Journal of Virological Methods

Performance of a six-methylation-marker assay on self-collected cervical samples – A feasibility study

In high-income countries, a high proportion of cervical cancers is diagnosed in screening non-attendees. One approach to improve screening coverage is to offer self-sampling for human papillomavirus (HPV) testing. However, especially young women are often HPV positive without having a precancerous lesion in need of treatment. To improve the rather low specificity of HPV testing additional markers such as DNA-methylation can be used. The aim of this feasibility study was to examine the performance of the methylation marker assay GynTect®, comprising six methylation markers, on dry self-collected cervico-vaginal samples compared to physician-taken samples. We recruited 89 patients from our colposcopy clinic of whom 87 qualified for the study. The women took a self-sample with the Evalyn-Brush. Afterwards the planned colposcopy was performed and smears for cytology and reference HPV testing were taken as well as a biopsy in cases of abnormalities. Physician-taken and self-collected samples were tested for HPV DNA and were analyzed with GynTect®. We obtained 95.5 % valid results for the self-collected samples which was very close to the physician-taken samples. Only about half of the self-collected samples were GynTect® positive in comparison to the physician-taken samples. GynTect® scores were significantly lower for self-collected than for physician-taken samples (p = 0.001, paired t-test). The overall concordance for GynTect® results was moderate (kappa 0.394; p < 0.001). For HPV testing we obtained a good concordance (kappa 0.586; p < 0.001). The GynTect® results for the self-collected samples showed a sensitivity for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+) of 26.1 % (95 %-CI: 0.13-0.46) and a specificity of 95.6 % (95 %-CI: 0.85-0.99), in comparison to a sensitivity of 45.5 % (95 %-CI: 0.27-0.65) and a specificity of 78.3 % (95 %-CI: 0.64-0.88) for the physician-taken samples. GynTect® methylation marker testing has a satisfactory amount of valid results on self-collected samples. However, the results of the self-collected samples differed clearly in comparison to the reference samples. To justify an application in screening, a larger study with more cases of high-grade cervical dysplasia and HPV positive patients will be needed.

Reference gene evaluation for digital PCR; Applications for RNA biomarker testing in cervical precancer

Persistent infection with high-risk human papillomavirus (hrHPV) causes almost all cases of cervical cancer. Despite the success of cervical screening in reducing cervical cancer incidence, novel tests are required to identify patients with HPV infection who do not have clinically significant disease, minimising unnecessary diagnosis and inappropriate treatment. Digital PCR (dPCR) is a technology that can support the identification and validation of mRNA biomarkers as it allows quantification with high precision. In gene expression studies, the use of reference genes is essential for accurate quantification of the target molecule. We investigated the suitability of a panel of eight reference genes (ACTB, GAPDH, RPP30, HPRT1, HMBS, MT-ATP6, UBE2D2 and GUSB) for normalisation of dPCR gene expression data in liquid-based cytology (LBC) samples representing low (CIN1) and high-grade (CIN3) cervical disease. To identify stable candidates, reference genes were compared using geNorm and NormFinder. Results of geNorm analysis indicated that inclusion of the four best performing reference genes (GAPDH, ACTB, GUSB and MT-ATP6) is optimal. GAPDH and ACTB were the most stable genes overall but were expressed at very high levels. Therefore, they may not be suitable for normalisation of dPCR data of putative biomarkers where expression levels are consistently much lower. Instead, we recommend the use of GUSB and HMBS as a stable reference gene pair. These are expressed at a suitable level for accurate normalisation of biomarker expression using dPCR.

Evaluation of HarmoniaHPV test for detection of clinically significant Human Papillomavirus infection using the VALGENT framework

The VALidation of HPV GENotyping Tests (VALGENT) is a framework for comparison and validation of HPV tests with genotyping capabilities. In this study, the clinical performance of a single tube HPV test -HarmoniaHPV- was assessed in SurePath™ samples and compared to a clinically validated reference test, the GP5+/6+ Enzyme ImmunoAssay (GP5+/6 + EIA). HarmoniaHPV test is a real-time, PCR based, limited genotyping HPV test which detects 14 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 with HPV16, and HPV 18 reported individually. Clinical performance was assessed using 998 unselected, cervical screening samples enriched with 297 cytologically abnormal specimens (100 atypical squamous cells of unspecified significance, 100 low-grade squamous intraepithelial lesions, 97 high-grade squamous intraepithelial lesions). Cases were defined as women diagnosed with histologically confirmed cervical intraepithelial neoplasia 2 or more (≥CIN2, N = 122). Using the manufacturer recommended (un-adjusted) cut-offs, HarmoniaHPV had non-inferior sensitivity for detection of ≥ CIN2 but showed inferior specificity. A cut-off optimisation exercise was therefore carried out and optimised cut-offs for each individual channel rendered a sensitivity and specificity of HarmoniaHPV that was non-inferior to GP5+/6 + EIA. Analytically, the test showed excellent intra- and inter-laboratory reproducibility, which improved further with the use of the optimised cut-offs. HarmoniaHPV when operated with optimised cut-offs fulfils the international clinical criteria for use in cervical cancer screening on SurePath samples. The optimised cut-offs warrant additional testing and independent validation.

Concordance of Anyplex™ II HPV HR assays with reference HPV assays in cervical cancer screening: Systematic review

Genital infection with certain types of human papillomaviruses (HPVs) is a major cause of premalignant dysplasia and cervical cancer globally. Identification of 14 high-risk human papillomaviruses (HR-HPV) is immensely important in elucidating molecular epidemiology, patient monitoring, and evidence-based treatment. HPVs testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. Thus, HPV assays are increasingly used for primary cervical screening and HPV genotyping, for monitoring vaccination-effect and determining changes in the epidemiology of viral genotypes across the globe. Testing for high-risk HPV is more effective in primary cervical cancer screening than in the cytological examination of a Pap smear. Separate genotyping may be useful for triage in both HPV-based and cytology-based screening. It should be used only for clinically validated tests. To evaluate the concordance of Anyplex™ II HPV HR with other HPV assays in cervical cancer screening. Validation studies of Anyplex™ II HPV HR assay from PubMed, google scholar, google database and Scopus were used to search articles. Articles published in English from 2013 onwards were systematically searched using keywords. Besides, other databases like Google Scholar and the Google database were searched manually for grey literature. The last search was done in December 2020. Then study eligibility, extracted data, and assessed risk of bias were assessed. Due to the great clinical heterogeneity of the included articles, the diagnostic performance of the anyplex II ™ HR HPV test was unlikely to be pooled. Rather, I did a descriptive presentation of the test performance to gather the best synthesis of evidence for the anyplex II ™ HR HPV test for the detection of CIN2+. Studies that evaluate the performance of the assay in terms of its sensitivity, specificity, reproducibility and positive and negative predictive values to comparator assays and/or histology were included in this review. Anyplex™ Ⅱ HPV HR showed consistently high absolute clinical sensitivity for CIN2+ and CIN3+, as well as comparative clinical sensitivity relative to the currently most widely used HPV test. Because of the significantly diff ;erent composition of the referral populations, Anyplex™ Ⅱ HPV HR absolute clinical specificity for CIN2+ and CIN3+ varied across studies but was comparable relative to reference assays. Five validation studies of Anyplex™ Ⅱ HPV HR performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, still comparative clinical sensitivity and specificity relative to HC2 and GP5+/6 + PCR. Anyplex™ Ⅱ has evaluated HPV HR in several settings and population groups. It is considered clinically validated for primary cervical cancer screening and triage in referral population settings.

Successful retrieval of human papillomavirus DNA after a 4.5 year storage on FTA elute cards

Efficient primary (vaccination) and secondary (screening) prevention strategies have the potential to eliminate cervical cancer worldwide. In this context, surveillance of HPV infections remains mandatory to assess the efficacy and the impact of screening and vaccination policies. Therefore there is a need to safely store cervical samples to conduct long-term studies in vaccinated and non-vaccinated subjects. Up-dated data on cervical specimen preservation on FTA® cards indicate that HPV DNA can be safely retrieved after 54 months of storage. A concordance of 97 % was achieved between HPV genotypes detected in initial cervical samples and on FTA® cards 4.5 years later. Even if a drop in HPV viral loads was observed in some cases at 4.5 years, using FTA® cards for safe and long-term storage of cervical samples represents an interesting option.

Clinical validation of the Roche cobas HPV test on the Roche cobas 6800 system for the purpose of cervical screening and qualification as a second-generation comparator test

This study assessed the relative clinical sensitivity and specificity for cervical precancer of the Roche cobas HPV test when processed using the Roche cobas 6800 system using the Roche cobas 4800 HPV test as comparator. Intra- and inter-laboratory reproducibility were evaluated as well. The cobas HPV test run on the cobas 6800 platform demonstrated a relative clinical sensitivity of 1.00 for histologically confirmed CIN2 + lesions in woman aged 30 years or older, with a relative clinical specificity of 1.001 (p for non-inferior accuracy < 0.0001). The intra- and inter-laboratory reproducibility were 99.6 % and 99.8 % respectively. The study is the third to show that cobas HPV testing on the 6800 platform consistently demonstrates a relative clinical sensitivity of ≥ 0.95 and a relative clinical specificity of ≥ 0.98 for CIN2 + . This would qualify it, according to a recently published criteria, as a second-generation comparator assay.

The value of HPV E6/E7 mRNA quantitative analysis in distinguishing high-grade cervical squamous intraepithelial lesions from low-grade cervical squamous intraepithelial lesions

A quantitative assay for HPV E6/E7 mRNA may be a valuable tool for cervical cancer screening. The purpose of this study is to compare the expression levels of HPV E6/E7 mRNA in high-grade cervical squamous intraepithelial lesion (HSIL) and low-grade squamous intraepithelial lesions (LSIL) and to determine a new method that can be used to distinguish cervical squamous intraepithelial lesions. Routine cytology, HR-HPV E6/E7 mRNA, histology, and p16 immunohistochemistry were performed in tissues from 142 patients with cervical squamous intraepithelial lesions. Significant differences were observed between the E6/E7 mRNA copy number values between the LSIL and HSIL cases (Mann-Whitney U-test, P < 0.001). The optimal cut-off value (≥9,222.00 copies/mL) was determined using the receiver operating characteristic curve to predict diagnostic performance. Out of the 161 samples tested in this study, four cases were classified cytologically as HSIL but had normal histology. The E6/E7 copy numbers in these cases were all higher than 9,222 copies/mL. Therefore, a quantitative assay for HPV E6/E7 mRNA may be a valuable tool that can be used to distinguish HSIL and LSIL, especially for those with HSIL, for which samples are not obtained by biopsy, or when HSIL is difficult to distinguish by morphology and p16 immunohistochemistry.

Optimization of droplet digital PCR assays for the type-specific detection and quantification of five HPV genotypes, including additional data on viral loads of nine different HPV genotypes in cervical carcinomas

The droplet digital PCR (ddPCR) system enables high-sensitivity detection of nucleic acids and direct absolute quantification of the targets. The aim of this research was to evaluate this system for viral load (VL) analysis of the human papillomavirus (HPV) genotypes HPV31, 35, 39, 51 and 56 measured in number of viral particles per cell. The sample types used for the optimization of the ddPCR assay were formalin-fixed paraffin-embedded (FFPE) tissues and cervical liquid cytology samples. The presently optimized ddPCR assays, together with assays optimized previously for HPV16, 18, 33 and 45, with the same ddPCR method, were used for the VL analysis of cervical tumor samples. Results published previously on the present study cohort showed that women with a cervical tumor containing multiple high-risk HPV genotypes had a worse prognosis compared to women with single-genotype-infected tumors. The VL was therefore analyzed in this study for the same cohort, as a possible explanatory factor to the prognostic differences. The results of the optimization part of the study, with analysis of VL using ddPCR in DNA from varying sample types (FFPE and liquid cytology samples), showed that each of the five assays demonstrated good inter- and intra-assay means with a coefficient of variation (CV) under 8% and 6% respectably. The cohort results showed no difference in VL between tumors with multiple and single HPV infections, and therefore did most likely not constitute a contributing factor for prognostic differences observed previously. However, tumors from women aged 60 years or older or containing certain HPV genotypes and genotype genera were associated with a higher VL.

Clinical and analytical performance of the CLART HPV 4S assay with SurePath screening samples from the Danish cervical cancer screening program using the VALGENT framework

The CLART HPV4S (CLART4S) is a novel full genotyping assay, based on PCR/microarray technology. We assessed the clinical accuracy of the CLART4S assays under the fourth installment of the VALGENT framework. The VALGENT cohort comprised 998 consecutive cervical samples from women participating in the Danish screening programme enriched with 297 samples with abnormal cytology (100 ASCUS, 100 LSIL, 97 HSIL). The CLART4S assay detects 16 HPV genotypes individually: 14 oncogenic and two non-oncogenic HPV types. The GP5+/6+ PCR Enzyme-Immuno-Assay (GP-EIA) and GP5+/6+ PCR with Luminex genotyping (GP-LMNX) were used as comparator tests for clinical accuracy and HPV genotype concordance, respectively. The sensitivity for ≥ CIN2 for the CLART4S assay was 96.7 % (GP-EIA: 92.6 %) with a relative sensitivity of 1.04 (1.00-1.09). The sensitivity for ≥ CIN3 was 98.8 % (GP-EIA: 94.0 %), with relative sensitivity of 1.05 (1.00-1.10). The specificity for <CIN2 was 88.6 % (GP-EIA: 89.2 %) with a relative specificity of 0.99 (0.98-1.01). The CLART 4S was found to be non-inferior to that of GP-EIA for both sensitivity (p < 0.0001) and specificity (p = 0.0452). The overall oncogenic HPV concordance between CLART4S and GP-LMNX was high, however when looking at individual genotype agreement the concordance was more diverse, with the highest agreement found in the Screening population.