Type-specific high-risk human papillomavirus viral load as a viable triage indicator for high-grade squamous intraepithelial lesion: a nested case– control study

Purpose Currently, the associations between type-specific high-risk human papillomavirus (HR-HPV) viral loads and cervical lesions are still inconsistent. We aimed to assess the type-specific HR-HPV viral load as a risk triage indicator for development of high-grade squamous intraepithelial lesion or worse (≥HSIL). Patients and methods A total of 19,446 women who underwent primary screening for cervical cancer using Cervista® HR-HPV and cytology assays were enrolled. The viral loads of 1,396 HR-HPV-positive specimens confirmed by Cervista® assay were detected by BioPerfectus Multiplex Real-Time PCR assay. The correlation between viral loads and cervical lesions was analyzed. The optimal cutoffs of individual HR-HPV viral loads used to predict ≥HSIL were determined from the receiver operating characteristic curve. A logistic regression model was used to analyze the relationship between covariates and the probability of ≥HSIL. Results The viral loads of HPV-16, -31, -33, -52, and -58 were positively correlated with the severity of the cervical lesion, which was significantly elevated in patients with ≥HSIL, whereas those of HPV-18, -45, -56, -59, and other types were not. The optimal cutoffs of the log10-transformed viral loads for HPV-16, -31, -33, -52, and -58 in identifying ≥HSIL were 4.26, 4.46, 4.48, 4.36, and 4.26 copies per 10,000 cells, respectively. Furthermore, multivariate analysis indicated that type-specific viral loads of HPV-16, -31, -33, -52, and -58 exceeding the cutoffs could be independent risk factors for the incidence of ≥HSIL. Conclusion The BioPerfectus Multiplex Real-Time PCR viral load assay provides viable triage for ≥HSIL when using appropriate cutoff levels.

The relationship between HR-HPV viral loads and cervical lesions remains unclear. The earliest measurements of HR-HPV viral load were performed semi-quantitatively using Hybrid Capture 2 11,12 and PCR-ELISA, 13 but the data generated regarding HR-HPV viral load were inaccurate and inconsistent. The use of reverse transcription-PCR (RT-PCR) 14 for HR-HPV viral load determination has become common. Although RT-PCR can absolutely quantify the viral load of HR-HPV, the results 14 are not accurate because the viral load is not normalized relative to the viral copies per cell or to the amount of an internal control gene. In recent years, some studies [15][16][17] have normalized the absolute viral load of HPV, but studies [15][16][17][18][19] of the relationship between HR-HPV viral load and cervical lesions have focused mainly on HPV-16 and -18 without considering non-HPV-16/18 types. Most studies have reported that the HPV-16 viral load varies directly with the grade of cervical lesions. [14][15][16][17] However, some studies have suggested that a single measurement of HPV-16 viral load is not a clinically useful biomarker. 19 HPV-18 viral load measurement is also questionable as a diagnostic tool for cervical lesions. It has been reported that high HPV-18 viral loads are correlated with the severity of cervical lesions, 15 but other studies have stated that HPV-18 viral load is not significant 12,16,19 and may, in fact, be lower in women with cervical intraepithelial neoplasia grade 2/3 (CIN2/3) than in those without it. 18 Similarly, the clinical relevance of the HR-HPV viral loads of non-HPV-16/18 types is dubious and inconsistent. [20][21][22][23] Moreover, no cutoffs have been established for the viral loads of individual types of HPV to distinguish between HR-HPV-positive women with high-grade squamous intraepithelial lesion or worse (≥HSIL) and those without it.
A previous study reported 24 the BioPerfectus Multiplex Real-Time PCR (BMRT) assay, which was a newly developed, highly automated approach for quantifying viral loads for 14 HR-HPV types. This method provides an absolute quantitative measure of individual types with normalization according to the amount of human DNA. 24,25 In order to confirm that type-specific HPV viral loads correlate with the grade of cervical lesions and determine whether the typespecific HPV viral load is a viable risk identification tool for ≥HSIL in HR-HPV-positive women, the present study analyzed type-specific viral loads of 14 HR-HPV types using the BMRT assay in a large-scale clinical trial.

Patients and study design
All clinical samples in this study were organized using the Fujian Province Cervical Lesions Screening Cohorts, Fujian, P.R. China. In one of these cohorts, 19,446 women assessed using Cervista ® HR-HPV assay and the Thinprep ® Cytologic Test at the Fujian Provincial Maternity and Children's Hospital between January 2012 and December 2016 were enrolled. The population eligible for this study involved two arms, one consisting of healthy patients undergoing routine physical examination and another consisting of patients visiting the outpatient clinic for any gynecologic conditions other than cancer. Qualified participants were those for whom HPV was positively detected by the Cervista ® assay, with definite cervical histopathology results, and who had a complete follow-up record ( Figure 1). In total, 698 eligible women who were histopathologically abnormal were classified in the case group. Another 698 women who were age matched with the case group and who were histopathologically normal were placed in the control group. All participants were examined, followed, and diagnosed using standardized protocols. All participants in this study provided written informed consent. The protocol was reviewed and approved by the Ethical Committees of the Fujian Provincial Maternity and Children's Hospital.

Cervical specimen collection
Cervical cells were collected from the cervical canal of all participants with plastic brushes, stored in ThinPrep ® Pap Test PreservCyt ® Solution (Hologic Inc., Madison, WI, USA), and immediately transported to the laboratory, where they were stored at 4°C. Thinprep ® Cytologic Test, Cervista ® HR-HPV, and HPV viral load testing were performed on ThinPrep cervical cells.

DNA preparations
DNA from the cervical specimens collected in Thinprep ® Pap Test PreservCyt ® solution was extracted and purified using a Genfind DNA Extraction Kit (Hologic Inc.) according to the manufacturer's instructions. The DNA was extracted by magnetic bead adsorption. The kit purified the DNA by removing blood, inflammatory cells, necrotic debris, HPV virions, and mucus. The concentration and purity of the cervical cell DNA were determined using a Q5000 spectrophotometer (Quawell Technology, Inc., San Jose, CA, USA).

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High-risk HPV viral load as a triage indicator

Liquid-based cytology
Cytological specimens were blindly evaluated, independent of the results of the other assays, by two experienced cytopathologists. If the diagnosis was different, the cervical samples were reviewed again and a consensus diagnosis was obtained. The results were evaluated using the Bethesda system. Samples were classified as: negative for intraepithelial lesion or malignancy (NILM); atypical squamous cells of undetermined significance; low-grade squamous intraepithelial lesion (LSIL); atypical squamous cells, not possible to exclude HSIL; HSIL; squamous cervical cancer (SCC); atypical glandular cells; and adenocarcinoma (ADC) in situ.

BMRT HR-HPV viral load assay
Type-specific HPV viral load measurement and genotyping were simultaneously conducted using a BMRT assay. The BMRT assay was performed with the fluorescence-based multiplex HPV DNA genotyping kit (Bioperfectus Ltd, Jiangsu, P.R. China). Specific primers and their corresponding TaqMan probes were designed to detect each of the 14 HR-HPV genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68), and the viral loads of the 14 high-risk genotypes were simultaneously quantified. A human single-copy gene encoding DNA topoisomerase III (TOP3) was tested to calibrate the standards for absolute quantification of the viral copies in a given sample. 24 The experimental procedure was conducted according to the kit manufacturer's instructions. PCR was performed on an ABI Prism 7500 Fast Dx System (Thermo Fisher Scientific, Waltham, MA, USA). Five-point standard curves for HPV and the cellular log-phase were established for absolute quantification. The standard curves for HPV and TOP3 were Y = -3.85633(log 10 X)+36.93833 and Y = -3.34656(log 10 X)+38.51644, respectively. The number of viral copies was normalized according to the cellular input and log 10 -transformed. In cervical specimens, the distribution of HPV infection is uneven. 21 Obtained specimens of cervical cells may, therefore, be partially infected with HPV, whereas other parts may not be infected, so the calculated viral load is the average viral load of all cells entering the test. Furthermore, a previous study has indicated that the viral load per 10,000 cells is considered the applicable unit of HPV load. 24 Therefore, the normalization of HPV type-specific viral loads was performed as follows: viral load = log 10 [Cn HPV/Cn TOP3)×10,000] copies/10,000 cells, where Cn HPV is the quantity of HPV DNA and Cn TOP3 is the number of human cells. Perfectus v1.0 was used for genotyping and quantitative analysis of HPV nucleic acids (Bioperfectus Ltd).

Histology
Women who were HPV positive and/or had an abnormal cytological result were referred for colposcopy and punch biopsy. Women with a punch biopsy diagnosis ≥HSIL underwent a loop electrosurgical excision procedure cone biopsy or conization by cold knife. Specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Subsequently, 4-μm-thick histological sections were cut and stained with H&E using the standard method. Cervical biopsy specimens were then histologically examined and classified according to the CIN system. The cervical lesions were diagnosed by two professional pathologists.

Statistical analysis
The target metric was the viral load of the 14 HR-HPV types at the first visit producing a positive test result. The mean and SD of the log 10 -transformed absolute virus copy numbers per 10,000 human cells were considered the type-specific HPV viral load. The viral loads of the 14 type-specific HR-HPVs were stratified by cytological and colposcopic grade. Pearson's correlation coefficient (r) was applied to determine the association between viral load and cervical lesion severity. One-way ANOVA was performed to compare type-specific

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High-risk HPV viral load as a triage indicator viral load with cervical cytology or histology. The Scheffé method was used for multiple pairwise comparisons of all HR-HPV types. Viral loads in single and multiple infections were separately correlated with cervical lesion severity. A paired t-test was used to compare type-specific HR-HPV loads between women with and without diagnoses of ≥HSIL. A receiver operating characteristic (ROC) curve was utilized to identify the optimal cutoff value of the type-specific HR-HPV viral loads for predicting ≥HSIL. A logistic regression model was used to analyze the relationship between covariates and the probability of ≥HSIL in HR-HPV-positive women; the OR was adjusted for covariates. SPSS v17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. All tests were run at a two-sided significance level of 0.05.

Performance and cutoffs of BMRT viral load assay for the incidence of ≥HSIL in HR-HPV infections
We analyzed the type-specific HPV viral load data using ROC curve models for all HR-HPV-positive women and determined the most useful cutoff for the identification of ≥HSIL (Figure 2). The area under the ROC curve (area under the curve [AUC]) for viral load was calculated for the ≥HSIL endpoints. According to the ROC curve analysis, the 14 HR-HPV viral load had an optimal cutoff of 3.17 copies/10,000 cells (log 10 -transformed). At this cutoff level, the viral load was able to identify ≥HSIL with a sensitivity ( According to the multivariate logistic regression model analysis (Table 4), the independent risk factors for the incidence of ≥HSIL in HPV infections following log 10

Discussion
In recent years, quantitative measurements of HR-HPV load have become common. However, associations between HPV viral loads and cervical lesions are inconsistent. Furthermore, previous studies 15, 16 focused mainly on HPV-16 and -18, regardless of the absolute load of individual types. 12 This study was conducted to evaluate the correlations between the absolute viral loads of the 14 type-specific HR-HPVs, as detected by BMRT HPV viral load assay, and the degree of cervical lesions. In addition, the optimal cutoffs for typespecific HR-HPV viral loads were defined and the ability of the assay to identify ≥HSIL in HR-HPV infections was evaluated according to the cutoffs.
This study showed that the viral loads of HPV-16, -31, -33, -52, and -58, as well as of the 14 HR-HPVs linearly increased as cervical cytological severity also increased. Similarly, for both single infection and multiple infection cases, there were significantly higher viral loads for these types among women with ≥HSIL than among those with NILM/LSIL. We noticed that these five genotypes were associated with a substantially higher risk of ≥HSIL. The present study indicates that the most useful cutoff levels for the log 10 -transformed viral loads of HPV-16, -31, -33, -52, -58, and the 14 HR-HPVs were 4. 26, 4.46, 4.48, 4.36, 4.26, and 3.17 copies per 10,000 cells, respectively, and that these cutoffs provided viable triage for the risk of ≥HSIL in HR-HPV infections (all P<0.05). This finding is consistent with previous studies reporting the effect of HPV-16 viral load on the development of cervical cancer. 15,26,27 In our research, the cutoff level for the log 10 -transformed viral load of HPV-16 was 4.26 copies per 10,000 cells, which is similar to the cutoff level reported by Segondy et al. 15 Moreover, Sun et al 24 also reported that using 16,600 copies/10,000 cells (log 10 -transformed: 4.22 copies/10,000 cells) as a cutoff value can predict CIN2+. Among non-16/18 HR-HPV genotypes, special attention should be given to HPV-31 and -33 due to their high carcinogenicity 6 and to HPV-52 and -58 due to their high prevalence in Asian women. 28 However, earlier studies have seldom or inconsistently reported correlations between HPV-31, -33, -52, and -58 viral loads and cervical lesions. 21,[29][30][31][32] Moreover, HPV-16, -31, -33, -52, and -58 are assigned to species 9, consistent with the ALTS trial, 21 and we found that the elevated risk of ≥HSIL for high viral loads appeared to be specific to alpha-9-associated types. In addition, because the HPV-16 viral load predominated, estimates of the viral load of the combined 14 HR-HPVs showed similar trends to those for HPV-16 in terms of cervical cancer disease progression.
In contrast, the viral loads of HPV-18, -45, -56, -59, and other high-risk types did not correlate with cervical lesion severity and were of limited clinical utility in this study. High

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High-risk HPV viral load as a triage indicator viral loads of HPV-18, -45, -56, and -59 were not effective at detecting ≥HSIL in HR-HPV-positive women. The data indicated that the viral loads of HPV-56 and -59 are relatively high in ≥HSIL cases with both single and multiple infections, but not significantly so. In contrast, the viral loads of HPV-18 and -45 in ≥HSIL cases were lower than those in NILM/LSIL cases. This result corroborates a previous study indicating that HPV-18 DNA loads were lower in women with HSIL than in those without. 18 This finding indicates that cervical lesions induced by HPV-18, -45, -56, and -59 and other high-risk types are more likely to be associated with increased HPV transcript levels rather than with viral copies 33 and with more integration rather than episomal status in invasive cancer. 34 Additionally, HPV-18, -45, and -59 as representative types of the alpha-7 species were found to be strongly associated with ADC. 35 The glandular epithelium of ADC does not support productive HPV infection and may limit intracellular viral replication; 36 therefore, the low viral loads in these types of malignancy result in difficulty in detecting HPV and underestimations of the association of HPV-18, -45, and -59 loads with glandular lesions. The HPV life cycle strictly follows the differentiation program of cellular DNA machinery. The HPV viral load, reflecting cytological changes in exfoliated cells, is closely correlated with the number of infected cells and the viral copies in these cells. 37 Therefore, a possible mechanism underlying the association between high viral loads and incremental incidence of cervical neoplasia is an elevated likelihood of cells with transforming infections and viral integration into the cellular genome, concurrent with an increased number of infected cells and/or increased number of viral copies in individual cells. 38 Cervical lesions often start from one or more cells and then develop by clonal expansion. The association between ≥HSIL and HPV-16, -31, -33, -52, and -58 viral loads may be explained by the possibility that the number of cells with transforming infections increases with the number of viral copies per cell and/or the number of infected cells.
The higher viral loads of HPV-16, -31, -33, -52, and -58 in cases of ≥HSIL should not be considered to be from ≥HSILs themselves, as cancer cells undergoing clonal expansion may contain fewer viral copes than proliferating cells undergoing productive viral replication. Cervical specimens were obtained by scraping maturing cells from broad areas of the cervical epithelium. Even when they are present, ≥HSILs constitute only a small proportion of the cells removed by scraping. 39 There are some limitations of this study. First, the lengths of the infections in the women participating in this study are unknown. Therefore, no conclusion regarding transient infection was drawn. Second, this study had a small number of women diagnosed with ADC. Consequently, no general conclusion could be drawn regarding the difference between control and ADC cases in terms of viral loads. Further studies are needed to confirm our findings and bridge these information gaps. Third, we did not assess the correlation between infection type (episomal, integrated, mixed) and viral load. Evaluation of this relationship will give us a deeper understanding of the biological significance of viral loads. This is the direction in which we plan to continue our efforts in the future. Finally, although HPV viral load testing has potential applications as an alternative to cytology in future clinical practice, the extent to which these findings can be transformed into clinical practice must be rigorously verified in the context of viral load measurement methods and variation in type-specific carcinogenicity among regions.

Conclusion
Our research demonstrates the potential value of type-specific HPV viral load assay. The viral loads of HPV-16, -31, -33, -52, and -58 were positively correlated with cervical disease progression. Optimal cutoffs of type-specific HR-HPV viral loads were established to screen for ≥HSIL. The type-specific viral load assay provides a viable triage tool for ≥HSIL in HR-HPV infections.