Clinical Impact of the Analytical Specificity of the Hybrid Capture 2 Test: Data from the New Technologies for Cervical Cancer (NTCC) Study

Abstract

The Hybrid Capture 2 (HC2) test targets 13 human papillomavirus (HPV) types. Here, cross-reactivity with non-HC2-targeted HPV types is described. We aimed to define the proportion of HC2-positive women who had negative results with HC2-targeted HPV types and estimate its determinants and impact on women's health management. The New Technologies for Cervical Cancer (NTCC) trial was followed in two predetermined phases. Women in the experimental arm were tested for the presence of HPV DNA by HC2 following a sample collection in PreservCyt (first phase) or Digene specimen transport medium (STM) (second phase). HPV genotyping was performed on DNA samples from HC2-positive women by PCR with GP5 ؉ /GP6 ؉ primers and reverse line blot (RLB) hybridization. Untyped samples were submitted to direct sequencing or restriction fragment length polymorphism. Multivariate logistic regression analysis estimated the adjusted odds ratios ( C ervical screening has the main purpose of decreasing the burden of cervical cancer by detecting and treating highgrade cervical intraepithelial neoplasia (CIN). Highly sensitive and specific tests have been established to identify the human papillomavirus (HPV) infections that are associated with detectable CIN. Double-testing studies (1) and randomized controlled trials (RCTs) (2-6) have highlighted Hybrid Capture 2 (HC2) as a highly sensitive test (Ͼ95%) (7) for detecting highgrade CIN. Conversely, the HC2 test showed lower clinical specificity than did cytology (90%) (7). Most of the false positives that make the test specificity so low are due to women who are actually infected by a high-risk HPV type but who have not developed high-grade lesions and, in most cases, will never develop cervical lesions (8). To date, this drawback has been overcome by the introduction of triage procedures that limit the referrals of HPV-positive women for unnecessary diagnostic procedures.It has been claimed that HC2 also has analytical false-positive results. HC2 includes a cocktail of probes designed to detect 13 HPV types, which are classified by the International Agency for Research on Cancer (IARC) (9) as carcinogenic with sufficient evidence (group 1), and HPV-68, which is classified as probably carcinogenic (group 2A). Some studies have shown cross-reactivity with other HPV types that are phylogenetically related to the targeted genotypes (10)(11)(12)(13)(14)(15)(16)(17)(18)(19).Increasing analytical false positives were observed with decreasing viral load, as measured by the ratio between the relative light units (RLU) of the specimen and the RLU of a positive cutoff (PC) consisting of 1 pg/ml of HPV DNA (RLU/PC ratio) (11,15,(20)(21)(22). In addition, lower reproducibility of the HC2 results between laboratories (23) and lower agreement between HC2 and PCR with the MY09 to MY11 primers when followed by dot blot hybridization (19) was reported with samples collected in PreservCyt (which is used for liquid-based cytology) than in those collected in the Digene standard transport medium (STM); this su...

Human papilloma virus genotyping for the cross‐sectional and longitudinal probability of developing cervical intraepithelial neoplasia grade 2 or more

“…Although GP5+/GP6+ PCR could in principle detect >40 HPV types, in this analysis, HPV genotyping was restricted to the 13 high‐risk HPV types targeted by HC2. In addition, although other methods were applied in samples where no HC2‐targeted HPV types were identified,20, 21 their results are not considered in the present analysis. Typing results were not used for women's management.…”

Cervical cancer screening by high risk HPV testing in routine practice: results at one year recall of high risk HPV-positive and cytology-negative women

“…52) would increase the specificity of the HPV test, and decrease the referral rate to colposcopy. In most crosssectional studies 5,6,16,[25][26][27][28] HPV infections characterized by 1.00-1.99 RLU/PC ratios appeared rarely/never associated with high-grade lesions. Our data suggest that some CIN2þ may be detected at baseline, or develop during follow-up (4/234 and 4/42, respectively, in our experience), in women who test HC2-positive at 1.00-1.99 ratio.…”

The Age Distribution of Type-Specific High-Risk Human Papillomavirus Incidence in Two Population-Based Screening Trials

“…Other laboratory test conducted in the context of the trials were described previously (3,(7)(8)(9).…”