2-5, 6-50, >50).