Human Papillomavirus Prevalence in Oropharyngeal Cancer Before Vaccine Introduction, United States

Martin Steinau; Mona Saraiya; Marc T. Goodman; Edward S. Peters; Meg Watson; Jennifer L. Cleveland; Charles F. Lynch; Edward J. Wilkinson; Brenda Y. Hernandez; Glen Copeland; Maria S. Saber; Claudia Hopenhayn; Youjie Huang; Wendy Cozen; Christopher Lyu; Elizabeth R. Unger


Emerging Infectious Diseases. 2014;20(5):822-828. 

In This Article

Materials and Methods

Cancer Tissue Specimens

As part of the Centers for Disease Control Cancer Registry Sentinel Surveillance System study (M. Saraiya, unpub data), a systematic review of cases of oropharyngeal cancer diagnosed during 1995–2005 was performed. The cases were selected from 7 participating registries, including 4 central cancer registries in Florida, Kentucky, Louisiana, and Michigan and 3 Surveillance, Epidemiology, and End Results program (SEER []) cancer registry-based residual tissue repositories in Los Angeles County, CA; Hawaii; and Iowa. The following anatomic regions (by ICD-O-3 codes) were included: C01.9 and C02.4 (base of the tongue and lingual tonsil); C09.0, C09.1, C09.8, C09.9, and c14.2 (tonsil); C14.0, C14.2, C14.8, C02.8, C10.2, C10.8, and C10.9 (other oropharynx).[13] Of 4,073 cases matching these criteria, we requested samples from 1,271 case-patients representative of the whole case-patient population regarding sex, age, and race/ethnicity. One archived, formalin–fixed paraffin-embedded tissue sample, representative of the primary tumor, was selected by the submitting pathology laboratory. If tissue from the primary tumor was unavailable, a sample from a metastatic lesion in a lymph node was accepted because HPV prevalence is usually maintained in OPSCC–positive lymph nodes.[14] With the exception of 32 cases from Hawaii and 11 from Los Angeles County, which had been sampled by Chaturvedi et al.,[12] cases were selected exclusively for this study. Each participating state and CDC received approval from their institutional review boards for the study; CDC approved the overall study.

DNA Extraction and HPV Typing

All laboratory methods were described previously.[15,16] Six consecutive 5-μm sections were cut from each selected tissue block; special precautions were used to avoid cross-contamination. The first and last sections were stained with hematoxylin and eosin and reviewed by a study pathologist (ERU) to confirm the presence of viable tumor tissue. DNA was extracted from two 5-μm sections by using high temperature–assisted tissue lysis[17] and further purification was carried out by automated extraction by using Chemagic MSM1 (PerkinElmer, Waltham, MA, USA). HPV types were determined from 2 commercial assays by using an algorithm which was evaluated earlier for this application.[18,19] First, all DNA extracts were tested by using the Linear Array HPV Genotyping Assay (Linear Array; Roche Diagnostics, Indianapolis, IN, USA) and a HPV-52-specific PCR to resolve ambiguous positive results from the XR probe of the Linear Array HPV test.[20] Samples that had negative or inadequate linear array results (negative for HPV and cellular β-globin controls) were retested with the INNO-LiPA HPV Genotyping Assay (Innogenetics, Gent, Belgium). HPV status was recorded for 40 types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, IS39, and 89 as tested; and HPV of an unknown type (HPV-X) for additional unspecified types as indicated by LiPA results.


Prevalence was assessed as percentage positive from the total number of cases with valid results. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered to have a high risk for oncogenic potential[21] and all other types, including HPV-X, to have a low risk, showing low or no known oncogenic potential.

Hierarchical categories for HPV status were assigned as follows: HPV-16 includes all cases positive for this type regardless of other results. HPV-18 includes all cases positive for HPV-18, but not for HPV-16; other 9-valent, high-risk HPV types included in the next generation of the HPV vaccine: HPV-31, -33, -45, -52, -58, but not HPV-16 or -18; other high-risk HPV cases positive for any high risk not included in the previous categories: HPV-35, -39, -51, -66, -68; and low-risk HPV: all other cases positive for any remaining low-risk HPV types.

Statistical analysis was restricted to case-patients that had confirmed invasive OPSCC. Case-patient age at diagnosis was stratified into 4 groups: <50, 50– 59, 60–69, and ≥70 years. Cancer stages were crudely classified as local, regional, or distant (metastatic) by SEER classifications. Differences in prevalence of positive results for high-risk HPV or HPV-16/18, categorized by patient's age, sex, race/ethnicity, and the anatomic location of cancer, were evaluated by using the χ2 or Fisher exact test whenever possible. Multivariate analysis was performed by using logistic regression with a step-down procedure, adjusting for age, sex, and race/ethnicity as appropriate. All statistical calculations were made by using SAS 9.3 (SAS Institute Inc., Cary, NC, USA).