Introduction

Cervical cancer is caused by persistent human papillomavirus (HPV) infection. However, HPV prevalence data and survival rates among HPV-infected women are scare in Saudi Arabia. This study assessed the prevalence of HPV genotypes between 2006 and 2016 and determined whether HPV presence predicted cervical dysplasia classification, cervical cancer, or survival among women attending a Saudi referral hospital.

Methods

Cervical biopsy specimens underwent HPV detection using PCR, HPV viral load quantification using quantitative real-time PCR, HPV genotyping using GenoFlow array testing, and tumor suppressor protein p16^INK4a expression measurement using immunohistochemistry. Kaplan-Meier plots were constructed to analyze overall survival rates and to assess survival based on age, HPV presence, cervical dysplasia, and cervical cancer.

Results

Of the 316 cervical specimens examined, HPV was detected in 96 (30.4%); 37% had cervical cancer; 14% cervical intraepithelial neoplasia (CIN) III, 5% CIN II, and 17% CIN I. The two most common types detected were HPV-16 (56.2%) and HPV-18 (8.3%). A significant association was found between HPV-16 viral load and disease progression (P <  .001, Mann-Whitney U) and between HPV presence and cervical cancer (χ2, 56.78; P < .001). HPV status was a significant predictor of survival: HPV-negative women had poorer survival rates than HPV-positive women (multivariate Cox regression, hazard ratio, 7.04; 95% CI, 2.03-24.45). In addition, overexpression of p16^INK4a was correlated with the severity of histologic abnormality (log-rank test, P = .049).

Conclusions

These findings suggest that implementing cervical cancer and HPV screening programs will decrease cervical cancer rates and improve survival rates of women in Saudi Arabia.

Introduction

The vaginal microenvironment plays a role in persistence of high-risk HPV (HR-HPV) and cervical carcinogenesis. The genital carriage of HR-HPV, common curable sexually transmitted infections (STIs), Mycoplasma spp., Lactobacillus spp., and bacterial vaginosis (BV)-associated pathogens was evaluated in asymptomatic African women.

Methods

Women were consecutively recruited at the outpatient clinic for women’s sexual health “La Renaissance Plus”, N’Djamena, Chad. Genital secretions were self-collected using veil (V-Veil-Up Gyn Collection Device, V-Veil-Up Pharma Ltd., Nicosia, Cyprus), and were tested by multiplex real-time PCR for HR-HPV by Anyplex™ II HPV28 genotyping test (Seegene, Seoul, South Korea), STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis) and Mycoplasma spp. (M. hominis, Ureaplasma parvum, U. urealyticum) by Allplex™ STI Essential Assay (Seegene), Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Candida albicans, other Candida spp. and BV by Allplex™ Vaginitis Screening Assay (Seegene).

Results

A total of 253 women (mean age, 35.0 years) was enrolled. The prevalence of HPV infection was 37.9%, including 62.5% of HR-HPV. Common STIs were infrequent (2.8%) [C. trachomatis (1.2%), N. gonorrhoeae (0.4%), M. genitalium (1.6%), T. vaginalis (0.4%)]. Prevalences of genital Mycoplasmas spp. were high (54.2%) [U. parvum (42.6%), U. urealyticum (19.8%), M. hominis (19.1%)]. Carriage of G. vaginalis (83.4%), A. vaginae (37.9%), Mobilincus spp. (89.7%), C. albicans (17.4%) and other Candida spp. (22.9%), were high. The Lactobacillus spp. flora was generally normal (62.8%). BV was diagnosed in 32.4%. By bivariate analysis, HR-HPV infection was associated with A. vaginae (P=0.050), M. hominis (P=0.01) and BV (P=0.04). By multivariate analysis, strong association between HR-HPV and M. hominis persisted (adjusted odd ratio: 2.5; 95%IC: 1.3-5.1; P<0.01).

Conclusions

Multiplex PCR testing allows easy diagnostic approach of female genital microbiota. HR-HPV infection was associated with M. hominis, which may play a role in HR-HPV induced cervical carcinogenesis in African women.

Introduction

To evaluate the performance of Ampfire HPV technology, a new isothermal amplification assay for primary cervical cancer screening, using both self- and clinician-collected samples.

Methods

This is a sub-study from the Chinese multi-center screening trial (CHIMUST). The self-collected and direct collected samples obtained from 3 of the 6 study sites (stored in PreservCyt at -4°C) were used. 6619 women participated in the trial from these three sites. Samples from women with complete data were tested with the Ampfire assay. These women had been previously tested with Cobas and SeqHPV assays, and the clinician-collected samples were also tested with cytology. In CHIMUST all patients, HPV positive (self- or clinician-collection assayed on Cobas or SeqHPV) were recalled for colposcopy and had directed and/or random biopsies plus ECC.

Results

Preliminary analysis includes 1,941 women with a mean age of 42.8 from 1 of the 3 sites. CIN 2 was 0.82% (16/1941); CIN 3 was 0.51% (10/1941); cervical cancer 0.1% (2/1941). The sensitivity of Ampfire for CIN2+ of self-collections and clinician-collections both were 92.9%; specificity was 92.9% and 93.2% respectively. The sensitivity of Cobas for CIN2+ of self-collections and clinician-collections both were 96.4%; specificity was 92.4% and 93.6% respectively. The sensitivity of SeqHPV for CIN2+ of self-collections and clinician-collections were 100% and 96.43% respectively; the specificity was 93.5% and 93.6 respectively. The sensitivity and specificity for CIN2+ were similar among the three assays.

Conclusions

The Ampfire HPV showed similar sensitivity and specificity to Cobas and SeqHPV for CIN2+ on both self- and clinician-collections(P＞0.05). The speed, cost, and simplicity and ability to do full genotyping as well as a full STD panel, will make the Ampfire assay particularly suited for middle and low resource settings. It’s accuracy with self-collection makes it applicable for mass screening programs. The full data will be reported.

Introduction

Due to the low sensitivity of cervical cytology the primary screening test has been replaced by HPV testing which have a higher sensitivity but does not provide as high specificity. In order to increase the specificity several methods have been explored as triage such as analysis of HPV viral load. A number of studies have evaluated the use of HPV viral load but results have been conflicting with respect to its clinical utility. This study was performed to evaluate the use of high-risk HPV (hrHPV) viral load in the screening test to predict persistent infection and presence of cervical CIN2+.

Methods

We followed women between 30-60 years of age in a population screening cohort who performed self-sampling of vaginal fluid followed by a hrHPV test. Women who were hrHPV positive in their screening test repeated the hrHPV test 3-6 months later and these were included in the present study.

Results

Results show that women with persistent infection had higher HPV viral load in the primary screening test than women with transient infections, both for HPV16 (p=5.33e-03) and the total viral load of all hrHPV (p=3.88e-07). In women with persistent HPV16 infection and histologically confirmed CIN2+ had 48% an increase in HPV16 titer in the follow-up test as compared to 20% of women with persistent infection without CIN2+ lesions. When analysing all hrHPV types together, 41% of women with persistent infection and CIN2+ had an increase in titer as compared to 26% of women without CIN2+.

Conclusions

Viral load of hrHPV in the primary screening test is associated with presence of CIN2+. Serial testing of HPV viral load has the potential to distinguish women with CIN2+ lesions from women with persistent infection without CIN2+ lesions.

Introduction

The High risk Human Papillomavirus (HR-HPV) is the etiologic agent of cervical cancer, second female cancer in Algeria. The objective of this work is to carry out a preliminary prospective study for the detection of HR-HPV infections in Tlemcen, where screening is based solely on cytology.

Methods

One hundred and thirty (130) cervical specimens were used in this study. HPV detection was performed by the Cobas® 4800 HPV test in a laboratory of medical analysis “Ibn Sina” (Constantine) using a real-time PCR. This test identifies specifically HPV16 and/or HPV18 types, and detects simultaneously the remaining HR-HPV types.

Results

A percentage of 21.5% represents positive HPV specimens (28/130) with 5 multiple infections (HPV16 associated) and 23 single infections. The rate of infection by HPV16 (alone or combined) was 28.6% (8/28). Risk factors that appear to be related to HPV infection were, in addition to HIV infection, the lack of early and regular screening; where the majority of patients performed their smears for the first time at an advanced age whose principal reason of gynecological consultations was hemorrhage on contact. In addition to abusive use of oral contraception, and early age of marriage.

Conclusions

These preliminary results encourage us to develop a study on a larger sample of patients, including the HPV test. This test will optimize the performance of early screening of this cancer in our population; to define the prevalence of HPV infection and the distribution of different genotypes, which is necessary in the introduction of vaccination.

Introduction

Automated evaluation of p16/Ki-67 dual stain (DS) with CYTOREADER, a cloud-based deep-learning application, leads to greater accuracy, efficiency, and substantially improved specificity compared to Pap cytology for triage of HPV-positive women. Here we conducted an evaluation of interobserver reproducibility and accuracy of assisted clinical evaluation of DS slides using CYTOREADER.

Methods

CYTOREADER comprises a fully-automated workflow combining whole-slide scanning with automated evaluation using a deep-neural network for the detection of DS-positive cells. In addition, it provides a computer-assisted mode that presents all DS-positive cells on a slide ranked by the likelihood that a cell is DS-positive. For this pilot study, a total of 160 DS-slides from HPV-positive women were evaluated by 6 readers (4 Cytotechnologists, 2 Pathologists), 40 slides each. Slides were evaluated manually and using both the CYTOREADER assisted- and fully-automated modes. The time to evaluate slides was recorded for both the manual and assisted approach. We assessed the reproducibility of assisted DS evaluation using the percent agreement and evaluated the sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or greater (CIN3+).

Results

The average time to evaluate 10 DS slides was 44 (±18) minutes for manual and 29 (±9.9) minutes for computer-assisted evaluation. The percent agreement was 74.2% between assisted and manual, 71.3% between fully-automated and manual, and 80.4% between assisted- and fully-automated evaluation. The interobserver agreement between two readers was 76.3% for manual and 77.5% for assisted DS evaluation. The sensitivity and specificity for detection of CIN3+ was 83.3% and 52.5% for manual, 94.4% and 52.0% for assisted, and 100% and 61.8% for fully-automated, respectively.

Conclusions

Computer-assisted evaluation is reproducible and achieves greater sensitivity at comparable specificity for CIN3+ compared to manual reading. Using CYTOREADER for either assisted or fully-automated evaluation of DS increases the efficiency and accuracy of slide interpretation for triage of HPV-positive women.

Introduction

Cervical cancer (CC) kills 330,000 people annually and requires persistent infection with high-risk Human Papillomavirus (hrHPV) for its development. European guidelines advocate the use of hrHPV DNA tests for CC prevention in women over the age of 30 years. Most presently available hrHPV tests however do not provide quantitative sample cellularity assessment, as a measure of sample adequacy, or hrHPV viral load. The aim of this study was to evaluate the relationship between normalized HPV type-specific viral load and cervical cytology grade using HPV OncoPredict, a new in-vitro diagnostic tool allowing accurate quantitative sample cellularity assessment and hrHPV genotype-specific viral load (E6/E7 DNA) determination, developed as part of a Horizon 2020 SME Instrument Project (SME Instrument Grant GA 806551).

Methods

Clinician-collected cervical (CCC) samples were collected from 100 women referred to colposcopy at San Gerardo Hospital, Monza, Italy, for a diagnosis of precancerous cervical disease, classified by cytological stage (undetermined, low or high grade [ASCUS, LSIL, HSIL] squamous intraepithelial lesions). CCC were collected using L-Shaped FLOQSwabs (Copan) and resuspended in 20 mL of ThinPrep (Hologic). Nucleic acid extraction from 1 mL of sample was performed using NucliSENS easyMAG (bioMérieux), further eluted in 100 μl of buffer. HPV OncoPredict prototype (Hiantis) was used to determine samples' cellularity and normalized hrHPV genotype-specific viral load.

Results

Preliminary data using HPV OncoPredict indicate an adequate cellularity for all samples (mean human cellularity values of 6.78E+04, 1.06E+05, 7.02E+04 cells/mL for ASCUS, LSIL and HSIL respectively). Mean normalized viral loads overall ranged from 1.27E+02 (ASCUS) to 1.85E+02 (HSIL) GU/human cell, with differences in viral loads observed among different hrHPV genotypes.

Conclusions

HPV OncoPredict preliminary evaluation has shown promising results, allowing the possibility to accurately define normalized genotype-specific viral loads. Differences in viral loads observed between hrHPV suggest that further studies are required to determine normalized genotype-specific clinical cut-off values.

Introduction

HPVs are responsible for 5% of all cancers, mostly at the cervical and anal canal sites. HPV16 is the most frequently genotype associated with (pre)-cancer development following a persistent infection. Since recent next generation sequencing (NGS) analyses revealed that HPVs are highly polymorphic, it can be hypothesized that within genotypes, HPV variants may present different carcinogenic properties. The aim of our study was to develop NGS methods for High Risk (HR)-HPVs in order to analyse HPV genome variations during natural history of cervical lesions.

Methods

First, we took advantage of the Hybrid Capture II screening test to capture DNA of 13 HR HPV genomes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and prepare libraries. An amplicon strategy was also developed to specifically sequence HPV16 using primers amplifying HPV16 whole genome. In-house NGS pipe-lines were developed and used for sequencing data analysis. DNA extracted from HPV infected cells and from previously genotyped cervical specimens were used to validate NGS protocols.

Results

The capture method permitted to retrieve concordant HPV genotypes in 90% (n=17/19) of cases. One sample (5%) presented partially concordant genotypes as one HPV51 was missed by NGS. One sample (5%) was discordant.

Concerning the amplicon method, HPV16 was correctly identified from 47 samples allowing a 100% concordance with the INNO-LiPA genotyping. HPV16 variant identification is still ongoing.

Conclusions

Two standardised and affordable NGS methods to sequence the whole genome of HR-HPV and HPV16 from clinical samples will be presented. By implementing these techniques on cervical samples representative of the natural history of cervical cancer, we show how our methods will help to identify HR-HPV variants and study their potential role in carcinogenesis.

Introduction

A cluster randomized cervical cancer screening study was performed in Butajira, south central Ethiopia, where acceptance of self-sampling for HPV DNA testing was compared to visual inspection with acetic acid. (1) As adjunct study, persistently HPV-positively tested women were retested with an innovative assay based on the QuantiGene 2.0 technology platform (Thermo Fisher). The aim was to compare feasibility and precision in detection of dysplasia.

Methods

Women self-sampled cervicovaginal smears using the Evalyn brush (Rovers Medical Devices, Oss, The Netherlands) and multiplexed genotyping (MPG) was performed on extracted DNA. HR-HPV positive women underwent VIA for triage and resampling into Thinprep/PreserveCyte 4-8 months later. MPG retesting and QuantiGene assay were performed on Thinprep samples for detection and quantitation of E7 oncogene and cellular biomarker mRNA expression (e.g. p16, Ki67, Stathmin/oncoprotein 18, MCM2, ALDH1A1, Birc5/Survivin).

Results

Out of 1020 women accepting self-sampling and HPV testing 144 (14.1%) tested HR-HPV positive and 122 attended VIA triage of whom 110 gave a second Thinprep sample. Upon retesting 4-8 months later 84 (76.4%) women had cleared the original HR-HPV genotype and 26 (23.6%) were persistently HR-HPV genotype positive. Colposcopy showed dysplastic changes in 2 women with CIN2+ alterations while the third had a TZ3. QuantiGene test analysis including strength of E7 mRNA and cellular biomarker expression identified these 3 samples with markedly increased expression. The relative screening burden by HPV test (110 positives) versus Quantigene mRNA detection (3 positives) was 36 fold reduced.

Conclusions

Acceptance of self-sampling and HPV screening was high. Clearance of prevalent HPV infections within appx. 6 months was high posing a problem for triage and follow up. Repeated HPV testing rounds may reduce over referral. QuantiGene-based mRNA detection including biomarker evaluation, which is a simple and robust technique, enhanced specificity remarkably.

References

1) Gizaw M, et al., Cancer Prev Res 2019 (9):609-616

Introduction

Sexually transmitted infections (STIs) have become increasingly prevalent and impose a major global health and economic burden for the patients, populations and healthcare. Many STIs are silent, hidden infections and can have serious impact on individual’s health. There is an urgent need for a comprehensive assay to detect a broad-spectrum of STIs simultaneously in a single reaction at an extremely low-cost.

Methods

Type/species-specific primers were designed for clinically relevant 27 HPVs and 13 STIs and two internal human gene controls, which are used to estimate copy number per cell and to monitor cross-contamination, respectively. Amplification and barcoding/indexing of each sample is performed in a single-tube PCR reaction and all the amplicons are pooled together and sequenced by NGS. The HPV samples used in this study were previously analyzed by Roche Cobas HPV test and all the HPV/STI samples were previously tested with other validated assays.

Results

Our results show that the new assay can detect all intended types/species with high sensitivity, specificity and uniformity. The entire workflow consists of four steps of DNA extraction, single-well and one-step amplification, sample pooling/library preparation and sequencing. The assay can be completed within 24 hours including 17 hours sequencing. Using this comprehensive assay, our results show that many samples are found to have more than one clinically important type/species present that go undetected in routine clinical and diagnostic testing.

Conclusions

We have developed a highly multiplex and comprehensive STI assay that uses low amount of DNA, detects and quantifies 27 HPVs and 13 STIs in one single-tube and one-step amplification reaction. Due to its simple procedure, the comprehensive STI assay is very low-cost and can be easily automated for low/medium and high throughput sample scales. The comprehensive STI assay can be used for screening, detection, research and epidemiological settings.

Introduction

Sexually transmitted infections (STIs) impose a major global health and economic burden. Globally, there are 1.1 billion existing infections and each year, there are over 376 million new cases. Many of these STIs are hidden and silent infections that can have serious impact on physical and psychological health. We have developed a comprehensive assay that detects 27 HPVs and 13 STIs uniformly in one single reaction with a simple workflow using next-generation sequencing (NGS) technology.

Methods

A set of 300 samples that were previously tested with Roche Cobas HPV assay and Reverse Line Blot Hybridization (RBL) assay were analyzed by our comprehensive NGS-based method. A panel of multiplex type/species-specific primers were designed to detect 27 HPVs and 13 STIs and two internal human gene controls. Amplification and barcoding/indexing of each sample is performed in a single tube reaction and all the amplicons are pooled and sequenced by NGS.

Results

Our results show that the STI-NGS assay has an excellent agreement with Roche Cobas HPV assay and RBL assay. We could detect HPV and STI types/species with high sensitivity, specificity and uniformity. The comprehensive STI NGS assay has a very simple workflow and the process consists of DNA extraction, single-tube amplification, sample pooling/library preparation and sequencing. The assay can be completed within 24 hours. Our results show that many samples have multiple HPV and STI infections that go undetected in routine clinical and diagnostic testing.

Conclusions

We have developed a highly multiplex and comprehensive STI assay that uses low amount of DNA, detects and quantifies 27 HPVs and 13 STIs in a single-tube and single-step amplification reaction. The assay is quantitative and generates viral load/copy number for all the types/species detected in a sample. The comprehensive STI assay has a simple workflow, easy to automate and is very low-cost.

Introduction

The negative predictive value of a screening test is important for determining the safety of negative results. Nowdays, numerous HPV screening tests are in use. Our objective was to determine the predictive value of a negative HPV test performed with an E6/E7 mRNA test.

Methods

After cotesting 5,053 women of ages 25-65 y.o. with liquid-based cytology (LBC) and APTIMA® HPV (AHPV), those with a positive AHPV or with LSIL+ cytology were adviced to undergo colposcopic biopsy. Those women without a high grade (CIN2+) cervical lesion were followed-up as per European and national recommendations. From the group of women with a negative cotest, an active recruitment by phone calls was established 3 years after the baseline cotest. Predictive values for CIN2+ of those women with adequate FU were calculated.

Results

The baseline cotest showed a 9% (454) AHPV prevalence. From those women, 265 had a 3 year follow-up (FU) with colposcopy and biopsy, yielding 100 CIN2+ lesions (risk of 37,7%). From those with a negative AHPV result, active 3-year recruitment was performed in 1,023 women and found 44 women with a new HPV infection. In 33 cases, a biopsy was performed, finding 3 new cases of CIN2+ lesions (9,1%). From 71 women with a negative AHPV and abnormal cytology at baseline, adequate FU was available in 45. Only 1 high-grade lesion, an endometrial adenocarcinoma at baseline, was found.

Therefore, the NPV for AHPV in our study was 99,6%, while the PPV was 37,7%.

Conclusions

Primary mRNA HPV testing with APTIMA® in a screening population provides a NPV higher than other HPV tests reported in the literature after 3 years of adequate follow-up. Therefore, the safety of this test is reassuring as a screening tool.

Introduction

The collection of the cervix is ​​mainly based on cells suspended in liquid-based media (LBC). In several studies, it has been proven that LBC analysed with Cobas® HPV test meets the criteria of the international guidelines for clinical validation. The indicating FTA card is a dry medium in which applied cervical cells are kept stable at room temperature. HPVIR is a multiplex real-time PCR test that detects and quantifies 12 high-risk HPV. The purpose of this study was to evaluate whether a strategy with cervical specimens collected on the FTA card and subsequently analysed with HPVIR test meets the criteria of the international guidelines for a clinically validated method of cervical cancer screening.

Methods

A non-inferiority test was used to compare the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a reference test (Cobas^® HPV test) based on LBC samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. We evaluated the clinical specificity in 799 women without ≥ CIN2, and the clinical sensitivity in 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples of which 51% previously tested HPV positive.

Results

The clinical sensitivity and specificity for samples collected on the FTA card and analysed with HPVIR test were non-inferior to the reference test (non-inferiority test score, p = 1.0 x 10^-2 and p = 1.89 x 10^-9, respectively). Adequate agreement of > 87 % was seen in both the intra- and inter-laboratory comparisons.

Conclusions

Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

Introduction

With a growing number of countries moving to large regional, or national, HPV-based screening programs there is a growing need for high volume (>1000 tests per day) clinically validated HPV assays to support these programs. The introduction of the Gardasil vaccine has also driven interest in understanding the role of HPV31,33,45,52, and 58 in screening programs and pathways.

Methods

The Abbott alinity m HPV assay run on the alinity m instrument was compared to the reference Roche cobas 4800 HPV test using the Meijer Criteria framework to examine sensitivity and specificity for histologically confirmed cervical intraepithelial neoplasia grade two or above (CIN2+). Intra- and Inter-laboratory reproducibility was also examined. The alinity m HPV assay is currently not approved for clinical use but this is expected to change in late 2019. Samples for this study are from the Compass Trial which is a consent-based trial and this current study has ethics approval.

Results

Relative Sensitivity for histologically confirmed CIN2+ is 96.7% (95%CI 88.5-99.6%). Currently (n = 300 of 805 tested to date) relative specificity for CIN2+ is over 99%. Final results, including sensitivity, specificity and inter- and intra-laboratory reproducibility will be presented.

Conclusions

Current data suggests that the Abbott alinity m HPV assay has sensitivity and specificity that is comparable to Roche cobas 4800 HPV reference test with the additional utility of producing a more detailed HPV genotyping profile (HPV16, HPV18, HPV45, HPV31/33/52/58, HPV35/39/51/56/59/66/68) which may be useful in monitoring Gardasil9 vaccine impacts on screening programs.

Introduction

Whilst most HPV-based screening programs focus on high volume HPV assays and accompanying instrumentation, there is also emerging interest in HPV assays which can be run on small, flexible platforms on which a wide range of other assays can be utilised. Such systems may have considerable usefulness in low and middle income settings.

Methods

The AusDiagnostics High-Risk HPV panel was compared to the reference Roche cobas 4800 HPV test using the Meijer Criteria framework to examine relative sensitivity and specificity for histologically confirmed cervical intraepithelial neoplasia grade two or above (CIN2+). Intra- and Inter-laboratory reproducibility was also examined. Samples for this study are from the Compass Trial which is a consent-based trial and this current study has ethics approval.

Results

Relative sensitivity for histologically confirmed CIN2+ was 100% (95%CI 94.8-100%). Relative specificity for CIN2+ is 98.9% (95%CI 97.8-99.5%). Intra-laboratory reproducibility had a lower confidence interval of >87% and inter-laboratory data will also be presented.

Conclusions

The AusDiagnostics High-Risk HPV panel is as sensitive and specific as the Roche cobas 4800 HPV reference test. The AusDiagnostics High-Risk HPV panel also presents individual genotyping for the twelve oncogenic HPV types and HPV66 and HPV68a/b.

Introduction

Screening based on the HPV test confers 60-70% more protection against CC than cytology-based screening. The finding that women with normal cytology and presence of 16 or 18 have a higher risk of having or developing a CC or precancer (HSIL/CIN2 +) compared to other genotypes HPV supports the need to incorporate the genotyping 16/18 in the first line screening.

Methods

CRYGEN study compared 10 different screening protocols. For that, 1988 women of 35 years old were included in the study. All were tested for HPV and cytology. The HPV test used was Roche Cobas 4800 HPV test®, wich detects 14 HR genotypes, genotyping specifically 16/18 and mantaining the remaining 12 HR-HPV as undifferentiated.

Results

26 CIN2 + lesions were diagnosed when HPV genotyping is performed in the first line with direct shunt to colposcopy compared to the 20 CIN2 + diagnosed if it is not genotyped and the shunt depends on the result of triage cytology.

HPV 16 was present in 17 of the 26 HSIL/CIN2 + detected after biopsy (65.38%), having been 6/26 reported as normal cytology rep resenting 23.07% of all HSIL/CIN2 + lesions detected and 35,29% (6of17) of HSIL/CIN2 + lesions with positive HPV16. Moreover, 14.63% of all women with positive result to HPV 16 and normal triage cytology had an HSIL/CIN2 + lesion. These women would only be diagnosed in that round of screening if all positive HPV16 are directly referred to colposcopy.

Conclusions

Cytological triage yields low sensivity due to high rate of false negatives compared to HPV test. It is considered essential to include the genotyping of HPV16 in the first line of screening and direct shunt to colposcopy if this specific genotype is detected, since it significantly increases the detection rate (in 23,07%) of HSIL/CIN2 + lesions.

Introduction

Cervical cancer is caused by persistent infection by at least one high-risk human papillomavirus (hrHPV) genotype. Cytology-based cervical cancer screening is switched to HPV screening in the majority of European countries. Cervical cancer screening program in the Czech Republic is still based on cytology with HPV triage. The objectives of the study were to compare the detection of hrHPV using three HPV screening methods and to investigate HPV prevalence in Czech screening population.

Methods

Five hundred cervical swabs were sampled from Czech women (age 30-64years) during regular gynecological examination. All samples were analyzed using digene HC2 HPV DNA Test (HC2, Qiagen), QIAscreen HPV PCR test (Qiagen) and cobas® 4800 HPV (Roche) HPV DNA detection methods.

Results

All samples were suitable for analysis using HC2, Qiascreen failed in 0.4% samples and cobas failed in 0.8% sample. HrHPV DNA was detected in 11.4% (57/500) samples using HC2, in 9.4% (47/500) samples using QIAscreen and in 10.8% (54/500) samples using cobas. All three methods gave concordant result in 94% (470/500) of samples. All three methods gave concordant result in 94% (16/17) samples from women with abnormal cytology findings (ASC-US +).

Conclusions

HrHPV prevalence in the screening population of Czech women is between 9.4% and 11.4% depending on the used HPV detection method. All three compared method gave concordant result in 94% of the analyzed samples.

This work was supported by grants: IGA LF UP 2019_003, CZ.02.1.01/0.0/0.0/16_019/0000868, LM2015064, and charity Cancer Research Czech Republic.

Introduction

HIV+MSM are at highest risk of developing squamous cell carcinoma of the anus. Over 80% of these cases are caused by high-risk Human Papillomaviruses (HR-HPV) types 16 or 18. Linear Array HPV Genotyping test (Roche), the gold standard for decades, is no longer available. An alternative was needed. We evaluated AmpFire HPV Genotyping Assay (Atila BioSystem) and compared results to Roche’s test.

Methods

150 anal swab-based specimens collected in PreservCyt media from consented HIV+MSM participants with >ASCUS grade cytology results, being referred for High Resolution Anoscopy, were tested for HPV by both assays according to each manufacturers’ protocol. Atila uses crude cell lysate, isothermal multiplex amplification, fluorescent detection of HPV16/18/31/33/35/39/45/51/52/53/56/58/59/66/68 in 96 well plate format; results available in 90 min. Roche requires DNA extraction, multiplex PCR amplification, probe hybridization, colorimetric detection with visual readout; results are available within 1-2 days.

Results

Comparisons were made for all 15 HR-HPV types, and for HPV16/18 only; the two HR-HPV types most highly associated with anal cancer. Agreement in the former comparison was 89.5% (95%CI; 0.8248-0.9326) (Kappa=0.6392); agreement in the latter was 90% (95%CI; 0.8404-0.9429) (Kappa=0.796). Analytical sensitivity, specificity, PPV and NPV for all 15 HR-HPV types were 62.2%, 96.3%, 80.6% and 91.1%, while those for HPV16/18 were 89.1%, 90.7%, 87.7% and 91.8%. HPV16 was most prevalent; detected in 32% (Atila) or 30.7% (Roche) of HIV+MSM participants. HPV16/18 were detected in 47.3% (Atila) or 46.7% (Roche) of participants. Detecting multiple HR-HPV types from anal specimens was common. Average [median] numbers of HR-HPV types detected per specimen was 3 [3] (Atila) and 2.2 [2] (Roche).

Conclusions

We validated anal specimens from HIV+MSM participants on Atila’s assay. Agreement between the two assays was good, especially for HPV16/18. Atila’s protocol takes less time to obtain results, is more economical and uses fewer specialized equipment to run.

Introduction

Persistent infection by Human Papillomaviruses (HPV) is characterized by viral oncogene expression and upregulation of cellular proteins that can be regarded as biomarkers for transformation. The oncoproteins E6 and E7 have pleiotropic effects and interact with cellular proteins regulating proliferation, tumor suppressors, (cancer) stem cell markers, and tumor markers. The strength of expression correlates to dysplasia stage and progression.

Methods

A multiplexed mRNA quantifying assay based on Luminex/QuantiGene 2.0 technology platform (Thermo Fisher Scientific) was developed. It combines detection of the E7 oncogene of 18 HPV genotypes and 18 cellular biomarker expression, routinely used for diagnosis, markers for cancer stem cells, tumor markers and housekeeping genes for normalization. All mRNA species are detected simultaneously from a crude lysate of a cervical smear sample taken into Thinprep/PreserveCyte. In a prospective trial 1400 consecutively collected samples were measured and data used for logistic regression and ROC analyses.

Results

The assay has a high sensitivity detecting less than 40 CaSki or HeLa cells. It genotypes 18 HR-HPVs and detects leading types in multiple infections with highest E7 expression. Logistic regression identified E7 expression strength as most informative biomarker discriminating low grade and high grade (CIN2+) dysplasia. P16, proliferation associated markers (Ki67, MCM2, Stathmin), and tumor markers (ALDH1A1, BIRC5, hTERT) contributed to detection and differentiation of dysplastic stages. Accuracy by ROC analysis to detect CIN3+ from the initial screening sample was 90%.

Conclusions

Multiplexed mRNA quantification of viral and cellular biomarkers by QuantiGene 2.0 Plex assay detects HPV infection and dysplasia with high accuracy. The assay could be used as a triage to colposcopy after primary HPV screening. The assay procedure is simple and robust and maybe used in LMIC settings to reduce further triage and avoid overreferral.

Introduction

Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection.

Methods

Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test.

Results

Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher’s exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001).

Conclusions

Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

Introduction

After the training program for the evaluation of dual p16/Ki-67 immunocytochemical staining (DS) on conventional smears, four evaluators had 111 of false positive (FP) and false negative (FN) results according to CIN2+ histopathological diagnosis. We aimed to identify the causes behind the false results.

Methods

Three evaluators (a student, senior cytotechnologist and cytopathologist) evaluated 497 DS slides with known CIN2+ result and reported 96 FP and 14 FN results. The consensus of five cytopathologists made the final decision about whether DS is present. An experienced cytopatologist examined FP cases in which the consensus found DS and evaluated morphological features of DS cells according to the Bethesda 2014. The cases were categorized into the group with low-grade (LG) morphology and the group with high-grade (HG) morphology (Figure 1). 128 true positive (TP) cases served as a control. Fisher's exact test was used to compare morphology between groups of FP with DS and TP cases (significance level α = 0.050).

Results

Consensus of five cytopatologists concluded that among the 96 FP results, DS was present in 73 (76.0%), while DS was misinterpreted as positive in 23 (24.0%). Among the 14 FN results, DS was not present in 11 (78.6%), while it was overlooked in 3 cases (21.4%). Among the 73 FP results with DS present, LG morphology was observed in 47 cases (64.4%) and HG in in 23 cases (31.5%), while 3 cases (4.1%) were morphologically negative. Among the 128 TP controls, LG morphology was less common (15 cases; 11.7%) while HG was more common (110 cases; 85.9%). Three cases (2.3%) were morphologically negative. The difference in distribution of morphology was statistically significant (p < 0.001).

Conclusions

DS with LG morphology was more common in FP cases while DS with HG morphology predominated in TP cases.

Introduction

Human papillomavirus 16 (HPV16) E6 seropositivity is a promising early marker of HPV-driven oropharyngeal cancer (HPV-OPC); yet, it is unclear if current imaging modalities can visualize early tumors within the oropharynx. The objective was to determine the sensitivity of ultrasound for detecting HPV-OPC compared to CT and PET/CT.

Methods

Fifty-one patients with known or suspected OPC, without prior treatment or cancer (other than non-melanoma skin cancer) were recruited from the Vanderbilt Head and Neck Clinic. Eight standard ultrasound images (transverse/sagittal views of tonsils, transverse/coronal views of tongue base [BOT], and bilateral long-axis lateral BOT) were obtained using the Lumify portable ultrasound system and mobile application (Philips Healthcare, Bothwell, WA). Blood was collected for HPV serologic analyses. Pathologic details, p16 status, final staging, and radiologic findings were abstracted from the medical record. The sensitivity of each imaging modality was compared to the final clinical diagnosis (gold-standard), as determined by the combined findings from the clinical and radiologic workup (excluding ultrasound), and biopsy of the primary site (as required).

Results

Following the clinical work-up, 24 (51%) BOT, 22 (43%) tonsil, and 2 (4%) unknown primaries were diagnosed; 3 patients (6%) had benign disease. 47 of 48 tumor and/or nodal specimens were tested for p16; all were positive. The presence or absence of primary oropharyngeal lesions were correctly identified in 90.2% (95%CI:78.6%-96.7%) using ultrasound; 69.4% (95%CI:54.6%-81.7%) using CT; 83.3% (95%CI:68.6%-93.0%) using PET/CT and 92.9% (95%CI:80.5%-98.5%) using biopsy. Ultrasound correctly identified 10 out of 14 tumors missed by CT and identified the absence of tumors in 3 cases where CT and/or PET/CT incorrectly identified a tumor. The smallest tumor was 0.51cm in greatest dimension; average size was 2.29cm. 76.1% (95%CI:61.2%-87.4%) of p16 positive patients had detectable HPV16 E6 antibodies.

Conclusions

Transcervical ultrasound and HPV16 E6 antibodies are sensitive for the diagnosis of HPV-OPC.

Introduction

Introduction: HPV18 is second only to HPV16 as an etiologic factor in cervical cancer but is not well represented in precancers. Whole genome sequencing of HPV18 may shed light on the molecular changes with cervical disease progression.

Methods

Methods: Whole genome sequencing following HPV target enrichment (eWGS) was performed with Illumina Hiseq 2500 and interpreted following published methods on samples from the Early Detection Research Network cervical cancer biorepository. HPV types were identified if ≥511 reads mapped to reference genome (CLC Genomics). Multiple sequence alignment of the HPV18 consensus and sublineage reference sequences with MUSCLE 3.8.31, and maximum likelihood phylogenetic analysis were used to determine HPV18 lineage/sublineages. HPV integration was identified by deletions (>50 bp) in the mapped reads with at least 10X depth of coverage.

Results

Results: eWGS identified HPV18 in 49 samples: 16 from women with no cervical disease (No-CIN), 11 from cervical intraepithelial neoplasia grade 1 (CIN 1), 13 CIN 2, 6 CIN 3 and 3 invasive cancer. HPV 18 was the only type detected in 5 samples, the remaining had 1 to 18 additional types (mean 5 types). HPV16 was co-identified with HPV18 in 16 samples. Most HPV18 belonged to A lineage (41), 8 were B, none in C lineage. Within A lineage, there were 8 A1 (16.3%), 25 A3 (51%); 7 A4 (14.3%) and 1 A5 (2%). B sublineages included B1 (4, 8%) and B3 (4, 8%). HPV18 lineage/sublineage was not associated disease. HPV18 was integrated in 5 samples, all with at least CIN2 (3 invasive cancer, 1 CIN 3 and 1 CIN 2) Integration of HPV18 genome resulted in deletions ranging from 224-3958 bp (mean, 2283bp), affecting E1, E2, E5 and L2 genes.

Conclusions

Conclusion: Using eGWS provides direct information on genetic variants and integration of HPV 18. This method will be used to study additional samples.

Introduction

Despite the widespread of HPV infections, only a small fraction of women with a hrHPV infection will progress to pre-cancer or cancer. This indicates that additional risk factors are important for carcinogenesis. HPV type-specific viral load has been associated to an increased risk of cervical cancer development, with its potential use as a molecular triage tool in self-sampling based screening programmes. This ongoing study aims to determine HPV viral load level among different sample types: cervical, vaginal and urine samples, as well as assessing the correlation between viral load and grade of cervical lesion.

Methods

Presently, clinician-collected cervical, self-collected vaginal (FLOQSwab, Copan), urine samples (Collipee, Novosanis), were obtained from 180 women attending the Colposcopy Clinic, San Gerardo Hospital (Monza, Italy). All samples were extracted using NucliSENS easyMAG (bioMerieux) and HPV detection carried out using AnyplexII HPV28 (Seegene). Viral load was evaluate using “in house” HPV type-specific assays able to detect 7 different HPV types (HPV16, 18, 31, 33, 45, 51, 52).

Results

A very good HPV test concordance was observed between vaginal and urine self-samples as compared to clinician-collected cervical samples (gold standard). Usually, HPV viral load detection resulted higher among cervical samples compared to both self-sample types. Median values are reported in Figure1. Preliminary results have shown a higher viral load median value for HPV16 and HPV 31 types in patients with a high-grade intraepithelial lesion than patients with low-grade dysplasia (HPV16: 7.36E+07 vs 3.94E+06 copies/10^4 cells; HPV 31: 1.98E+05 vs 3.83E+04 copies/10^4 cells).

Conclusions

Higher HPV viral loads may indicate viral persistence, progression to cervical dysplasia, and may even serve as a prognostic biomarker in cervical cancer screening based on self-collection; however, longitudinal studies are needed to confirm these preliminary data.

Introduction

Persistent infection with oncogenic human papillomavirus (HPV) has been linked to cervical cancer. European Guidelines advocate the use of HPV testing as primary cervical cancer screening method in organized population-based programs. Furthermore, HPV-genotyping and viral load are able to distinguish persistent infections and to stratify the risk related to cancer development in the infected population, as individual oncogenic genotypes have different carcinogenic potential. Good performance and reproducibility of laboratory results is fundamental for safe HPV-based screening and requires quality assessment to monitor the entire workflow, including nucleic acid extraction, amplification, and detection.

The aim of this study was to evaluate a novel panel of HPV type-specific controls (MICROBIX) on testing with AnyplexII HPV28 (Seegene), a clinically validated full-genotyping assay, and with HPV OncoPredict viral load and RNA assays, two full-genotyping IVD prototypes developed as part of an ongoing Horizon 2020 Project (SME Instrument Grant GA 806551).

Methods

MICROBIX controls, available for hrHPV types 16, 18, 31, 33, 39, 45 and HPV 67, contain components found in HPV-infected clinical samples, such as integrated and episomal viral DNA, viral RNA as well as host epithelial cells.

All samples were extracted using NucliSENS easyMAG (bioMerieux) and HPV detection carried out using AnyplexII HPV28 (Seegene), HPV OncoPredict viral load (Hiantis) and HPV OncoPredict RNA (GeneFirst) assays, according to manufacturers’ instructions. Absolute HPV viral loads were further evaluated by means of “in house” HPV genotype-specific assays using Droplet Digital PCR (BioRad).

Results

MICROBIX controls showed an excellent compatibility with all evaluated HPV genotyping assays. MICROBIX controls tested in combination with HPV OncoPredict viral load were shown to provide accurate quantitative assessment of genotype-specific viral loads, as compared to the absolute values obtained using Droplet Digital PCR.

Conclusions

The novel MICROBIX HPV genotype-specific controls showed promising results for their future application as QC samples in laboratories performing HPV testing.

Introduction

Human Papillomavirus is associated with cervical, anogenital and oropharyngeal cancers. Multiple subtypes, persistent and concurrent HPV infections are common among HIV+ women.However, there is a paucity of data regarding the concordance of HPV genotypes between the cervical and anal region. Therefore, this study aims to investigate the association of cervical HPV infection, with genotype-concordant anal HPV infection among women with CIN in Durban South Africa.

Methods

A longitudinal cohort of 148 women aged 18-65 years with high grade cervical CIN at King Edward Hospital, Durban South Africa were followed-up prospectively from October 2016- March 2017. Behavioural, clinical and demographic data were collected. Cervicovaginal and rectal swab samples were collected from women (n=30) over the age of 30 years to detect DNA of 37 HPV subtypes using Roche Linear array. Descriptive statistics and multivariable regression to describe the relationship between genotype-concordant HPV infection identified from anal and the cervical region.

Results

Majority of participants 97% (n=145) were African black women, with an average age of 38 years. Of which 94.6% (n=140) were HIV positive with a mean CD4 count of 481. Participants Anal HPV infection was 93.3% (n=28), with HPV 16 and 18 detected in 64.3% (n=18). Overall, oncogenic HPV subtypes were concordant between the two anatomical sites, with an avergare genotype specific concordance of 45.6%; HPV16 was 55% and HPV 18 31.2,% respectively.

Conclusions

In this population of women cervical and anal HPV infections occur consecutively. The high degree of genotype-specific concordance, therefore, suggests that the cervix and anus may serve as reservoirs for HPV infection at other either anatomical site. Thus more studies are needed to further explore this phenomenon to eliviate the burden of HPV-related disease. This data may have implications in informing public health guidelines, advocacy for cervical and anal screening of HIV positive women.

Introduction

Cervical cancer is the second most common cancer in women between 35 and 64 years of age worldwide. In Argentina around 4,000 new cases are diagnosed each year, and 1,800 women die from this disease. The aim of this study is to assess the prevalence of HPV infection in a group of women screened for cervical cancer with Co-test (Hybrid Capture – HCII – and Cytology).

Methods

A retrospective, descriptive and cross section study which included 2304 women between 30 and 65 years of age, recruited from a Community Private Health Care setting (CEMIC), who were screened for cervical cancer with Co-test between 08/2013 and 07/2018.

Results

The negative rate of Co-tests was 85.3%. Six women (0.26%) showed a negative HCII and ASCUS cytology; they were instructed to perform a new test in 3 years’ time. 331 women (14.4%) had a positive HCII, of which 56 (2.43%) showed a positive Co-test and were referred for evaluation by the Lower Genital Tract Disease Section. 275 (11.9%) showed a positive HCII and a negative cytology; they remained on follow-up.

Conclusions

The positive rate of HCII was 14.4%. HPV screening allows us to identify a minority of women with CIN2+ risk.

Introduction

Persistent infection with high-risk human papillomavirus (hr-HPV) is an important co-factor in cervical cancer development and is associated with DNA methylation on both human and viral genes. The S5 DNA-methylation classifier, based on target CpG sites of the human gene EPB41L3, and viral late gene regions of HPV-16,18,31 and 33 has demonstrated better performance for detection of cervical intraepithelial neoplasia grade 2/3 (CIN2/3) women than either HPV16/18 genotyping, cytology or combination. We tested the performance of S5 in detecting invasive cancers versus pre-cancers and quantified the degree of separation between normal, CIN3 and invasive cancer S5 scores.

Methods

Methylation status of the S5 selected CpG sites was tested in DNA extracted from exfoliated cervical cells from the UK (n=138), Spain (n=100), Colombia (n=96), Philippines (n=50), Georgia (n=42), Ethiopia (n=79), India (n=60), South Africa (49), Bhutan (n=60) and USA – New Mexico (n=200). Samples were histologically defined as normal (healthy patients), CIN3 and invasive cancer. DNA bisulfite conversion was carried out and followed by pyrosequencing for the 6 components of the classifier.

Results

Methylation at all sites increased proportionally with disease severity (Cuzick trend of z=9.2933, p,0.0001). The separation of normal from CIN3 and from invasive cancer was highly significant (Mann Whitney tests, all p<0.0001). ROC curves were used to assess the diagnostic potential of S5 in differentiating CIN3 and cancers from normal patients. The AUC was 0.94 (CI 95%: 0.92 to 0.96, p<0.0001) with a sensitivity of 93% and a specificity of 75%, based on a cut-off at highest Youden J-index. We found a strong correlation between S5 scores and disease lesion r = 0.64 (95% CI 0.59 - 0.68, p<0.0001).

Conclusions

The S5 methylation classifier may be useful in cervical screening programs for differentiating normal and pre-cancers from invasive cervical cancers in women across different countries..

Introduction

Men who have sex with men (MSM), especially MSM with HIV, have increased risk for anal cancer; however, screening for either anal precancers or anal cancers is not widely recommended. The Prevent Anal Cancer (PAC) Palpation Study will recruit MSM and transwomen, aged ≥25 years in two cities in the United States (Chicago and Houston) to test anal self-exams (ASE) and anal companion exams (ACE) that seek to reduce morbidity and mortality from anal cancer.

Methods

The PAC Palpation Study (1R01CA232892) will recruit 800 HIV+ and HIV- persons through 2021 with oversampling of Black MSM given their underrepresentation in HPV research and high risk for HIV. Participants will be taught about anal anatomy, pathology, and the procedure for an ASE or ACE, and then conduct the exam in private and record a result of either abnormal or normal. The primary objective is to compare the participant’s ASE or ACE result at baseline with the clinician’s gold-standard Digital Anal Rectal Exam (DARE) to determine concordance, sensitivity and specificity. The secondary objectives are to test the effect of practice on concordance after one year, and, using mathematical modeling, estimate the cost-effectiveness of ASE, ACE, and DARE and their impact on survival and health-related quality of life.

Results

Enrollment has not begun. Implementation lessons learned include harmonization of human protections, community engagement, and web-based/app-based recruitment and eligibility screening across multiple sites. Quality of life and other questions related to cost-effectiveness have been included in computer-assisted self-interviews.

Conclusions

The PAC Palpation Study will test the ability of ASE and ACE to detect early anal cancer tumors when they are much more treatable. Results could propel the development of a low-resource screening for rapid dissemination to populations with high anal cancer risk and no currently proven screening options.

Introduction

Screening for cervical cancer using an HPV-based assay is currently being implemented in many countries worldwide. The advantage of HPV-based screening is increased sensitivity compared to cytology, but the specificity is inferior. To avoid overtreatment reliable triage of HPV-positive women is required. DNA methylation could be a feasible method for this triage.

Methods

This study is based on a cohort of 305 women from a referred population. From each woman a physician-taken cytology sample, a self-collected vaginal sample, a urine sample and a histology sample was collected. The first 44 HPV-positive physician-taken cytology samples have been tested with a DNA methylation assay, GynTect®. Two-three ml of ThinPrep material was used for the analysis following the manufactures instructions. QPCR was performed on the QuantStudio 12K Flex instrument. A Gyntect score is calculated, and the sample considered positive if the score is equal to or higher than 6. The test result was compared to the histology diagnoses.

Results

In this pilot study 20 samples were tested GynTect positive and 24 were test negative.

The sensitivity and specificity for CIN2+ and CIN3+ were calculated, and for CIN2+ the sensitivity was 64% and the specificity was 88%, while for CIN3+ it was 94% and 85%, respectively.

Conclusions

These preliminary results indicate that the GynTect® assay could be an option for triage of HPV positive samples. Accordingly, the remaining physician-taken samples will be tested to strengthen these initial results, and updated results will be presented.

In addition, the self-collected vaginal samples are also expected to be tested with this methylation assay, as these samples cannot be triaged with cytology.

Introduction

Human Papiloma Virus (HPV) infection and the development of anal cancer (AC) is increasing in HIV-positive men who have sex with men (MSM). Most subjects who screen positive by HPV-DNA testing or cytology do not have concurrent precancer. The challenge is differentiating the screen-positive subjects with benign, transient HPV infections from those that have precancerous lesions with a high risk to progress to cancer. We aim to estimate the anal HPV prevalence and the performance of three HPV detection tests.

Methods

The ELAVI67 project, a prospective longitudinal study, includes samples from 347 HIV-MSM recruited at baseline in Barcelona. Baseline samples have already been collected and follow-up samples are currently being collected every 6/12 months after the baseline during minimum 24 months. The ELAVI67 cohort underwent anal smear and HRA with biopsy of suspected dysplasia areas. Baseline anal smear samples were tested by anal liquid-based cytology, HPV DNA detection performed by both Linear Array (LA) (37 HPV genotypes) and Hybrid Capture®2 (HC2) (13 high-risk (HR-HPV) genotypes), and E6/E7-mRNA test using Aptima® (14 HR-HPV genotypes).

Results

Prevalence HR-HPV was 40.9% by HC2, 79.3% by LA and 50.1% by Aptima. The overall agreement and ki between LA and HC2 for the common genotypes was poor (66%, 0.325). Whereas, the concordance and ki for shared genotypes between LA and Aptima was higher (74%,0.475) and increasingly better as the genotypes compared were restricted to HPV16/18/45 or to HPV16. For the identification of high-grade lesions (HSIL), the detection of HR-HPV by LA showed the most sensitive values (95%), although the specificity dropped to a 28% (AUC,0.61). The test that displayed a better performance was Aptima, showing a sensitivity of 80% and a specificity of 63% (AUC,0.71).

Conclusions

Preliminary results indicate that E6/7-mRNAtest perform better than HC2 and LA and could be considered for the detection of HSIL.

Introduction

Human papillomavirus (HPV) is the principal cause of anal squamous cell carcinomas (ASCC) (>90%). Recently, our team reported the potential interest of HPVctDNA to monitor post-treatment high grade anal intraepithelial neoplasia. No study evaluates HPVctDNA to monitor patients with low grade/high grade anal intraepithelial neoplasia (LGAIN/HGAIN) potentially at risk to develop ASCC.We evaluated HPVctDNA as a predictive and prognostic biomarker in HIV MSM patients particularly affected by LGAIN/HGAIN or ASCC.

Methods

55 HIV MSM with anal lesion followed at Hôpital Européen George Pompidou (Paris) for HIV infection have been enrolled. We characterized oncogenic HPV genotype in anal biopsies. According to it, HPVctDNA detection by droplet-based digital PCR (ddPCR) was performed on plasma samples collected at the time of anal lesion diagnosis. So far, we have developed ddPCR to detect the most prevalent oncogenic HPV namely HPV16, -18, -31 and -33.

Results

In all biopsies, we reported 22 different HPV genotypes and as expected, HPV16 was the most prevalent (49%). Finally, regarding HPV16, -18, -31 or -33 genotype detected in anal lesion biopsy, corresponding HPVctDNA by ddPCR could have been performed on 30 plasmas. No HPVctDNA was detected in plasma of LGAIN/HGAIN patients. For 7 ASCC patients , 5 have undetectable HPVctDNA. For the 2 positive patients, a longitudinal analysis of HPVctDNA from plasma collected one year before and after diagnosis has been performed. In one case, HPVctDNA was already detected in anterior plasma and in both HPVctDNA became negative following treatment, correlating with a clinical cure.

Conclusions

Our result confirmed the capacity of HPVctDNA as a non-invasive biomarker to detect ASCC and its specificity regarding anal lesion stages. Moreover, the possibility to early detect HPVctDNA before anal cancer diagnosis leads the way for a wider evaluation of HPVctDNA as a complementary tool to monitor ASCC development in at risk HIV MSM population.

Introduction

INTRODUCTION :Primary high risk human papillomavirus (HR-HPV) screening is being widely adopted in national programs for cervical cancer screening. However since majority of the HPV infections are transient, most suitable triage strategies for the HR-HPVpositive women are still being explored. This study evaluated the association between HR-HPV viral load and cervical intraepithelial neoplasia (CIN) lesions and its potential for triage after primary HPV screening.

Methods

METHODS: We conducted a retrospective review of 229 HPV positive women in the age group of 30-60, who underwent primary screening for HR HPV DNA testbetween January 2017 to December 2018 at the tertiary cancer centre. HR HPVwas detected by Hybrid Capture 2 assay and viral load was measured by the ratio of relative light units to standard positive control (RLU/CO).Histopathology established the pathological grades of CIN.The clinical performance to detect CIN 2 and above lesions (CIN2+) at different viral load cut-off values was calculated.

Results

RESULTS :The prevalence ofCIN2+ among HPV positive women was 30.6 % (70/229).The mean RLU/CO values for histopathology grades ofnegative CIN, CIN 1, CIN 2, CIN 3, and invasive cancer were 330.65, 610.72, 694.35, 910.85 and 778.39 respectively. The severity of cervical lesions increased with the increasing viral load(P < 0.001).The algorithm using RLU/CO value cut offs at >=10, >=100, and>= 1000 for detecting CIN 2+ lesions had sensitivity of 0.91 (0.82 - 0.97), 0.73(0.61 - 0.83) and 0.33 (0.22 - 0.45)and specificity of 0.26 (0.19 - 0.33), 0.56 (0.48 - 0.64) and 0.86 (0.79 - 0.91) respectively. Increasing the cut-point of the HC2 viral load assay improved the specificity and decreased the false positive ratesat the cost of loss in sensitivity.

Conclusions

CONCLUSIONS: Increase in the HR-HPV viral load increases the risk of cervical cancer and precancerous lesions. Quantifying viral load can be effectively utilised for strengthening cervical screening programs.

Introduction

HPV detection is one the main screening strategies for cervical cancer. Worldwide, 12% of women with a normal cytology are HPV-positive. In the Caribbean and Eastern Africa, such prevalence is high (35%), whereas in North America the prevalence is low (4%). In Mexico high-risk HPV-HR prevalence is around 14%. Differences in the sensitivity of the detection methods used could be the reason for potential differences between populations.

Methods

Cervical scrapes from patients who attended the IMSS cervical cancer-screening program from different regions in Mexico were analyzed, including Puebla, Veracruz, Morelos, Guerrero, Mexico City, Campeche, and Quintana Roo. The samples were collected in PreservCyt medium and processed with CobasÒ4800 HPV Test. The clinical data were collected. Descriptive statistics were performed in the IBM SPSS program.

Results

2526 samples were collected. 20.1% were positive for HPV-HR and only 0.3% were invalid. The presence of HPV-HR was more frequent in Campeche (25.3%), followed by Morelos (23.8%), Veracruz (21.5%), Quintana Roo (18.3%), Mexico City (17.5%), Puebla (16.7%), and Guerrero (15.0%). Of the valid samples, 3.6% of the cases were HPV16, 2.0% HPV18 and 17% positive for other high-risk genotypes. Regarding the HPV16 positive samples, 55% were identified as single genotype, 42.6% in multiple infection with other genotypes. Multiple infections of HPV16 with HPV18 were uncommon (2.2%). The frequency of HPV-HR seems to decrease with age, from 35% in the group <= 24 years of age to 14% in the group of >=50 years of age.

Conclusions

Our results indicate that there are differences in the prevalence of HPV-HR in the populations studied, which could be associated to changes in the risk factors of particular regions in the Mexican territory. It is important the access to molecular HPV test for improve cervical cancer screening.

Introduction

Worldwide, HPV16 is the most common genotype in patients with cervical cancer (CeCa) and samples negative for intraepithelial lesions or malignancy (NILM). Although HPV18 is the second in frequency in most countries, there are regions where the frequencies of other genotypes are higher than HPV18, both in CeCa and NILM. The frequency of HPV genotypes has been poorly studied in the Yucatan Peninsula (southeastern Mexico). This geographical region has particular characteristics, such as a significant population of Mayan origin.

Methods

One thousand three hundred and ninety-seven cervicovaginal samples of women who attended the IMSS cervical cancer screening program were HPV testing by Hybrid Capture 2® (HC2). The genotyping of positive samples was done by HPV direct-Flow Chip system. The cervical cancer samples were Formalin-Fixed Paraffin-Embedded and genotyped by Inno-Lipa HPV Genotyping Extra.

Results

We found that 26.7% of samples were positive for HPV. The frequency of HPV decreased according to age: in the group under 24 years, the frequency was 44% and in women >= 50 years the frequency was 17.4%. The genotypes identified in the screening samples were (in descending order): HPV51, 52, 59, 58, 31, 66, 16, 18, 45, and 39. While in the cervical cancer samples, the genotypes identified were (in descending order): HPV16, 18, 39, 52, 58, 35, 45, 31, and 51. Interestingly, in CeCa, HPV39 was as frequent as HPV18 (20%), while HPV52 and HPV58 were particularly frequent (17% and 9%).

Conclusions

Although HPV16 and HPV18 were the most common genotypes in women with CeCa from the Yucatan Peninsula, they were not the most frequent genotypes in screening samples. HPV52 and 58 were particularly frequent in this Mexican population. Interestingly, these two genotypes are also common in some regions of Asia.

Introduction

Among HPV infections (HPVI), there is a plentiful literature about cervical swabs, but a poor epidemiology about different body districts. Nowadays, anal cancers are screened for the presence of HPV and the pathological role of this virus is well known. We use mRNA test to distinguish transient HPVIs from persistent or progressive ones even in male population

Methods

From May 2015 to January 2019 we analyzed our database comprehensive of a consecutive pool of 4100 patients (401 male, M) who routinely came to our attention for a HPVI screening. We offered swab tests to look for HPVI at cervical, oral, anal and seminal/urethral area

Results

Cervical: 3649 patients, 2524 HPVI detections, 1336 co-infections, 1256 High risk HPV types detected. Anal: 148 patients (56M), 94 infections (35M), 54 co-infections, 38 High risk HPV types. Oral: 52 patients (20M) 1 infection. Seminal/Urethral: 399 patients, 209 infections (92 co-infections, 86 High risk HPVI). 998 tests patients were followed by E6/E7-mRNA qualitative expression analysis. 206 were positive (15M) with 18 co-infections.
Anal cytology listed 62 patients with a swab test. Among them, 12 patients had a high risk HPVI, and 2 of them had anal carcinoma

Conclusions

Since the male population have a poor compliance in term of awareness about HPVI, we hope to improve screening for general population. Our HPV DNA/mRNA data are not satisfying, but the mRNA analysis is effective to assess the aggressiveness of the infection. Genotyping analysis remains the main screening tool for the evaluation of HPV infection

Introduction

A change of the current screening algorithms to a HPV-based screening setting is discussed in several countries due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs.

Methods

A series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (n = 5), cervical intraepithelial neoplasia grade 1–3 (n = 74), and normal cytology (n = 201; further n = 352 collected in SureThin) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas HPV and CINtec Plus biomarker results.

Results

All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV (all HPV types) and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%).

Conclusions

GynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases.

Introduction

HPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. Methylation markers are presently discussed as a suitable tool for triaging HPV positive women. We compared the two assays GynTect and QIAsure.

Methods

In a retrospective setting 210 samples from the colposcopy clinic of the university hospital in Jena were tested with GynTect, comprising 6 (ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671) different methylation and QIAsure, comprising 2 (FAM19A4, mir124) methylation markers. The cohort comprises 2 cervical cancer scrapes, 5 CIS, 38 CIN3, 18 CIN2 and 15 CIN1 and 92 no CIN samples, all tested HPV positive. In addition, 40 HPV negative Pap I samples were tested.

Results

A subset of 2 cervical cancer scrapes, 43 CIN3 and CIS, 8 CIN2 and 5 CIN1 and 82 no CIN, all HPV positive, have already been tested with both assays. Detection rates of cancer and CIN3 cases were similar with 100% detection of cancer and CIS with both assays and 68.4 vs 60.5% CIN3 detection for QIAsure and GynTect, respectively. Regarding ≤CIN2 samples, detection rates are more different with 37.5 vs 25.0% for CIN2, 40.0 vs 20.0% for CIN1 and 37.5 vs 13.4% for no CIN samples.

Conclusions

Analyses for more ≤ CIN2 samples are still ongoing and will be completed for the meeting. This subset already indicates a difference between both assays regarding the detection rates of mild lesions and HPV positive, but unsuspicious samples.

Both assays intend to detect HPV-positive women with clinically relevant disease, thus being a suitable tool for the use in triage screening settings. So both, sensitivity for relevant lesions and adequate specificity is required for such a triage assay.

Introduction

As HPV is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical, head and neck cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention, as a diagnostic complement and evaluation of the effectiveness of the vaccines available. In this context, this study compares four methodologies for HPV detection and genotyping: HPV-HR+GT 16/18 test on cobas® 4800 HPV System (real-time PCR based), nested-PCR followed by conventional Sanger sequencing, High+Low PapillomaStrip kit (reverse hybridization based) and rolling circle amplification (RCA) followed by next generation sequencing (NGS) at Illumina HiSeq2500 platform.

Methods

Cervical samples from 2,076 patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil were collected and analyzed for HPV infection using cobas HPV test. Of those, 114 samples were randomly included in this study and grouped according to the results obtained by cobas. Sanger sequencing was performed on a nested MY09/GP05 PCR fragment, reverse hybridization using High+Low Risk PapillomaStrip kit was based on a PCR of HPV E6-E7 genes to identify 37 HPV types and NGS was performed from RCA product.

Results

Our data shows heterogeneity among the four methods used for HPV identification with concordance rates ranging from 66.7 to 100%. Reverse hybridization and cobas showed limitations for genotyping as they restrict the HPV types identified based on probes or type-specific primers, however they were more efficient to identify the presence of high-risk HPVs with clinical relevance. On the other hand, Sanger sequencing and NGS preceded by non-biased assays would be indicated for HPV prevalence and genotyping studies because they can identify each HPV type present in the samples, including novel HPVs.

Conclusions

The four methodologies studied have advantages and disadvantages, and their use should vary according to the purpose of each study.

Introduction

Some populations are more vulnerable to the development of anogenital cancer due to HR-HPV infection, such as HIV+ individuals and those who have limited access to health services as ethnic and racial groups, who live in semi-isolated Brazilian territory (quilombola communities). The incidence of cervical cancer remains high in the Brazilian population despite control actions adopted (cytopathology and vaccination 4vHPV), while there are no clinical trials to provide suitable screening for anal cancer. Therefore, biomarkers emerge as promising tools for anogenital cancer screening. We aimed to investigate whether the viral load corresponding to nonavalent vaccine (9vHPV) types is related to cervical and anal lesions in vulnerable populations (MSM and women HIV+, and quilombola women). This study obtained approval by the Ethical Research Council of the Federal University of Espírito Santo, Brazil.

Methods

Viral DNA was extracted from 267 cervical and anal samples (QIAamp DNA Mini Kit™-QIAGEN). HPV was screened with sets of PGMY09/11 primers and genotyped by Reverse Line Blot (RLB). The viral load of HR-HPV was determined by Real-time PCR (TaqMan® protocol) from 158 patients wihout lesions, 62 with LSIL and 47 with HSIL.

Results

The median viral load was always higher for HIV+ than for HIV- individuals. The HR-HPV 16/18 viral load was significant in HSIL for HIV- patients (p=0.02), but not for HIV+. HPV16-infected HIV- individuals had always low viral load (≤10 copies/cell) in cases of normal cytology, and at least moderate (>10 copies/cell) in cases of the lesion (p=0.04). HPV16 and HPV31 viral load behaved differently regarding HIV serological status: it was statistically significant in cases with lesion for HPV16 in HIV- (p=0.002), and for HPV31 in HIV+ (p=0.001).

Conclusions

The median viral load increased according to the “no lesion”, “LSIL” to “HSIL” cytology, regardless of HIV serological status. HPV16 and HPV31 viral load behaved differently according to HIV serological status.

Introduction

Head and neck carcinomas (HNC) are the world's sixth most common cancer. Most of HNC are associated with tobacco and other environmental factors but a growing part of oropharyngeal tumors are caused by persistent infection of human papillomavirus (HPV). Patients with HPV positive cancers have a better prognosis with fewer recurrences. This may be caused by different anti-tumor immune response and immune profile of patients. Multispectral fluorescent immunohistochemistry (fIHC) is a powerful tool for a detailed analysis of the tumor microenvironment. This method allows to access the phenotype and calculate cells in different compartments of the tumor since in comparison to flow cytometry, an architecture of the tissue remains preserved. fIHC is uniquely suited to study interaction of immune and cancer cells in situ.

Methods

In this study four different panels consisting of five antibodies each were optimized and tested on formalin-fixed paraffin-embedded (FFPE) slides of the human tissue using Opal™ 7-Color Fluorescent IHC Kit (PerkinElmer). These panels include antibodies against markers for phenotyping of immune cells (CD3, CD4, CD8, FOXP3) as well as for the description of their function (PD1, CTLA4, ICOS, CCR4). The presence and quantity of immune cells with different phenotypes were evaluated in stroma and tumor compartment using InForm™ software (PerkinElmer) in retrospective samples with known etiology. For all patients the demographic and clinical data were available and these patients were followed for up to 18 years.

Results

Preliminary analyses have shown statistically significant differences in number of cells of different phenotypes in tumors of different etiology. Besides HPV etiology some population of immune cells in the tumor microenvironment predict independently better survival of patients. More detailed survival analyses with inclusion of other clinical and demographic data will be presented.

Conclusions

Detailed analyses of the tumor infiltrating lymphocytes allows for selection of prognostic markers in HNC of different etiology.

Introduction

Persistent infection with high-risk human papillomavirus (HR-HPV) is the necessary cause of cervical carcinoma. HPV DNA testing is used for primary screening and co-testing. A primary focus to identify HPV infection at stage of integration of HPV viral genome to cervical epithelial cell. The activity of E6/E7 mRNA needs to be quantified within infected cervical epithelial cell.

Methods

Women presenting with intermenstrual bleeding, postcoital bleeding, prolonged vaginal discharge or an unhealthy cervix underwent VIA and collection of cervical samples in ThinPrep® PreservCyt® Solution (Hologic MA, USA) for HR-HPV DNA testing (HC2, Qiagen), cytology HPV mRNA (HPV OncoTect 3Dx (IncellDx, CA, USA), colposcopy and biopsy.

Results

302 women underwent screening. Lesions detected were: CIN1 (n=39), CIN2 (n=3), CIN3 (n=3) and invasive cancer (n=3). VIA and HC2 showed the best sensitivity. HPV OncoTect 3Dx test standardization was done. Samples were hybridized with oligonucleotide probes for E6/E7 mRNA and counterstained with a nuclear dye for cell cycle analysis. Cells were analyzed on a CytoFLEX (Beckman Coulter, Inc. CA). Ectocervical cells were differentiated from endocervical cells, inflammatory cells and debris using forward and side light scatter properties, depending on the size of cell and cytoplasmic complexity. It quantitatively detected both E6/E7 mRNA overexpression and cell proliferation in intact cervical cells

Conclusions

E6/E7 mRNA detection allows identification of a transcriptionally active virus genome. A larger study is needed for standardisation of the morphological cut-off to differentiate between transient and persistent HPV infections, recognize high-grade CIN and establish its role in the screening strategy

Introduction

Cervical cancer remains a significant cause of morbidity and mortality in women worldwide and is the leading cause of cancer-related death in Botswana. Persistent HPV infection leads to cervical cancer. Host epigenetic changes have also been recognized as important players in carcinogenesis.

Methods

We assessed hr-HPV prevalence and genotype distribution in tissue specimens from confirmed invasive cervical cancer cases and non-cancers using Real-Time PCR. We also assessed the methylation status in invasive cancer vs. non-cancers in bisulfite-treated DNA by Methylation-Specific PCR using primers designed to distinguish methylated from un-methylated DNA in order to evaluate DAPK1 promoter methylation as an epigenetic marker.

Results

126 cervical cancer cases and 86 non-cancers were analyzed. 88 (69.8%) women with cervical cancer were HIV-infected. Fifty-seven (64.8%) of the HIV-infected women had a baseline CD4⁺ count ≥ 350 cells/μl, and 82 (93.2%) were on ART at the time of cervical cancer diagnosis. The median age of HIV-infected patients was significantly younger than that of HIV-uninfected patients (p < 0.001). Hr-HPV genotypes identified included the HPV-16 (75.4%), HPV-18 (28.6%), and other hr-HPV genotypes (16.7%). HIV infection was positively associated with the presence of the HPV-16 genotype (p = 0.036), but not with HPV-18 or with other hr-HPV genotypes. Overall methylation analysis was assessed in 85 cervical cancer cases and 86 non-cancer tissue blocks. DNA methylation status in cervical cancer patients was 64.7% compared to the non-cancer patients (25.6%). A significant strong association between DAPK1 promoter methylation and cervical was shown compared to non-cancer (p<0.001). HIV status was not associated with DAPK1 promoter methylation (p=0.87) in cervical cancer group.

Conclusions

These results highlight the importance and potential impact of large-scale HPV vaccination programs covering HPV-16 and HPV-18 genotypes in countries like Botswana with high burden of HIV infection. Furthermore methylation markers may have valuable role in early detection of invasive cervical cancer.

Introduction

Human Papillomavirus (HPV) is now recognized as a causative agent of oropharyngeal squamous cell carcinoma (OSCC) which is associated to a better prognosis and affects young men mostly. Among the different mechanisms involved in clinical outcome of OSCC, a deregulation in the immune response avoids the recognition and elimination of neoplastic cells. AKNA, an AT-hook transcriptional factor, is regulated by HPV, nevertheless, little is known about AKNA status in HPV positive OSCC.

Methods

Through the analysis of tumor biopsies from the National Cancer Institute from Mexico, a retrospective study was conducted to determine the levels and localization of AKNA by Immunohistochemistry Image Analysis.

Results

The results indicated only a 17.9% of positivity to HPV. AKNA expression was heterogeneous in tumor cases being positive in the nucleus in a 46%. After Kaplan Mayer analyses, the presence of HPV did not show an impact in overall survival, whilst AKNA nuclear localization was close to significance, indicating that AKNA could be active in these tumors.

Conclusions

The results suggest that loss of AKNA might be contributing to a better overall survival. Still, it is necessary to determine the significance of HPV in the modulation of AKNA levels in OSCC.

Introduction

In precancerous lesions of the cervix and in cervical cancer (CC), a state of immunosuppression has been identified, characterized by an increase in the expression of type II interleukins (IL-4, IL-10, suppressors of the cellular immune response) and a concomitant reduction of interleukins type I (IL-2, INF-γ), which favors the persistence of HPV infection. The objective of this study was to evaluate the association of genetic polymorphisms of the -590C>T (IL-4); −573G>C (IL-6); −592C>A, −819C>T and −1082A>G (IL-10); −509C>T (TGF-β1); −308G>A (TNF-α) and -1615C>T (IFN-γ) with persistence and clearance of HPV in the cervix.

Methods

Dynamic cohort study in 267 HPV positive women in the cervix and who were included in the baseline study and evaluated at 12 months to determine persistence or viral clearance. HPV molecular test and evaluation of allelic discrimination polymorphisms with Taqman probes in peripheral blood mononuclear cell samples was realized. Bivariate and logistic regression analysis was performed to determine the statistical association of these polymorphisms with persistence and clearance-HPV adjusted by potential confusers.

Results

We found to be a carrier of polymorphisms -573G>C (IL-6), -380G>A (TNF-α), -1615C>T (IFN-γ) is a risk factor for persistence of HPV infection, while being carriers of polymorphisms -590C>T (IL-4) and -509C>T (TGF-β1) confer protection against persistence and elimination of HPV infection, respectively.

Conclusions

The associations found for the polymorphisms of IL-6, TNF-α and IFN-γ suggest that these SNPs are potential predictors of persistent HPV infection in the cervix. It is necessary to evaluate its potential use and thus reduce the current care burden of diagnosis and treatment of premalignant lesions and CC.

Introduction

Urine sampling is an interesting solution to increase the uptake of cervical screening. Previous studies pointed out good accuracy for HPV detection and the feasibility of cervical cancer detection by urinary DNA methylation analysis. Urine can be separated in different fractions: full void urine, urine sediment and urine supernatant. This study aims to determine which urine fraction is most competent to distinguish between healthy controls, high grade cervical intraepithelial neoplasia (CIN3) and cervical cancer by DNA methylation analysis.

Methods

Urine samples were collected from 17 women with cervical cancer, 30 women with CIN3 and 30 healthy female controls. Each urine sample was processed into 3 fractions and tested for 5 methylation markers (ASCL1, GHSR, LHX8, SST, ZIC1) by quantitative methylation specific PCR. Spearman correlation coefficients between paired urine fractions were determined and methylation levels between disease categories were compared. Receiver operating characteristic curves were made and area under the curve (AUC) was calculated for CIN3 and cervical cancer detection.

Results

In general strong correlations (r>0.60) were found between the different urine fractions for all markers. Significant difference between CIN3 patients and controls was found for 2 out of 5 markers in full void urine, 4 out of 5 markers in urine sediment and 2 out of 5 markers in urine supernatant (AUC 0.55- 0.79). All markers demonstrated a significant increase in DNA methylation levels and an excellent performance (AUC 0.87- 0.99) in all urine fractions to discriminate between cervical cancer and controls.

Conclusions

This study shows a good performance of all urine fractions for cervical cancer detection. Our results also indicate the potential of CIN3 detection by urinary methylation analysis and demonstrate best performance of urine sediment to detect CIN3.

Introduction

There is growing interest in methods of triaging HPV-positive women in terms of risk. Extended genotyping is one of the methods of assessment of an increased risk of developing cervical carcinoma. This study aims to assess in a private practice scenario the validity of utilising extended genotyping as a method of choice.

Methods

We undertook an investigation of HPV genotype prevalence within specific subsets of cervical cancer screening patients within South Africa. There were three patient cohort arms included in this study: (i) high-risk HPV positive (non-16/18) patients; (ii) high-grade cytology cases; and (iii) invasive cervical cancer. All specimen were collected in BD SurePath vials and HPV genotyping analysis was conducted using the BD Onclarity HPV Assay. All biopsy specimens were analysed from FFPET.

Results

The first arm involved triage of Roche cobas “other 12” positive cases irrespective of the Pap test results.

Preliminary data from the Roche cobas “other 12” positive cases (N=113) were generated by genotype analysis using the Onclarity HPV assay. Surprisingly, approximately one-third of the Roche cobas cases were negative to high-risk HPV genotypes using the Onclarity assay. Of the remaining positive cases (N=75): 56/59/66 (32%); 35/39/68 (24%); 31 (18%); 52 (16%); 33/58 (16%); 45 (11%); and 51 (9%).

The second arm is investigating extended genotyping on HSIL, ASC-H, AGC and malignant pap tests.

The third arm includes biopsy specimens of cervical malignancies to establish the prevalence of high risk HPV genotypes..

Additional data will be presented on the prevalence of HPV genotypes within these different categories.

Conclusions

Emerging clinical evidence in the peer-reviewed literature has identified HPV extended genotyping as a potential triage method for high-risk HPV positive patients and atypical cytology cases. We have undertaken a systematic investigation of extended HPV genotyping as a triage tool applicable to the South African cervical cancer screening.

Introduction

Improving the specificity and accuracy for anal hHSIL screening minimizes healthcare costs.

Methods

431 HIV-infected/-uninfected MSM (332), cis-women (96) and transfemine-women (3) were evaluated using Dacron-cytology (aCyt), hrHPV-DNA, hrHPVE6/E7, and HRA/biopsy to predict histological HSIL (hHSIL). Sensitivity, specificity, and area under Receiver-Operating Characteristic (AUC) curves were evaluated for chemiluminescence compared to control (hrHPV-DNA=RLU/Co and hrHPVE6/E7=S:CO) as positivity cut-points: >2, >4, >6, >8, >10 S:CO or RLU/Co, respectively, compared to manufacturer recommended cut-point, >0.5 S:CO and >1 RLU/Co. Gender- and HIV(⁺)-adjusted logistic regression models estimated odds of hHSIL for each cut-point. Statistical contrasts evaluated differences between cut-points, overall.

Results

Mature adult (54.6 (σ=11.4) years), ever-smokers (61%) comprised the cohort; 44% reported HIV-infection. Men showed 2-fold higher odds of >ASC-US cytology (p<0.05) and nearly 3-fold higher prevalence of hHSIL than women (45% vs. 16% p<0.0001).

Adjusted analyses showed aCyt sensitivity to predict hHSIL at any alternative cut-point did not significantly vary using either test across these cut-points (p>0.05). However, using either hrHPV test, at all cut-points, specificity for hHSIL was statistically significantly greater than aCyt: hrHPV-DNA=69%-83%; hrHPVE6/E7=77%-82% vs. 51%. All single-test hrHPV-DNA cut-points >4 RLU/CO showed higher specificity than >1 RLU/CO (78%-83%, p-values <0.02). Single-test hrHPVE6/E7 specificity improved for cut-points >8 vs. >0.5 S:CO (81%-82% vs. 73%, p-values<0.05).

Conclusions

Adjusting positivity cut-points for anal hHSIL screening using hrHPV-DNA or hrHPVE6/E7 assays improves specificity appreciably over aCyt, while retaining similar sensitivity. Employing molecular tests as a primary screen for anal hHSIL opens opportunities to evaluate targeted, cost-effective population-based cancer prevention strategies.

Introduction

Low-grade cervical dysplasia is frequently managed by observation because most dysplasia will resolve without intervention. Currently there are no clinical tests that will identify women at risk for progression to high-grade dysplasia from among those that will resolve naturally. A molecular biomarker that predicts the outcome of low-grade CIN could be used clinically to triage women for definitive care.

Methods

We conducted a retrospective case-control study to reveal microRNAs that could predict the outcome of low-grade CIN. Women were identified through electronic medical records as having a diagnosis of low-grade CIN. Women with a subsequent diagnosis of high-grade CIN were designated as cases (n=22). Women with a significant history of normal cervical tests following the low-grade CIN diagnosis and without evidence of clinical intervention were included as controls (n=29). Total RNA was isolated from the archived diagnostic specimen with the low-grade CIN diagnosis. MicroRNA expression was analyzed using arrays with probes for all known human microRNAs (LC Sciences, Houston, TX). Differential microRNA expression between cases and controls was calculated.

Results

Twenty-nine microRNAs were differentially expressed at the p<0.01 level when comparing cases to controls. Notably, miR-638 was significantly downregulated and miR-1260a was significantly upregulated in women with low-grade CIN that progressed to high-grade CIN. Literature supports a role for miR-638 as a tumor suppressor miRNA, while miR-1260a has been shown to promote migration and invasion properties. These findings are consistent with a possible role for miR-638 and miR-1260a in the pathophysiology of cervical dysplasia progression.

Conclusions

MicroRNA testing, either alone or in combination with other biomarkers, may have a role in future clinical tests to direct management of women with low-grade CIN. Dysregulated microRNAs may also play a role in the pathophysiology of dysplasia progression.