High-risk Human Papillomavirus Viral Load Research Articles

Molecular triaging options for women testing HPV positive with self-collected samples.

We review developments in molecular triaging options for women who test positive for high-risk human papillomavirus (hrHPV) on self-collected samples in the context of cervical cancer elimination. The World Health Organization (WHO) recommends hrHPV screening as the primary test for cervical screening due to its high sensitivity compared to other screening tests. However, when hrHPV testing is used alone for treatment decisions, a proportion of women of childbearing age receive unnecessary treatments. This provides the incentive to optimize screening regimes to minimize the risk of overtreatment in women of reproductive age. Molecular biomarkers can potentially enhance the accuracy and efficiency of screening and triage. HrHPV testing is currently the only screening test that allows triage with molecular methods using the same sample. Additionally, offering self-collected hrHPV tests to women has been reported to increase screening coverage. This creates an opportunity to focus health resources on linking screen-positive women to diagnosis and treatment. Adding an additional test to the screening algorithm (a triage test) may improve the test's positive predictive value (PPV) and offer a better balance of benefits and risks for women. Conventional triage methods like cytology and visual inspection with acetic acid (VIA) cannot be performed on self-collected samples and require additional clinic visits and subjective interpretations. Molecular triaging using methods like partial and extended genotyping, methylation tests, detection of E6/E7 proteins, and hrHPV viral load in the same sample as the hrHPV test may improve the prediction of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and invasive cancer, offering more precise, efficient, and cost-effective screening regimes. More research is needed to determine if self-collected samples are effective and cost-efficient for diverse populations and in comparison to other triage methods. The implementation of molecular triaging could improve screening accuracy and reduce the need for multiple clinical visits. These important factors play a crucial role in achieving the global goal of eliminating cervical cancer as a public health problem.

The features of high-risk human papillomavirus infection in different female genital sites and impacts on HPV-based cervical cancer screening

BackgroundThe causal role of high-risk Human papillomavirus (HR-HPV) in the pathogenesis of anogenital cancers is well established. In contrast, information on HR-HPV distribution of continuous anatomic sites within the female genital tract is limited, and the impact of sample type on the clinical performance in HPV-based cervical cancer screening warrants investigation.MethodsA total of 2,646 Chinese women were enrolled in the study from May 2006 to April 2007. We analyzed the infection features by infection status and pathological diagnoses of 489 women with complete HR-HPV type and viral load data on the cervix, upper vagina, lower vagina, and perineum samples. Additionally, we assessed the clinical performance for detecting high-grade cervical intraepithelial neoplasia of grade two or worse (≥ CIN2) among these four types of samples.ResultsHR-HPV positivity rate was lower in the cervix (51.53%) and perineum (55.83%), higher in the upper (65.64%) and lower vagina (64.42%), and increased with the severity of cervical histological lesions (all P<0.001). Single infection was more dominant than multiple infections at each anatomic site of the female genital tract. The proportion of single HR-HPV infection decreased successively from the cervix (67.05%) to the perineum (50.00%) (Ptrend=0.019) in cervical intraepithelial neoplasia grade 1 (CIN1) and was higher in samples of the cervix (85.11%) and perineum (72.34%) in ≥ CIN2. In addition, the highest viral load was observed in the cervix compared to the other three sites. The overall agreement of the cervical and perineum samples was 79.35% and increased continuously from normal (76.55%) to ≥ CIN2 (91.49%). As for the detection of ≥ CIN2, the sensitivity was 100.00%, 97.87%, 95.74%, and 91.49% for the cervix, upper vagina, lower vagina, and perineum samples, respectively.ConclusionsSingle HR-HPV infection predominated throughout the female genital tract, but the viral load was lower compared to multiple HR-HPV infections. Despite the decreasing viral load from cervix to perineum, the clinical performance for detecting ≥ CIN2 of the perineum sample was comparable to that of the cervix.

High-risk human papillomavirus genotype distribution among women living with HIV; implication for cervical cancer prevention in a resource limited setting

BackgroundFor women living with HIV (WLHIV), the burden of persistent HPV infection, cervical pre-cancerous lesions and cancer have been demonstrated to be higher than among HIV-negative women. As Ghana and other lower-middle-income countries (LMIC) work toward developing national cervical cancer programmes, it is essential that local scientific evidence be provided to guide policy decisions, especially for such special populations. The objective of this study was to determine the distribution of high-risk HPV genotype and related factors among WLHIV and its implication for the prevention of cervical cancer prevention efforts.MethodsA cross-sectional study was conducted at the Cape Coast Teaching Hospital in Ghana. WLHIV, aged 25–65 years, who met the eligibility criteria were recruited through a simple random sampling method. An interviewer-administered questionnaire was used to gather socio-demographic, behavioural, clinical and other pertinent information. The AmpFire HPV detection system (Atila BioSystem, Mointain View, CA was used to detect 15 high-risk HPV genotypes from self-collected cervico-vaginal samples. The data collected were exported to STATA 16.0 for statistical analysis.ResultsIn all, 330 study participants, with mean age of 47.2 years (SD ± 10.7), were involved. Most (69.1%, n = 188/272) had HIV viral loads < 1000 copies/ml and 41.2% (n = 136) had ever heard of cervical screening. The overall hr-HPV prevalence was 42.7% (n = 141, 95% CI 37.4–48.1) and the five commonest hr-HPV types among screen positives were HPV59 (50.4%), HPV18 (30.5%), HPV35 (26.2%), HPV58 (17%) and HPV45 (14.9%). Most infected women (60.3%, n = 85) had multiple hr-HPV infections, with about 57.4% (n = 81) having 2–5 h-HPV types, while 2.8% (n = 4) had more than five hr-HPV types. A total of 37.6% (n = 53) had HPV16 and/or18, while 66.0% (n = 93) had the hr-HPV genotypes covered by the nonavalent vaccine. Women with HIV viral load ≥ 1000copies/ml (AOR = 5.58, 95% CI 2.89–10.78, p < 0.001) had a higher likelihood of being co-infected.ConclusionThis study found out that the prevalence of hr-HPV still remains high in women with HIV, with a notable occurrence of multiple infections and infection with genotypes 16 and/or18. Additionally, an association was established between hr-HPV and infection HIV viral load.. Therefore, comprehensive HIV care for these women should include awareness of cervical cancer, consideration of vaccination and implementation of screening and follow-up protocols. National programmes in LMIC, such as Ghana, should consider using HPV-based screen-triage-treat approach with partial genotyping.

Positive Linear Relationship between Nucleophosmin Protein Expression and the Viral Load in HPV-Associated Oropharyngeal Squamous Cell Carcinoma: A Possible Tool for Stratification of Patients

Most oropharyngeal squamous cell carcinomas (OPSCCs) are human papillomavirus (HPV)-associated, high-risk (HR) cancers that show a better response to chemoradiotherapy and are associated with improved survival. Nucleophosmin (NPM, also called NPM1/B23) is a nucleolar phosphoprotein that plays different roles within the cell, such as ribosomal synthesis, cell cycle regulation, DNA damage repair and centrosome duplication. NPM is also known as an activator of inflammatory pathways. An increase in NPM expression has been observed in vitro in E6/E7 overexpressing cells and is involved in HPV assembly. In this retrospective study, we investigated the relationship between the immunohistochemical (IHC) expression of NPM and HR-HPV viral load, assayed by RNAScope in situ hybridization (ISH), in ten patients with histologically confirmed p16-positive OPSCC. Our findings show that there is a positive correlation between NPM expression and HR-HPV mRNA (Rs = 0.70, p = 0.03), and a linear regression (r2 = 0.55; p = 0.01). These data support the hypothesis that NPM IHC, together with HPV RNAScope, could be used as a predictor of transcriptionally active HPV presence and tumor progression, which is useful for therapy decisions. This study includes a small cohort of patients and, cannot report conclusive findings. Further studies with large series of patients are needed to support our hypothesis.

Changes in the Cervical Microbiota of Women with Different High-Risk Human Papillomavirus Loads

The cervical microbiota is essential in female sexual health, and its altered states seem to have a central role in the dynamic of high-risk papillomavirus (hrHPV) infections. This study aimed to evaluate the variation in bacterial communities' compositions according to hrHPV. We collected two samples per woman, with a difference of 12 ± 1 months between them, and performed a follow-up on 66 of these women. The viral load (VL) of the hrHPV was estimated by quantitative PCR (qPCR), then it was normalized (using the HMBS gene as reference) and transformed to the Log10 scale to facilitate the interpretation. The VL was categorized as Negative, without hrHPV copies; Low, less than 100 hrHPV copies; Medium, between 100 to 102 hrHPV copies; and High, >102 hrHPV copies. The microbiota composition was described through the Illumina Novaseq PE250 platform. The diversity analyses revealed changes regarding the hrHPV VL, where women with low VL (<100 hrHPV copies) presented high diversity. The community state type (CST) IV was the most common. However, in women with high VL, a lower association with Lactobacillus depletion was found. Lactobacillus gallinarum and L. iners were the most abundant species in women with high VL, whereas women with low VL had a 6.06 greater probability of exhibiting Lactobacillus dominance. We identified conspicuous differences in the abundance of 78 bacterial genera between women with low and high VL, where 26 were depleted (e.g., Gardnerella) and 52 increased (e.g., Mycoplasma). A multilevel mixed-effects linear regression showed changes in the diversity due to the interaction between the measurement time and the VL, with a decrease in diversity in the second follow-up in women with low VL (Coeff. = 0.47), whereas the women with medium VL displayed an increase in diversity (Coeff. = 0.58). Here, we report for the first time that the cervical microbiota is influenced by the number of copies of hrHPV, where a decrease in the abundance of Lactobacillus, greater diversity, and enrichment of bacterial taxa is relevant in women with low VL.

Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNat® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs.

Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt® (HOLOGIC®, USA) medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries. This pilot study aimed to evaluate the analytical performance of eNat ® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 (Seegene, Korea), Papilloplex® High Risk HPV (GeneFirst, UK), and HPV OncoPredict (Hiantis, Italy). 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 μL using Microlab Nimbus platform (Seegene, Korea) and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman. hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667 <k <0.796). Moreover, the discordances between vaginal self-samples collected from the same woman were found only in cases of normal cytology or low-grade cytological lesions and were generally related to low hrHPV viral loads as indicated by the quantitative HPV OncoPredict assay (6.24E+02 copies/10,000 cells). The study's preliminary findings demonstrated a very good agreement between cervical and vaginal self-collected samples suspended in ThinPrep®PreservCyt® and eNat®, suggesting that the latter could represent a good alternative medium in HPV screening programs based on self-collection.

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

ObjectiveTo 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.MethodsWe 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.ResultsA 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%.ConclusionHPV 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.

Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNat® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs

Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt® (HOLOGIC®, USA) medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries. This pilot study aimed to evaluate the analytical performance of eNat® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 (Seegene, Korea), Papilloplex® High Risk HPV (GeneFirst, UK), and HPV OncoPredict (Hiantis, Italy). 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 μL using Microlab Nimbus platform (Seegene, Korea) and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman. hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667&lt;k&lt;0.796). Moreover, the discordances between vaginal self-samples collected from the same woman were found only in cases of normal cytology or low-grade cytological lesions and were generally related to low hrHPV viral loads as indicated by the quantitative HPV OncoPredict assay (6.24E+02 copies/10,000 cells). This study demonstrated a very good agreement between cervical and vaginal self-collected samples suspended in ThinPrep®PreservCyt® and eNat®, suggesting that the latter could represent a good alternative medium in HPV screening programs based on self-collection.

Clinical Performance of the BD Onclarity Extended Genotyping Assay for the Management of Women Positive for Human Papillomavirus in Cervical Cancer Screening.

Among women whose cervical specimens tested positive for high-risk human papillomaviruses (hrHPV) via the Hybrid Capture 2 assay in the Canadian Cervical Cancer Screening Trial (CCCaST), we assessed hrHPV genotype concordance between BD Onclarity HPV Assay and Roche's Linear Array, overall and stratified by hrHPV viral load. We also evaluated the performance of cytology, cytology combined with hrHPV genotyping (Onclarity assay) for HPV16/18 and non-HPV16/18 types, and hrHPV genotyping triage strategies for the detection of cervical intraepithelial neoplasia grade 2 or 3 and worse (CIN2+/CIN3+). Standard measures (expected agreement, agreement, and κ values) were used to compare Onclarity to the reference test, Linear Array. Twenty-four triage strategies were evaluated by calculating their sensitivities, specificities, and positive and negative predictive values for CIN2+ and CIN3+ detection. Among 734 hrHPV+ samples tested, there was near perfect concordance irrespective of viral load between the Onclarity and Linear Array assays for the individual genotypes [human papillomaviruses (HPV) 16, 18, 31, 45, 51, 52] by Onclarity (κ values ranged from 0.92-0.98). Strategies with adequate specificity (>75%) and the highest sensitivities to detect CIN3+ among 617 women positive for hrHPV, were positivity to HPV16 and/or 31 (Sensitivity: 65.2%, Specificity: 76.9%) and HPV16 and/or 18 (Sensitivity: 58.7%, Specificity: 81.6%). While confirming the importance of HPV16, we found that HPV31 was comparable with HPV18 for the detection of CIN2/3+ in the triage of women positive for hrHPV. HPV31 may be an important genotype in the triage of women positive for hrHPV.

Significance of the viral load of high-risk HPV in the diagnosis and prediction of cervical lesions: a retrospective study

BackgroundThe significance of HPV viral load in the detection of cervical lesions is still controversial. This study analyzed the correlation between the high-risk HPV viral load and different cervical lesion degrees.MethodsThis retrospective study included women positive for high-risk HPV DNA and screened for cervical lesions between 01/2015 and 06/2018. The high-risk HPV DNA load was measured by the second-generation Hybrid Capture technology and classified as low, moderate, and high. Colposcopy and biopsy were performed in all patients. The patients were grouped as normal, cervical intraepithelial neoplasia (CIN) grade 1, CIN grade 2, CIN grade 3, and cervical cancer. Multivariable logistic regression was performed to explore the association between high-risk HPV DNA load and cervical lesions. The odds ratios (ORs) represent the odds for increasing from low to high viral load.ResultsFinally, 265 patients were grouped as normal (n = 125), CIN 1 (n = 51), CIN 2 (n = 23), CIN 3 (n = 46), and cervical cancer (n = 20). Among them, 139 (52.5%) had a low viral load, 90 (34.0) had a moderate viral load, and 36 (13.4%) had a high viral load. Taking the normal control group as a reference, a high viral load was an independent factor for CIN 1 (OR = 3.568, 95% CI: 1.164–10.941, P = 0.026), CIN 2 (OR = 6.939, 95% CI: 1.793–26.852, P = 0.005), CIN 3 (OR = 7.052, 95% CI: 2.304–21.586, P = 0.001), and cervical cancer (OR = 8.266, 95% CI: 2.120–32.233, P = 0.002).ConclusionsAmong women who underwent cervical biopsy, higher high-risk HPV viral load in cervical lesions was associated with a higher risk of high-grade cervical lesions.

Viral load and high prevalence of HR-HPV52 and 58 types in black women from rural communities

BackgroundThe high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer development, and the most common types were included in the last approved nonavalent vaccine (9vHPV). Geographical, socioeconomic and ethnic barriers in developing countries challenge primary and secondary prevention measures of cervical cancer. We aimed to determine the prevalence of HPV infection and the viral load of HR-HPV 9vHPV-related types black women resident in rural semi-isolated communities.MethodsA descriptive study was conducted with 273 cervical samples of women from rural communities of Southeastern Brazil. Viral DNA was amplified by PCR, the genotype was identified by Reverse Line Blot (RLB) and Restriction Fragment Length Polymorphism (RFLP), and real-time PCR was applied to determine the viral load.ResultsHPV frequency was 11.4% (31/273), associated with the presence of cytological abnormalities (32.3%; p < 0.001). Thirty-one distinct genotypes were detected; HR-HPV occurred in 64.5% (20/31) of the samples and the most prevalent type were HPV52 > 58, 59. Multiple infections occurred with up to nine different genotypes. The viral load of HR-HPV 9vHPV-related types was higher in lesions than in normal cytology cases (p = 0.04); “high” and “very high” viral load occurred in HSIL and LSIL, respectively (p = 0.04).ConclusionsWe highlight that despite the low HPV frequency in the black rural women population, the frequency of HR-HPV was high, particularly by the HR-HPV52 and 58 types. Moreover, the HR-HPV viral load increased according to the progression from normal to lesion, being a potential biomarker to identify those women at higher risk of developing cervical lesions in this population.

The treatment strategy of patients with positive margins after cervical cold knife conization-A 7-year retrospective study in China.

To explore treatment strategies for patients with positive margins after cervical cold knife conization (CKC) by estimating the risk of residual or recurrent CIN2 or worse (CIN2+). A retrospective study included 569 patients receiving CKC for CIN3 in Xiangya Hospital from January 2013 to December 2017. Demographic characteristics and test results were obtained before CKC, after CKC, at 6, 12, and 24months, then annually thereafter. The primary end point was residual/recurrent CIN2+ post-CKC. Fourteen (2.46%) patients had residual/recurrent CIN2+ with a median time of occurrence at 12months post-CKC. Taking the average age and hrHPV viral load tested by Hybrid Capture 2 (HC2) as thresholds, the risk of residual/recurrent CIN2+ was higher in women aged over 40years or with a baseline HC2 of 300 or more for the ratio of relative light units to positive cut-off values. Patients with positive margins were at higher risk of residual/recurrent CIN2+ (hazard ratio 3.66, 95% confidence interval 1.25-10.71), especially when endocervix was involved. A total of 536 (94.20%) patients received HPV testing within 6months after CKC. Patients with both positive HPV testing results and positive margins were at the highest risk of residual/recurrent CIN2+. Patients with positive endocervical margins are at high risk for residual/recurrent CIN2+, independent of the severity of margins. HPV testing within 6months after CKC may be a feasible triage strategy for these patients.

Diagnostic value of high-risk human papillomavirus viral load on cervical lesion assessment and ASCUS triage.

This study aims to evaluate HR‐HPV viral load in the cervical lesion assessment and its diagnostic value on the triage of ASCUS. The three‐step protocol for cervical cancer screening was carried out in 5171 patients from June 2017 to August 2019, and 1620 histopathological results were obtained. The positive rate of HR‐HPV and TCT increased with the aggravation of pathological grades of cervical lesions. The sensitivity and specificity of HR‐HPV (DH3) to detect CIN II+ were 91.91% and 84.46%, respectively. In comparison, the corresponding results of the cytology test were 80.51% and 83.12%. HPV16/18 viral load was positively correlated with the grade of cervical lesions (p < 0.001, r = 0.321). The diagnostic efficiency of AUC by applying HPV16/18 viral load was 0.682 for the diagnosis of CIN II+. The optimal HPV16/18 viral load for predicting CIN II+ was 6.80 RLU/CO (relative light units/cut‐off), with corresponding sensitivity of 48.6%, specificity of 79.7%, and Youden index of 0.283. In the ASCUS population, viral loads were statistically different in HPV16/18 and the other 12 HR‐HPV when compared cervicitis group with CIN I group and CIN II+ group (all p < 0.05). Statistical differences were detected concerning HPV16/18 viral load, contact bleeding status, and smoking status when compared cervicitis group with CIN I group and CIN II+ group (p < 0.05), with a corresponding odds ratio of 1.004, 1.533, and 5.513, respectively. Our findings suggest that HR‐HPV viral load can be regarded as a useful tool to predict the grade of cervical lesions for ASCUS triage.ClinicalTrials.gov ID: NCT03178136.

Detection of Cervical Lesions and Cancer in Air-Dried Cytologic Smears by Combined Analysis of mRNA and miRNA Expression Levels

Cervical cancer screening is based on cytologic analysis and high-risk human papillomavirus (HR-HPV) testing, each having their drawbacks. Implementation of new biomarker-based methods may improve screening accuracy. Here, the levels of 25 microRNAs (miRNAs, miRs) and 12 mRNAs involved in cervical carcinogenesis in 327 air-dried Papanicolaou-stained cervical smears from patients with cervical precancerous lesions, cancer, or without the disease were estimated by real-time PCR. Using logistic regression analysis, small-scale miRNA-based, mRNA-based, and combined molecular classifiers were built based on paired ratios of miRNA or mRNA concentrations; their ability to detect high-grade cervical lesions and cancer was then compared. Combined mRNA-miRNA classifiers manifested a better combination of sensitivity and specificity than miRNA- and mRNA-based classifiers. The best classifier, combining miR-375, miR-20, miR-96, CDKN2A, TSP4, and ECM1, predicted high-grade lesions with diagnostic sensitivity of 89.0%, specificity of 84.2%, and a receiver-operating characteristic area under the curve of 0.913. Additionally, in a subsample of the same specimens, the levels of MIR124-2 and MAL promoter methylation, HR-HPV genotypes, and viral loads were analyzed. The relative high-grade lesion risk estimated by the classifier correlated with the frequency of MAL and MIR124-2 methylation but not with the HR-HPV genotype or viral load. The results support the feasibility of cellular biomarker-based methods for cervical screening and patient management.

Analysis of influencing factors of viral load in patients with high-risk human papillomavirus

BackgroundHigh-risk human papillomavirus (HR-HPV) load is thought to be influenced by many factors, and the relationship between viral load and the degree of cervical lesion is controversial. This study explored the possible influencing factors of HR-HPV viral load in the uterine cervix.MethodsA total of 605 women who needed colposcopic evaluation for abnormal cervical screening at the Affiliated Hospital of Weifang Medical University, China, between November 2017 and September 2018 were enrolled. Cervical specimens were collected from the endo- and ectocervix separately using two different cervical brushes. The hybrid capture II test was used to measure HR-HPV load. Age, histological severity, number of viral types, and area and location of cervical lesions were recorded. The correlations between viral load and influencing factors were analysed using univariate and multivariate analyses.ResultsHR-HPV load was positively correlated with age, histological severity, multiple HPV types and area of cervical lesions (P < 0.05). Viral load with the combination of endo- and ectocervical sampling was significantly higher than simple endocervical sampling (P < 0.001). Multivariate analysis showed that age, multiple HPV types and area of cervical lesions were independent factors for HR-HPV load with a combination of endo- and ectocervical sampling (P < 0.05). However, only age and area of cervical lesions were independent factors for viral load with simple endocervical sampling (P < 0.05). No significant association was found between viral load and lesion severity in multivariate analysis (P > 0.05).ConclusionHR-HPV load is influenced by age, histological severity, multiple viral types, area of cervical lesion and sampling methods. Age and area of cervical lesions are independent factors for viral load.

Distribution of cervical lesions in high-risk HPV (hr-HPV) positive women with ASC-US: a retrospective single-center study in China

BackgroundTo investigate distributions of cervical lesions and factors associated with the severity of the cervical lesions in high-risk HPV (hr-HPV) positive women with atypical squamous cells of undetermined significance (ASC-US) cytology.MethodsClinical information of 250,000 women who underwent HPV and cytological test was collected from January 2012 to January 2019. The association between the severity of the cervical lesions and hr-HPV genotypes, hr-HPV viral load, and ages, were analyzed in hr-HPV-positive/ASC-US women.Results3459 hr-HPV-positive/ASC-US women were enrolled in this study. Overall, 43.51% of women with ASC-US had normal histological results, 34.35% had high-grade squamous intraepithelial lesion (HSIL), and 1.30% had cervical cancer. The rate of HSIL or worse (HSIL+) in women with single HPV16 infection (63.09%) was the highest, followed by HPV33 (57.50%), HPV51 (36.11%), HPV58 (36.11%), HPV52 (28.28%), HPV18 (26.37%), HPV66 (19.35%), HPV39 (18.92%), HPV53 (15.00%), and HPV56 (8.51%). Detection rate of HSIL+ in low, intermediate and high viral-load groups were 15.87% (n = 30), 34.91% (n = 74) and 40.68% (n = 214) (Cochran-Armitage Trend test χ2 = 35.03, p < 0.0001). Compared with the 51–60-year-old group (21.65%), the women in ≤ 30 (40.52%), 31–40 (39.67%), and 41–50 (34.22%) year-old groups had significantly higher risk of HSIL+. The women in ≤ 51–60 (2.68%) and > 60 (3.41%) year-old groups were at increased risk for cervical cancer, compared with the ≤ 30-year-old group (0.61%).ConclusionsASC-US women with HPV 16/18/33/51/52/58 single infection and multiple infections, as well as high HPV viral loads, have high risk of HSIL+.

Abstract 4330: HPV double capture NGS method in cervical cancer: Identification of MACROD2 gene as HPV hot spot integration site and viral load status as a prognostic factor

Abstract BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide and constitutes the second most common malignancy in women. CC exhibits differences in clinical behavior; infection by high-risk Human Papilloma Virus (HPV) remains an important initiating event in tumorigenesis and the most important risk factors for CC. As HPV integration types may be specific biomarkers for prediction of clinical outcomes, we analyzed the association between the different viral integration signatures and clinic-pathological parameters in CC patients. EXPERIMENTAL DESIGN: Patients included in this study were enrolled in the EU-funded RAIDs Network (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) prospective CC BioRAIDs study [NCT02428842]. HPV double capture method followed by NGS was used to define the different integration signatures. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed in a large series of 272 CC BioRAIDs patients. RESULTS: The distribution of HPV signatures differed from that previously described in HPV-positive anal squamous cell carcinoma (p&amp;lt;0.001). We identified more than 250 HPV insertions sites (inter-genic or intra-genic) and reported MACROD2 gene as the most frequent integration site followed by the MIPOL1/TTC6, TP63, ERBB2, KLF12. The seven patients with HPV integration in the MACROD2 gene tend to have a worse progression free survival (PFS) compared to other patients. HPV integration signatures were not associated with treatment type, histological subtype or FIGO staging, but were associated with PIK3CA mutational status (p=0.023) and HPV genotype status (p&amp;lt;0.0001). HPV45 and HPV18 genotypes were always integrated in our patients' population, as compared to other HPV genotypes. There was no significant association between the different HPV integration signatures and PFS. High viral load was associated with longer PFS (p=0.011). CONCLUSION: This is the first study assessing the prognostic value of HPV integration in a prospectively annotated CC patient cohort, which reports a hot-spot of HPV integrations at the MACROD2 gene, known to be implicated in impaired PARP1 activity and chromosome instability. Citation Format: Maud Kamal, Sonia Lameiras, Adeline Morel, Charlotte Lecerf, Sophie Vacher, Celia Dupain, Emmanuelle Jeannot, Marc Deloger, Nicolas Servant, Elodie Girard, Sylvain Baulande, Gemma Kenter, Ekaterina Jordanova, Els Berns, Roman Rouzier, Wulfran Cacheux, Christophe Le Tourneau, Alain Nicolas, Suzy Scholl, Ivan Bieche. HPV double capture NGS method in cervical cancer: Identification of MACROD2 gene as HPV hot spot integration site and viral load status as a prognostic factor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4330.

HR-HPV viral load quality detection provide more accurate prediction for residual lesions after treatment: a prospective cohort study in patients with high-grade squamous lesions or worse.

The relationship between high-risk-human-papillomavirus (HR-HPV) viral loads and residual/recurrence lesion is uncertain. This study aimed to evaluate the clinical value of HR-HPV viral loads to predict the residual/recurrence lesions among women with high-grade squamous lesions or worse (≥ HSIL) after surgery. Finally, 301 women who underwent primary screening of cervical cancer using polymerase-chain-reaction-(PCR)-reverse-dot-blot-(RDB) human papillomavirus (HPV) genotyping and cytology assays were enrolled. They received surgery and took HR-HPV viral loads with a BioPerfectus Multiplex Real-Time PCR assay. Colposcopy biopsies were performed in patients with HPV-16/18(+) and/or TCT ≥ ASCUS with HR-HPV(+). The risk of HR-HPV viral loads and potentials factors for residual/recurrence lesions were analyzed and the optimal cut-off values of HR-HPV viral loads were calculated. The significant differences were found in residual/recurrence lesions among patients with different ages, margin status, cytology and HR-HPV at 6months (all P < 0.05). Interestingly, HPV viral loads were observed significant differences in the group of residual lesions, not recurrence group. Furthermore, except for HPV-31/33, the viral loads of HP-16/52/58 were significant differences in residual lesions. The cut-off level of HR-HPV viral loads was 5.22 copies/10,000 cells, providing viable triage for the risk of residual lesions. Compared with different follow-up methods, the HR-HPV viral loads ≥ 5.22copies/10,000 cells (HR 3.39, 95% CI 1.57-7.35) had a higher risk for developing residual lesions. HR-HPV viral loads can be a reliable predictor of residual lesions. Furthermore, women with viral loads ≥ 5.22 copies/10,000 cells may have higher risk for residual disease and should be give a more aggressive treatment and follow-up strategy.

Increased High-Risk Human Papillomavirus Viral Load Is Associated With Immunosuppressed Microenvironment and Predicts a Worse Long-Term Survival in Cervical Cancer Patients.

To evaluate the correlation between tumor-infiltrating lymphocytes (TILs) and the viral load of high-risk human papillomavirus (HR-HPV) in cervical cancer patients. A total of 62 cervical cancer patients were recruited during 1993-1994 and assigned into four groups treated with radiotherapy alone or radiotherapy combined with chemotherapy and/or thermotherapy. Ki67+ tumor cells, CD4+, CD8+, FoxP3+, OX40+ and granzyme B+ TILs were detected by immunohistochemistry. The viral load of HR-HPV in biopsy tissues before therapy was detected by in situ hybridization. The patients with high HPV viral load showed a significantly lower 15-year survival rate and an advanced International Federation of Gynecology and Obstetrics (FIGO) stage and increased recurrence rate. The distribution of Ki67+ tumor cells, FoxP3+ TILs, and CD8+/FoxP3+ ratio was obviously different between low and high HPV viral load groups. A worse clinical outcome was also implicated with increased HPV viral load tested by Cox regression analysis. Patients with increased HR-HPV viral load tend to be resistant to therapy with decreased immune surveillance in the immune microenvironment. Thus, HR-HPV viral load would influence the local immune microenvironment, and then further affect the survival of cervical cancer patients.

HPV viral load in self-collected vaginal fluid samples as predictor for presence of cervical intraepithelial neoplasia

ObjectiveThis study was performed to evaluate the use of high-risk HPV (hrHPV) viral load in screening tests for cervical cancer to predict persistent infection and presence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+).MethodsWe followed women between 30 and 60 years of age who performed self-sampling of vaginal fluid and subsequently a hrHPV test. Women who were hrHPV positive in their screening test repeated the hrHPV test 3–6 months later and were included in the present study.ResultsOur results show that women with a persistent HPV16 infection had higher HPV viral load in their primary screening test than women with transient infections (p = 5.33e-03). This was also true for sum of viral load for all hrHPV types in the primary screening test (p = 3.88e-07). 48% of women with persistent HPV16 infection and CIN2+ had an increase in HPV16 titer in the follow-up test, as compared to only 20% of women with persistent infection but without CIN2+ lesions. For the sum of all hrHPV types, 41% of women with persistent infection and CIN2+ had an increase in titer as compared to 26% of women without CIN2 + .ConclusionsThe results show that hrHPV viral load in the primary screening HPV test is associated with the presence of CIN2+ and could be used in triaging hrHPV positive women for different follow-up strategies or recall times. Serial testing of hrHPV viral load has the potential to distinguish women with CIN2+ lesions from women with persistent infection but without CIN2+ lesions.