Human Papillomavirus Genotype Specificity of Hybrid Capture 2

Abstract

Hybrid Capture 2 (hc2), a clinical test for carcinogenic human papillomavirus (HPV) DNA, has proven to be a sensitive but only modestly specific predictor of cervical precancer and cancer risk. Some of its nonspecificity for clinical end points can be ascribed to cross-reactivity with noncarcinogenic HPV genotypes. However, the reference genotyping tests that have been used for these comparisons are also imperfect. We therefore sought to describe further the HPV genotype specificity of hc2 by comparing the hc2 results to paired results from two related PGMY09/11 L1 primer-based HPV genotyping assays: Linear Array (LA) and its prototype predecessor, the line blot assay (LBA). LA and LBA results were considered separately and combined (detection by either assay or both assays) for 37 individual HPV genotypes and HPV risk group categories (carcinogenic HPV > noncarcinogenic HPV > negative). Carcinogenic human papillomavirus (HPV) DNA testing in conjunction with cytology is now approved in the United States for use in cervical cancer screening (36, 48) because of the causal role of persistent carcinogenic HPV infection in the development of cervical cancer and its immediate precursor lesions. The prescribed use of HPV testing is for triage of equivocal cytology, atypical squamous cells of undetermined significance (ASCUS), to determine which patients are referred to colposcopy, and as an adjunctive screening test to cytology in women who are Ն30 years old. Recent, randomized clinical trials have highlighted the sensitivity of HPV testing for cervical precancer and cancer, which might lead to even greater use of these assays in the near future (5,24,26,30).One test, Hybrid Capture 2 (hc2; Digene Corporation, Gaithersburg, MD), has received FDA approval for use in cervical cancer screening as described above; hc2 has proven to be reliable (6,9,14,45) and sensitive for detection of cervical precancer (16,17,31,32,43). However, hc2 has exhibited cross-reactivity with untargeted, noncarcinogenic HPV genotypes (12,28,29,33,41,42,45,49), which further reduces the already suboptimal clinical specificity and positive predictive value of HPV testing (12). Its cross-reactivity is most pronounced in women with cytologic changes who often harbor multiple, noncarcinogenic HPV genotypes concurrently and/or have higher viral loads (12). The HPV genotypes most commonly found to cause hc2 cross-reactivity are HPV genotype 53 (HPV53) and HPV66, the latter of which has recently been deemed to be a carcinogenic HPV genotype (15) and therefore probably increased the clinical sensitivity of hc2. Earlier studies of hc2 (12,28,29,33,41,42,45,49) were based on single tests by research and prototype HPV genotyping assays, raising the possibility that some of the cross-reactivity resulted from misclassification by the HPV genotyping assay related to: (i) single testing of small aliquots for genotyping; (ii) modest analytic sensitivity; (iii) testing errors; and (iv) "drop out" of genotypes in multigenotype HPV infections due to PCR primer comp...

High-risk Human Papillomavirus Genotype Detection by Electrochemical DNA Chip Method

“…Among phylogenetic species, HPV 34 and HPV 67 were found within α11 and α9, respectively with carcinogenic genotypes and thus, had been found in the cancer group (Matsukura et al, 2004;Castle et al, 2008). Furthermore, the genotyping method in this study (Electrochemical DNA chip, Toshiba) was designed to detect only 13 HR-HPV genotypes and the INNO-LiPA kit does not include either HPV 34 or HPV 67 and thus, some uncommon genotype may have been missed.…”

Evaluation of oncogenic human papillomavirus RNA and DNA tests with liquid‐based cytology in primary cervical cancer screening: The FASE study

“…22,24 In this study, the estimated probability of a false positive result for CIN2þ was 13.6% for HC2 compared to only 8.2% for AHPV. This could be explained in part by HC2's propensity to crossreact with some low-risk HPV genotypes, 26 and/or the ability of AHPV to identify clinically significant cervical precancer. Thus, using HC2 alone for screening would lead to a high rate of false positives, resulting in unnecessary referrals to colposcopy with unwanted patient burden and health care costs.…”

Commercially available assays for multiplex detection of alpha human papillomaviruses

“…It has been recognized that the hc2 high-risk probe cocktail detects, in addition to the 13 HPV types included in the high-risk probe cocktail, at least 28 other HPV types, many of them considered to be lr-HPV types. A genotyping study of 3179 specimens of women participating in an ALTS trial (a clinical trial to evaluate management strategies for women with ASC-US or low-grade squamous intraepithelial lesions) showed that 7.8% of all hc2-positive results in this population were false-positive due to the cross-reactivity of hc2 with untargeted, noncarcinogenic HPV types [32]. Some recent studies have also shown that hc2 has an additional 5% false-positive rate due to positive hr-HPV results when no HPV DNA is present in the clinical specimen based on the use of different usually highly sensitive and broad-range PCR tests [32,33].…”

“…A genotyping study of 3179 specimens of women participating in an ALTS trial (a clinical trial to evaluate management strategies for women with ASC-US or low-grade squamous intraepithelial lesions) showed that 7.8% of all hc2-positive results in this population were false-positive due to the cross-reactivity of hc2 with untargeted, noncarcinogenic HPV types [32]. Some recent studies have also shown that hc2 has an additional 5% false-positive rate due to positive hr-HPV results when no HPV DNA is present in the clinical specimen based on the use of different usually highly sensitive and broad-range PCR tests [32,33]. The relatively high false-positivity rate of the current hc2 hr-HPV cocktail has important clinical consequences as the 2006 American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines state that it is 'unacceptable' for an HPV assay to test for nononcogenic HPV types if used in cervical cancer screening programs because false-positive results may result in unnecessary colposcopy procedures [34].…”