Verification of the association of the cycle threshold (Ct) values from HPV testing on Cobas4800 with the histologic grades of cervical lesions using data from two population-based cervical cancer screening trials
Authors: Zhang Yi , Du Hui , Xiao Aimin , Zhang Wei , Wang Chun , Huang Xia , Qu Xinfeng , Wang Jianliu , Wu Ruifang 
Published: June 2022

Abstract
Objective
To verify the association of high-risk human papillomavirus (hrHPV) viral load reflected by cycle threshold (Ct) values from HPV testing on Cobas4800 assay with the histologic grades of cervical lesions via analysis on the combined data from two cervical cancer screening trials and to explore the referability of Ct values in management of the abnormalities from cervical cancer primary screening.

Methods
We analyzed the data from Chinese Multi-Center Screening Trial (CHMUST) and BUJI Cervical Cancer Screening Study Project (BUJI Study). All data to be analyzed in this paper were related to provider-collected samples. One-way ANOVA was performed to compare the Ct values among different histological groups, and Kendall’s tau-b correlation was applied to examine the association between Ct values and cervical lesion grades. The stepwise incidence of CIN2+ and CIN3+ in every 100 HPV positive individuals were calculated according to the descending of the genotype specific Ct values. The highest Ct values related to CIN3+ incidence rate 4% (or 25%) were used as the cutoffs to distinguish low-Ct value cases from the high-Ct value ones.

Results
A total of 1376 women in CHUMUST and BUJI Study who were aged 30–59 and positive of hrHPV for provider-collected samples on Cobas4800 with complete data in terms of the relevant Ct values (CtV) and the histological diagnosis were included for analysis. Our data showed significant difference among different histological grades of cervical lesions in the CtV of hrHPV, HPV16-plus (positive of HPV16 only or HPV16 plus 18 and/or pooled 12-HPV), and pooled 12-HPV (P < 0.05). No significant difference was found among different grades of lesions in term of correlated CtV of HPV18-plus (positive of HPV18 only or HPV18 plus pooled 12-HPV) (P > 0.05). The CIN2+ or CIN3+ incidence per 100 positives significantly increased corresponding to the descending of the CtV of hrHPV, HPV16-plus and pooled 12-HPV. Compared with high-CtV groups (CtV > 33.2 for hrHPV, CtV > 29.6 for pooled 12-HPV), the relevant risks (RRs) of CIN2+ for hrHPV and pooled 12-HPV positive groups with low-CtV (CtV ≤ 33.2 and ≤ 29.6, respectively) were 3.2 (95%CI 2.18–4.80) and 2.3 (95%CI 1.50–3.45). Similarly, the RRs of CIN3+ for hrHPV and pooled 12-HPV positive groups with low-CtV were 6.5 (95%CI 2.83–14.80) and 2.7 (95%CI 1.15–6.39), respectively. The RRs of CIN2+ for medium- (30.3 < CtV ≤ 37.4) and low- (≤ 30.3) CtV HPV16-plus positives were 5.1 (95%CI 0.68–38.38) and 20.6 (95%CI 2.96–143.92) related to high-CtV (> 37.4) groups, and the CIN3+ incidence in low-CtV value group was nine-fold higher of that in medium-CtV ones [RRs, 9.0 (95%CI 2.89–28.10)]. In comparing with the algorithms of “HPV16-plus/18-plus + cytology ≥ ASCUS for pooled 12-HPV”, triage algorithm “HPV16-plus/18-plus + Ct value ≤ 33.2 for pooled 12-HPV” could achieve a comparable sensitivity of 93.2%.

Conclusion
HPV viral loads reflected by Ct values for hrHPV, HPV16-plus and pooled 12-HPV from Cobas4800 HPV testing were directly associated with the severity of cervical lesions. A lower HPV genotype-specific Ct value prompted a significantly high CIN3+ risk of 4% or higher in women positive of hrHPV, HPV16-plus or pooled 12-HPV, indicating that HPV viral load reflected by Ct values on Cobas4800 may be a promising risk indicator in management of abnormalities from primary cervical cancer screening.