HPV Viral Load Research Articles

Human papillomavirus type 16 and 18 viral loads as predictors associated with abnormal cervical cytology among women in Saudi Arabia

The detection of HPV viral DNA is regularly conducted with cervical screening. However, using a molecular marker such as the viral load may serve as a predictor associated with disease detection and progression. The present study aimed to screen for and genotype HPV among women in Saudi Arabia, develop and validate sensitive quantitative polymerase chain reaction (qPCR) assays to detect viral load for the two most common HPV types, namely 16 and 18, and assess whether HPV viral load could be used as a marker for cervical abnormality and disease progression. This study examined 733 specimens (both formalin-fixed paraffin embedded specimens and PAP smear samples) from women who underwent cervical screening. The specimens and samples were processed for DNA extraction and then tested for HPV DNA using nested PCR. Approximately 165 specimens (18%) were positive for HPV. Those specimens were genotyped using a reverse line blotting hybridization assay. The results indicated that the most common HPV types detected were a single infection with HPV 16 (51%) or with HPV 18 (28%) followed by infections with multiple HPV types (~7%). A qPCR TaqMan assay developed and validated in-house was used to determine viral load for HPV genotypes 16 (n = 80) and 18 (n = 45). Viral loads for both HPV types were significantly associated with cervical cytology grade (P < 0.05). The odds ratio (OR) for the HPV 16 viral load was high for specimens with cervical cancer (OR, 18.8; 95% CI, 4.3–82.9) or for those with high-grade squamous intraepithelial lesions (OR, 14.7; 95% Cl, 2.43–88.49). For the HPV 18 viral load, the OR was significant only for specimens with cervical cancer (OR, 11.1; 95% Cl, 2.2–54.9). Logistic regression models for HPV 16 and for HPV 18 viral load levels were significant, with higher viral load associated with cervical abnormalities. These findings indicate that viral load is a predictor significantly associated with cytology abnormality in women who are positive for high-risk HPVs and suggest that integrating a viral load test into current clinical screening practices for HPV-positive women is warranted in Saudi Arabia.

Влияние нормальной микрофлоры влагалища на течение папилломавирусной инфекции

Objective. To study the influence of normal vaginal microbiota on papillomavirus (HPV) infection. Patients and methods. We examined 160 women with HPV infection. We assessed the state of normal flora, the presence of sexually transmitted infections (STIs), and the HPV elimination rate. The correlation of living, apoptotic and necrotic epithelial cells depending on the levels of lactobacilli in native and stress-influenced specimens was studied in 140 HPV-positive and 20 HPV-negative patients. Results. We have found a stronger relation between HPV viral load and deficiency of lactobacilli (r = 0.498; р = 0.021) than between the former and the presence of STIs (r = 0.121; р = 0.072). High baseline levels of vaginal lactobacilli increase the likelihood of HPV elimination. Normal levels of lactobacilli enhance the survival of noninfected epithelial cells and reduce the levels of infected ones, which ensures a predominantly apoptotic form of cell death. HPV enhances the survival of infected cells and shifts the cell death structure toward necrosis. Conclusion. Lactobacilli counteract the influence of HPV on the vital conditions of vaginal epithelial cells. This accounts for better outcomes of HPV infection in patients with normal levels of flora. Key words: apoptosis, human papillomavirus, vagina, lactobacilli, cervix, epithelial cells

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.

Quantitative Oral HPV16 and HPV18 Detection in Persons Attending Dental Clinics.

This study aimed to assess quantitative human papillomavirus (HPV) type 16 and HPV18 detection in oral rinses obtained in dental offices in Seattle, Washington. We evaluated oral rinses collected during dental visits from 2016 to 2018. Multiplex TaqMan quantitative polymerase chain reaction was used to determine HPV16 and HPV18 viral load (VL). Of 15,313 persons, 152 (1%) had detectable oral HPV16/18. Men were at higher risk of oral HPV16/18 infection than women (1.6% vs. 0.6%; odds ratio, 3.2; 95% confidence interval, 2.1-4.4). Compared with women, men with HPV16 were older (median, 55 vs. 48 years; P < 0.001) and had higher VL (39.7 vs. 1.1 copies/mL, P < 0.001). Of 39 with HPV16 at baseline and a second oral rinse, 13 remained positive at subsequent rinse; of 8 with HPV18 at baseline, 2 remained positive at subsequent rinse. Persons with consecutive positive test results were all men and had higher baseline VL compared with those with first positive and second negative samples. Oral rinse is an acceptable method of HPV testing, and persons are interested in testing. Overall HPV16/18 prevalence was low, and detection was more frequent among men than women, especially at higher copy numbers. HPV16 persistence was more common in men with high VL at baseline test. Future studies are needed to evaluate the feasibility of an effective secondary prevention strategy for oropharyngeal cancer using quantitative oral HPV detection.

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.

Combining HPV DNA load with p16/Ki-67 staining to detect cervical precancerous lesions and predict the progression of CIN1\u20132 lesions

BackgroundHuman Papilloma Virus (HPV) DNA tests are highly sensitive and can triage women with mild lesions, improving the prognosis and diagnosis of cervical lesions. However, additional efficient strategies should be developed to improve the specificity of these tests.MethodsThis study aimed to evaluate the clinical value of HPV DNA load in improving the diagnosis and prognosis of cervical lesions by p16/Ki-67 testing. Histological samples were collected from 350 women with HR-HPV genotyping and analyzed by qRT-PCR. Immunohistochemical staining was used to assess p16 and Ki-67 expression and clinical performance characteristics were calculated.ResultsOf the cases, 271 had detectable HR-HPV infection, in which HPV-16 was most prevalent (52.0%), followed by HPV-58 (22.5%). P16/Ki-67-positivity increased with histological severity but not for HR-HPV infection. Amongst the 13 HR-HPV genotypes, only HPV-16 (P = 0.016) and HPV-58 (P = 0.004) viral loads significantly correlated with lesion severity. The P16/Ki-67/HPV DNA load co-test indicated an increased sensitivity for the detection of cervical intraepithelial neoplasia (CIN) lesions compared to p16/Ki-67 staining in HPV-16 and/or 58 positive cases. Viral load did not improve the sensitivity of p16/Ki-67 co-test in non-HPV-16 or 58 positive cases. The clinical performance of the p16/Ki-67/HPV DNA load co-test was limited for the prediction of the outcome of CIN1 lesions. However, amongst the 12 HPV-16 and/or 58 positive CIN2 cases in which return visit results were obtained, the behavior of the lesions could be predicted, with a sensitivity, specificity, positive prediction rate (PPV), and negative prediction rate (NPV) of 0.667, 1, 1 and 0.5, respectively.ConclusionCombination of the assessment of HPV DNA load with the intensity of p16 and Ki-67 staining could increase the sensitivity of CIN lesion diagnosis and predict the outcome of CIN2 in patients with a HPV-16 and/or 58 infection.

Association of Human Papillomavirus Genotype 16 Viral Variant and Viral Load with Cervical High-grade Intraepithelial Lesions.

Human papillomavirus genotype 16 (HPV16) is by far the genotype most strongly associated with cervical cancer; viral variant and/or viral load of HPV16 could modulate this association. The objective was to determine the association between the viral variant and viral load of HPV16 and the presence of cervical high-grade lesions. This cross-sectional study included all women in whom HPV infection was found by cervical smear during routine gynecologic health checks. Women with single or multiple HPV16 infections (n = 176) were selected for viral variant and viral load analysis. Smear results were classified using the Bethesda system. HPV types were classified according to the International Agency for Research on Cancer. Odds ratios (OR) with their 95% confidence intervals (CI) were estimated by logistic regression, adjusted for age, immigrant status, and coinfection with other high-risk genotypes. No statistically significant associations were found regarding the detected viral variants. A viral load above the median (>1,367.79 copies/cell) was associated with a significant risk of high-grade epithelial lesion or carcinoma, after adjusting for age, immigrant status, coinfections, and viral variant: (adjusted OR 7.89; 95% CI: 2.75-22.68). This relationship showed a statistically significant dose-response pattern after categorizing by viral load tertiles: adjusted OR for a viral load greater than the third tertile was 17.23 (95% CI: 4.20-70.65), with adjusted linear P trend = 0.001. In patients infected with HPV16, viral load is associated with high-grade intraepithelial lesions or cervical carcinoma. This could be useful as prognostic biomarker of neoplastic progression and as screening for cervical cancer.

HPV infections and flat penile lesions of the penis in men who have sex with men

BackgroundFlat penile lesions (FPL) in heterosexual men are thought to play a role in the transmission of HPV. We investigated the association between FPL and penile HPV, and explored determinants of FPL in men who have sex with men (MSM). MethodsIn 2015–2016, MSM were recruited based on HIV and penile HPV status in a previous cohort. MSM self-completed a questionnaire. Peniscopy was performed after application of acetic acid to visualize FPL. Penile physician-collected samples were tested for HPV-DNA using the highly sensitive SPF10-PCR DEIA/LiPA25 system. HPV viral load (VL) was determined using a quantitative type-specific (q)PCR targeting the L1-region. Presence of HPV and HIV, HPV VL and circumcision status were compared between MSM with and without FPL. ResultsWe included 116 MSM, of whom 59/116 (51%) MSM were HIV-positive and 54/116 (47%) had FPL. A penile HPV infection was present in 31/54 (57%) MSM with FPL and 34/62 (55%) MSM without FPL (p = 0.8). There was no difference between MSM with and without FPL regarding presence of penile HPV infection, HPV VL, HIV status or circumcision status (p > 0.05 for all). ConclusionAmong MSM in Amsterdam, we found no association between FPL and penile HPV, HPV VL, HIV status or circumcision status.

HPV detection and genotyping of head and neck cancer biopsies by molecular testing with regard to the new oropharyngeal squamous cell carcinoma classification based on HPV status

Recently, both the World Health Organization/International Agency for Research on Cancer (WHO/IARC) and the American Joint Committee on Cancer (AJCC) have classified oropharyngeal squamous cell carcinoma (OPSCC) on the basis of HPV status. For this purpose, the WHO/IARC recommended direct molecular HPV testing. In practice, formalin-fixed, paraffin-embedded (FFPE) biopsy specimens are frequently the only available samples. We herein compared in parallel two commercially available molecular assays that were first designed for cervical HPV detection and genotyping: Inno-Lipa HPV Genotyping Extra II (IL) and Anyplex II HPV28 (AP28). A total of 55 samples were tested. By IL assay, chosen as reference assay, 27 (49.1%) biopsies were positive for HPV16, 10 (18.2%) were positive for HPV but negative for HPV16, and 18 (32.7%) were negative for HPV. A valid result with AP28 was obtained for 51 biopsy samples (92.7%). Among 37 HPV positive samples by IL, 33 (89.2%) were positive by AP28. The agreement between both assays was good (Cohen's κ= 0.78). Among the six discrepancies between assays, always associated with low HPV16 viral load, four biopsies positive for HPV16 by IL could not be detected by AP28. Taken together, these observations demonstrate that both assays could be used in routine HPV detection and genotyping on FFPE biopsy samples of head and neck tumours.

Unlocking the Potential of Saliva-Based Test to Detect HPV-16-Driven Oropharyngeal Cancer.

The incidence of human papillomavirus (HPV)-positive oropharyngeal cancer (OPC) is rising in high-income countries, including Australia. Increasing evidence suggests that accurate HPV testing is pivotal for clinical decision making and treatment planning in these patients. Recently, the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor–node–metastasis (TNM) staging system for OPC (based on the p16INK4a (p16) status) was proposed and has been implemented. However, the applicability of this new staging system is still far from clear. In our study, n = 127 OPC patients from Queensland, Australia were recruited, and the tumor p16 expression in these patients was examined using immunohistochemical (IHC) analysis. HPV-16 genotyping, viral load, and physical status (episomal versus integrated) in the saliva samples of OPC patients were determined using the qPCR method. A good inter-rater agreement (k = 0.612) was found between tumor p16 expression and oral HPV-16 infection in OPC. Importantly, according to the eighth edition staging system, HPV-16 DNA viral load (>10 copies/50 ng) was significantly associated with the advanced stages of OPC. In concordance with previous studies, a mixed HPV-16 form (partially or fully integrated) was predominately found in OPC patients. Taken together, our data support HPV-16 detection in saliva as a screening biomarker to identify people within the community who are at risk of developing OPC.

PV-0533 HPV16 viral load may explain gender differences in treatment outcome of anal squamous cell carcinoma

PV-0533 HPV16 viral load may explain gender differences in treatment outcome of anal squamous cell carcinoma

Human Papillomavirus Status in Primary Lesions and Pelvic Lymph Nodes and Its Prognostic Value in Cervical Cancer Patients with Lymph Node Metastases.

BackgroundThe aim of this study was to assess the presence of HPV DNA in cervical tissues and lymph nodes in patients who have uterine cervical neoplasms with lymphatic metastases and who underwent surgery for invasive cervical cancer and pelvic lymphadenectomy, to establish the utility of HPV type and viral load in predicting disease progression.Material/MethodsWe retrospectively assessed 88 patients with uterine cervical neoplasms with lymph node metastases. All 88 patients were in FIGO stage IA-IIB. A total of 316 paraffin-embedded archival tissues (88 cervical samples and 228 pelvic lymph node specimens) were acquired. All the samples were analyzed using real-time PCR to determine HPV DNA presence/type and to quantify viral load.ResultsIn total, 17 HPV genotypes were detected in the cervical lesions and pelvic lymph nodes of the patients. The most common HPV type in all samples was HPV16, followed by HPV18. The existence of HPV16 DNA and low HPV16 viral load in cervical lesions were also significantly associated with disease recurrence. Furthermore, lymphovascular space involvement was also correlated with worse disease outcome.ConclusionsHPV16 DNA presence and low viral load in primary lesions can be used to predict disease recurrence. HPV DNA is a favorable prognostic indicator in patients with uterine cervical neoplasms who have lymphatic metastases.

Experimental research on the therapeutic effect of MMR vaccine to juvenile-onset recurrent respiratory papillomatosis.

To evaluate the efficacy of MMR vaccine in the treatment of juvenile-onset recurrent respiratory papillomatosis as adjuvant therapy by experimental research. Thirty-one children with RRP were enrolled and assigned randomly to intervention group or control group. Fifteen subjects in intervention group were treated with local application MMR vaccine on the lesion after surgery; sixteen subjects in the control group were treated with surgical excision alone. The quantity of virus of positive specimens was measured by fluorescence quantitative polymerase chain reaction. After treatment with MMR vaccine, viral load of intervention group was (9.56 ± 11.03) × 108 copies/ml, that of control group was (22.01 ± 17.78) × 108copies/ml, and there was significant difference between the two groups (P = 0.040). Local application MMR vaccine as adjuvant therapy can reduce HPV viral load significantly. It is suggested that the MMR vaccine may inhibit replication of HPV DNA, but the curative effect needs further confirmation.

HPV16 viral characteristics in primary, recurrent and metastatic vulvar carcinoma

Vulvar carcinoma is the fourth most common gynecological malignancy. Two separate carcinogenic pathways are suggested, where one is associated with the human papillomavirus (HPV) and HPV16 the most common genotype.The aim of this study was to evaluate HPV-markers in a set of primary tumors, metastases and recurrent lesions of vulvar squamous cell carcinomas (VSCC). Ten HPV16-positive VSCC with metastatic regional lymph nodes, distant lymphoid/hematogenous metastases or local recurrent lesions were investigated for HPV genotype, HPV16 variant, HPV16 viral load, HPV16 integration and HPV16 E2BS3 and 4 methylation.In all 10 analyzed case series, the same HPV genotype (HPV16), HPV16 variant and level of viral load were detected in all lesions within a patient case. Primary tumors with a high E2/E6 ratio were found to have fewer vulvar recurrences and/or metastases after diagnosis and treatment. Also, a significantly lower viral load was evident in regional lymph nodes compared to primary tumors.The data presented strengthens the evidence for a clonal HPV-induced pathway for vulvar carcinoma

22 - Virological and serological predictors of anal high-grade squamous intraepithelial lesions among HIV-positive men who have sex with men

Background Our objective was to identify virological and serological predictors of anal high-grade squamous intraepithelial lesions (HSIL) in HIV-positive men-who-have-sex-with-men (MSM). Methods HIV-positive MSM were recruited from a longitudinal study (2010–2013), during which anal self-swabs and serum were collected at up to five bi-annual visits. Swabs were HPV genotyped, and type-specific HPV viral load in anal swabs was determined. Serum antibodies to E6, E7, E1, E2 and L1 proteins of 7 hrHPV-types and HPV6 and 11 were analyzed. 193 participants had a high-resolution anoscopy (HRA) after the last study visit and were included in the current analysis. Anal HSIL was diagnosed by histopathological examination of anal biopsies. Causative HPV-type of anal HSIL was determined in whole tissue sections (WTS) and by laser capture micro-dissection if more than one HPV-type was found in WTS. Multivariable logistic regression was used to study whether persistent anal HPV infection, HPV viral load and seropositivity for HPV were predictors of anal HSIL, in general and for concordant causative HPV-type. Results Of 193 HIV-positive MSM, 50 (26%) were diagnosed with anal HSIL. HrHPV persistence in anal swabs was common, varying by hrHPV-type between 3–21%. Neither anal hrHPV viral load, nor seropositivity for L1, E6, E7, E1, or E2 was associated with anal HSIL. Only anal HPV persistence was independent associated with anal HSIL, in general and by concordant causative HPV-type. Conclusion Persistent HPV infection was strongly associated with anal HSIL, in general as well as for concordant HPV-type.

HPV-16 viral load in oropharyngeal squamous cell carcinoma using digital PCR

Conclusions: We did not identify any strong associations between HPV-16 viral load and any of the clinical or lifestyle factors.Objective: The epidemiology of oropharyngeal SCC is changing, with an increasing proportion of HPV-positive cases seen in the last decade. It is known that a high viral load is linked to the development of cervical cancer, the relation between viral load and oropharyngeal SCC is less clear. We sought to determine HPV-16 viral load in HPV-positive oropharyngeal SCCs using highly sensitive digital PCR and to identify clinical and lifestyle factors associated with viral load.Subjects and methods: We analysed 45 HPV-16 positive oropharyngeal SCCs diagnosed between 2013 and 2015. All patients completed a lifestyle questionnaire and clinical data were extracted from medical charts. Viral load was determined using digital PCR assays for HPV-L1 and RNAseP.Results: We found large variations in HPV-16 viral load from 1 to 930 copies per cell (median 34 copies per cell).

Role of HPV viral loads in random biopsy under normal colposcopy

Objective: To explore the role of HPV viral loads in random biopsy under normal colposcopy. Methods: 908 atypical squamous cells of undetermined significance (ASC-US) and HPV positive women, recruited in cluster sampling in 9 provinces including 5 urban areas and 9 rural areas in China from 1999 to 2008 and meeting the inclusion and exclusion criteria were included in this analysis. According to relative light units/cutoff (RLU/CO) value, subjects were stratified as low (286 cases), intermediate (311 cases) and high (311 cases) viral load groups. Risks of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) among different viral load groups were compared with linear trend Chi-square test. Results: Detection rate of CIN3+ in low, intermediate and high viral load groups were 2.1% (6 cases), 2.6% (8 cases) and 6.8% (21 cases) (Chi-square test for trend χ(2)=8.91, P=0.003) and were 60.3, 74.0 and 201.3 times higher than ASC-US and HPV negative women, respectively. Among 908 subjects, 27.0% (245 cases) were abnormal under colposcopy and 68.6% (623 cases) diagnosed as normal. Under normal colposcopy, detection rate of CIN3+ in low, intermediate and high viral load groups were 0.9% (2 cases), 0.9% (2 cases) and 3.8% (7 cases) (χ(2)=6.42, P=0.040). Conclusion: HPV viral loads display satisfactory risk stratification ability among ASC-US and HPV positive women under normal colposcopy. Women with high HPV viral loads show a significantly increased detection rate of existing CIN3+ and could be recommended to perform random biopsy for histologic diagnosis.

Diagnostic value of human papillomavirus (HPV) 16 and HPV18 viral loads for the detection of high-grade cervical intraepithelial neoplasia (CIN2+) in a cohort of African women living with HIV

BackgroundAfrican women living with HIV (WLHIV) are at high risk of cervical cancer but rarely adequately screened. Better strategies enabling identification of WLHIV with high-grade cervical intraepithelial lesions (CIN2+) are required. ObjectivesTo investigate the diagnostic value of HPV16 and HPV18 viral loads in a cohort of African WLHIV. DesignHPV16 and HPV18 viral loads were determined by quantitation of the E6 gene DNA by real-time PCR in cervical specimens collected at baseline and endline (16 months) from 245 African WLHIV positive for HPV16 or/and HPV18. Cervical biopsies were graded using the histopathological CIN classification. ResultsWomen with CIN2+ had higher viral load for HPV16 (p < 0.0001) or HPV18 (p = 0.03) than those without CIN2+. HPV16 viral load ≥3.59 log copies/1000 cells detected CIN2+ with sensitivity and specificity of 93.5% (95%CI: 81.7–98.3%) and 74.1% (95%CI: 66.3–80.6%), respectively, whereas HPV18 viral load ≥1.63 log copies/1000 cells detected CIN2+ with sensitivity and specificity of 59.1% (95%CI: 38.7–76.7%) and 66.9% (95%CI: 58.8–74.1%), respectively. A high baseline HPV16 viral load was significantly associated with persistence of, or progression to CIN2+ at endline; these findings were not observed for HPV18. ConclusionsHPV16 viral load is a powerful marker of CIN2+ in African WLHIV. HPV18 viral load is of lower diagnostic value in this population.

Detection of high-grade neoplasia in air-dried cervical PAP smears by a microRNA-based classifier.

Recent studies have shown that changes in the expression levels of certain microRNAs correlate with the degree of severity of cervical lesions. The aim of the present study was to develop a microRNA-based classifier for the detection of high-grade cervical intraepithelial neoplasia (CIN ≥2) in cytological samples from patients with different high-risk human papillomavirus (HR-HPV) viral loads. For this purpose, raw RT-qPCR data for 25 candidate microRNAs, U6 snRNA and human DNA in air-dried PAP smears from 174 women with different cervical cytological diagnoses, 144 of which were HR-HPV-positive [40 negative for intraepithelial lesion or malignancy (NILM), 34 low-grade squamous intraepithelial lesions (L-SIL), 57 high-grade squamous intraepithelial lesions (H-SIL), 43 invasive cancers], were statistically processed. The expression level changes of various individual microRNAs were found to be significantly correlated with the cytological diagnosis but the statistical significance of this correlation was critically dependent on the normalization strategy. We developed a linear classifier based on the paired ratios of 8 microRNA concentrations and cellular DNA content. The classifier determines the dimensionless coefficient (DF value), which increases with the severity of cervical lesion. The high- and low-grade CINs were better distinguished by the microRNA classifier than by the measurement of individual microRNA levels with the use of traditional normalization methods. The diagnostic sensitivity of detecting high-grade lesions (CIN ≥2) with the developed microRNA classifier was 83.4%, diagnostic specificity 81.2%, ROC AUC=0.913. The analysis can be performed with the same nucleic acid preparation as used for HPV testing. No statistically significant correlation of the DF value and HR-HPV DNA load was found. The DF value and the HR HPV presence and viral DNA load may be regarded as independent criteria that can complement each other in molecular screening for high-grade cervical intraepithelial neoplasia. Although it has several limitations, the present study showed that the small-scale analysis of microRNA signatures performed by simple PCR-based methods may be useful for improving the diagnostic/prognostic value of cervical screening.

Comparison of HPV testing by real-time fluorescent PCR and hc2 for detection of high-grade cervical intraepithelial neoplasia and cancer: a case-control study in high-risk patients

Introduction: HPV testing is more sensitive than cytology for cervical cancer screening but leads to over referral to colposcopy. This study was designed to evaluate the correlation of development of high-grade cervical intraepithelial neoplasia and cancer with HPV infection tested by real-time fluorescent polymerase chain reaction and conventional hc2 respectively and to find a cut-off value of HPV tests to increase the specificity of HPV testing and facilitate the triage before colposcopy. Material and Methods: Retrospective analysis the clinical data of 21,550 outpatients receiving cervical cancer screening, real-time PCR and hc2 testing were randomly chosen to test samples. Colposcopy with biopsies was performed to high-risk patients. Histopathology was taken as the diagnostic gold standard. Univariate ANOVA and multivariate binary logistic regression were adopted for data analysis. Results: The sensitivity and positive predictive value was 98.9% and 40.4% of hc2, 93.8% and 42.6% of RT-PCR respectively. Spearman rank correlation coefficient of viral load and lesions is 0.246, P=0.000. Comparing with HPV negative cases, the risk of having HSIL and cervical cancer for 16/18 genotypes positive cases was 11.7 fold-higher (OR=11.71, 95% CI: 3.75-36.61). The risk for other 11 genotypes positive cases was 3.1 fold-higher (OR=3.10, 95% CI: 0.97-9.85). The risk for high-level of viral load group (1 × 105~107) was significantly higher than the negative group (OR=1.98, 95% CI: 1.43-2.74). Conclusion: Real-time fluorescent polymerase chain reaction HPV detection method has better specificity and positive predictive value than conventional hc2. There is no optimal cut-off value of HPV viral load by hc2 to predict the development of cervical lesions. Patients with positive HPV 16/18 and high-level viral load by RT-PCR test have more risk to develop high-grade cervical lesions and carcinoma. Screening specificity is increased when taking these two cut-offs as the criteria to triage before colposcopy.