Comparing the Costs and Diagnostic Outcomes of Replacing Cytology with the QIAsure DNA Methylation Test as a Triage within HPV Primary Cervical Cancer Screening in The Netherlands.

Detecting hypermethylation of tumour suppressor genes could provide an alternative to liquid-based cytology (LBC) triage within HPV primary cervical screening. The impact of using the QIAsure® FAM19A4/mir124-2 DNA Methylation Test (QIAGEN, N.V, Hilden, Germany) on CIN3+ diagnoses, retention, unnecessary colposcopies, and programme costs is unknown. A decision-tree model was developed to compare LBC with the QIAsure Methylation testing to guide colposcopy referral. Incorporating clinician- and self-sampling pathways the model was informed by the Dutch cervical cancer screening programme, published studies, and manufacturer data. Clinical and cost outcomes were assessed using two scenarios for DNA methylation testing and LBC relative performance. Sensitivity analyses (deterministic and probabilistic) were performed to assess model and parameter uncertainty. A range of self-sampling uptake was assessed in scenario analyses. For the screening cohort (n = 807,269) where 22.1% self-sampled, the number of unnecessary colposcopies and CIN3+ diagnoses varied according to the relative performance of methylation testing and LBC. Irrespective of relative performance, the cost per complete screen was lower and fewer people were lost to follow-up when using DNA methylation testing. The results indicate that, within an HPV primary screening programme that incorporates self-sampling, using the QIAsure Methylation Test for triage reduces the cost per screen compared to LBC.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer.

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

FAM19A4/miR124-2 Methylation Testing and Human Papillomavirus (HPV) 16/18 Genotyping in HPV-Positive Women Under the Age of 30 Years.

BACKGROUND: High-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (CIN) grade 2/3 lesions in human papillomavirus (HPV)-positive women <30 years of age have high spontaneous regression rates. To reduce overtreatment, biomarkers are needed to delineate advanced CIN lesions that require treatment. We analyzed the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in HPV-positive women aged <30 years, aiming to identify CIN2/3 lesions in need of treatment. METHODS: A European multicenter retrospective study was designed evaluating the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in cervical scrapes of 1061 HPV-positive women aged 15-29 years (690 ≤CIN1, 166 CIN2, and 205 CIN3+). A subset of 62 CIN2 and 103 CIN3 were immunohistochemically characterized by HPV E4 expression, a marker for a productive HPV infection, and p16ink4a and Ki-67, markers indicative for a transforming infection. CIN2/3 lesions with low HPV E4 expression and high p16ink4a/Ki-67 expression were considered as nonproductive, transforming CIN, compatible with advanced CIN2/3 lesions in need of treatment. RESULTS: FAM19A4/miR124-2 methylation positivity increased significantly with CIN grade and age groups (<25, 25-29, and ≥30 years), while HPV16/18 positivity was comparable across age groups. FAM19A4/miR124-2 methylation positivity was HPV type independent. Methylation-positive CIN2/3 lesions had higher p16ink4a/Ki-67-immunoscores (P = .003) and expressed less HPV E4 (P = .033) compared with methylation-negative CIN2/3 lesions. These differences in HPV E4 and p16ink4a/Ki-67 expression were not found between HPV16/18-positive and non-16/18 HPV-positive lesions. CONCLUSIONS: Compared with HPV16/18 genotyping, the FAM19A4/miR124-2 methylation test detects nonproductive, transforming CIN2/3 lesions with high specificity in women aged <30 years, providing clinicians supportive information about the need for treatment of CIN2/3 in young HPV-positive women.

Evaluation of FAM19A4/miR124-2 methylation performance in the management of CIN3 diagnosed pregnant women.

Pregnant women diagnosed with CIN3 have high regression rates after delivery. Biomarkers are needed to only identify pregnant women with progressive CIN requiring treatment to reduce overreferral and overtreatment. In our study we evaluated the performance of the FAM19A4/miR124-2 methylation test for molecular triage on FFPE samples of CIN3+-diagnosed pregnant women with known clinical course over time as well in a cross-sectional setting. In this German multicenter retrospective study biopsy material was collected from pregnant women diagnosed with cervical cancer (n = 16), with CIN3 that progressed to cancer during pregnancy (n = 7), with CIN3 that regressed to CIN1 or less within 6 months after delivery (n = 41), without CIN (n = 16), CIN3 covering 3-4 quadrants (n = 14) and randomly selected CIN3 (n = 41). FAM19A4/miR124-2 methylation analysis was performed blinded on first diagnosis. All pregnant women with cervical cancer and with CIN3 progressing to cancer tested positive for FAM19A4/miR124-2 methylation (100%, 22/22). In the regressing CIN3 group 47.5% and in the group without CIN 21.6% tested methylation positive. High-volume CIN3 and random selected CIN3 were methylation-positive in 91.7% and 82.1%, respectively. Methylation levels were significantly higher in progressive CIN3 and cancer compared to the controls (P < .0005). The likelihood ratio of a negative methylation test (LR-) for progressive CIN3+ was 0 (95% CI: 0-0.208). A negative FAM19A4/miR124-2 methylation test can rule out progressive CIN disease in pregnant women diagnosed with CIN3. This can help the clinician by managing these pregnant women with conservative follow-up until after delivery.

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening.

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p < 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9−97.3; kappa value 0.90 (95% CI: 0.86−0.94); and 94.5%; 95% CI: 92.6−96.2; kappa value 0.87 (95% CI: 0.82−0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.

FAM19A4/miR124-2 methylation in invasive cervical cancer: A retrospective cross-sectional worldwide study.

Widespread adoption of primary human papillomavirus (HPV)-based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124-2 genes has shown promise for the triage of high-risk (hr) HPV-positive women. In our study, we assessed the consistency of FAM19A4/miR124-2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation-specific PCR (qMSP)-based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7-99.2) tested FAM19A4/miR124-2 methylation-positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124-2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV-negative carcinomas. These results indicate that a negative FAM19A4/miR124-2 methylation assay result is likely to rule out the presence of cervical cancer.

Selection of women at risk for cervical cancer in an HIV-infected South African population.

OBJECTIVE: Cervical cancer is the leading cause of cancer-related death in women in South Africa. This study evaluates DNA methylation levels in cervical (pre)cancer and aims to assess the value of high-risk human papillomavirus (hrHPV) testing and methylation analysis, alone or in combination, on physician-taken cervical scrapes to detect cervical cancer, and cervical intraepithelial neoplasia grade 3 (CIN3) in an HIV-infected South African population. DESIGN: Prospective observational multicentre cohort study. METHODS: Women from a cohort of women living with HIV (n = 355) and a referral cohort (n = 109, 60% HIV seropositive) were included. Cervical scrapes were collected for hrHPV testing and methylation analysis of cell adhesion molecule 1, T-lymphocyte maturation-associated protein, and microRNA124-2 genes. Histologic endpoints were available for all participants. Performance for detection of CIN3 or worse (CIN3+) was determined in the cohort of women living with HIV and different testing strategies were compared. RESULTS: HrHPV and methylation positivity rates increased with severity of cervical disease in the two study cohorts, each reaching 100% in samples of women with carcinoma. HrHPV testing showed a sensitivity for CIN3+ of 83.6%, at a specificity of 67.7%. Methylation analysis showed a comparable CIN3+ sensitivity of 85.2%, but a significantly lower specificity of 49.6%. HrHPV testing with reflex methylation analysis showed a CIN3+ sensitivity of 73.8%, at a specificity of 81.5%. CONCLUSION: In this HIV-infected South African population, stratifying hrHPV-positive women with reflex methylation analysis detects all cervical carcinomas and yields an acceptable sensitivity and specificity for CIN3+.

Higher HPV16 and HPV18 Penile Viral Loads Are Associated With Decreased Human Papillomavirus Clearance in Uncircumcised Kenyan Men.

BACKGROUND: Whether higher penile human papillomavirus (HPV) viral load is associated with a lower rate of HPV clearance remains unknown. OBJECTIVES: We examined the association between penile HPV16 and HPV18 viral load and subsequent HPV clearance in uncircumcised Kenyan men. STUDY DESIGN: Participants were human immunodeficiency virus (HIV)-seronegative, sexually active, 18- to 24-year-old men randomized to the control arm of a male circumcision trial in Kisumu, Kenya. Men provided exfoliated penile cells from two anatomical sites (glans/coronal sulcus and shaft) every 6 months for 2 years. GP5+/6+ polymerase chain reaction was used to identify 44 HPV-DNA types. Human papillomavirus viral load testing was conducted using a LightCyler real-time polymerase chain reaction assay; viral load was classified as high (>250 copies/scrape) or low (≤250 copies/scrape), for nonquantifiable values. The Kaplan-Meier method and Cox regression modeling were used to examine the association between HPV viral load and HPV clearance. RESULTS: A total of 1097 men, with 291 HPV16 and 131 HPV18 cumulative infections over 24 months were analyzed. Human papillomavirus clearance at 6 months after first HPV detection was lower for high versus low viral load HPV16 infections in the glans (adjusted hazard ratio [aHR], 0.65; 95% confidence interval [CI], 0.46-0.92)] and shaft (aHR, 0.44; 95% CI, 0.16-0.90), and HPV18 infections in the glans (aHR, 0.05; 95% CI, 0.01-0.17). DISCUSSION: High versus low HPV viral load was associated with a reduced HPV clearance for HPV16 infections in the glans and shaft, and for HPV18 infections in the glans, among young uncircumcised men. Reduced clearance of high viral load HPV16 and HPV18 infections in men may increase HPV transmission to their female partners as well as enhance the development of penile lesions in comparison to men with low viral load HPV infections.

Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women.

OBJECTIVES: DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples. METHODS: We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined. RESULTS: Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4-80.6) at a specificity of 67.8% (95%CI: 62.7-73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8-80.1) at a 76.4% (95%CI: 70.2-82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4-95.6) and 46.0% (95%CI: 40.4-51.5) for lavage self-samples, and 84.7% (95%CI: 76.4-93.0) and 54.9% (95%CI: 47.7-62.2) for brush self-samples. CONCLUSIONS: FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity.

Methylation Levels of CADM1, MAL, and MIR124-2 in Cervical Scrapes for Triage of HIV-Infected, High-Risk HPV-Positive Women in Kenya.

OBJECTIVES: To evaluate the value of cervical cell methylation markers in screening HIV-infected women also positive for high-risk human papillomavirus (hrHPV). DESIGN: Cross-sectional and prospective. METHODS: Two hundred forty-eight HIV-infected hrHPV-positive women enrolled in a cervical cancer screening study in Nairobi, Kenya, had colposcopy-directed biopsy and histological diagnoses. Exfoliated cervical cells were used to measure methylation levels of the CADM1, MAL, and MIR124-2 genes using quantitative methylation-specific polymerase chain reaction. Methylation levels were summarized as cycle threshold (Ct) ratios compared with the ß-actin gene. Median Ct ratios were compared across histological diagnoses, with 95% confidence intervals calculated by bootstrapping. Methylation levels at 6 months were assessed in 128 women who remained hrHPV positive. RESULTS: All 3 methylation markers showed significantly (P < 0.001) raised median Ct ratios in women with cervical intraepithelial neoplasia (CIN) grade 3 compared with women with a normal cervix. When markers were combined into a single test, the area under the receiver operating characteristic curve for prediction of CIN2 or worse (CIN2+) was 0.80. When the test was calibrated to have similar specificity, sensitivity of the combined tri-marker test for CIN2+ was comparable with cytology [atypical squamous cells of undetermined significance or worse] (89% and 95%, respectively) and superior to visual inspection with acetic acid (85% vs 70%) and HPV16/18 genotyping (65% vs 40%). Among women with no CIN2+ at baseline and persistent hrHPV at 6-month follow-up, MAL-m1 and MIR124-2 Ct ratios increased significantly. CONCLUSIONS: Methylation markers in combination with HPV testing may offer a full molecular screening strategy to the many HIV-infected women who are also hrHPV positive.

Follow-up of high-risk HPV positive women by combined cytology and bi-marker CADM1/MAL methylation analysis on cervical scrapes.

OBJECTIVES: Triage of HPV screen-positive women is needed to identify those with underlying cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+). Presently, cytology on a physician-taken cervical scrape is mostly accepted as triage test, but needs follow-up testing in order not to miss severe disease. Here, we evaluated the performance of combined cytology and bi-marker CADM1/MAL-methylation analysis as triage test on physician-taken cervical scrapes of HPV positive women. METHODS: In this post-hoc analysis, we used 364 left-over HPV positive cytology triage samples of participants of a randomized controlled trial (PROHTECT-3: n=46,001) performed in population-based cervical screening. Study endpoints were CIN2+ and CIN3+ detection. Cytology testing with and without methylation marker analysis was evaluated with regard to sensitivity, specificity, positive and negative predictive value, and referral rate. RESULTS: Bi-marker CADM1/MAL-methylation positivity increased proportionally with severity of underlying lesions. Overall, cytology and bi-marker CADM1/MAL-methylation analysis yielded similar performances with regard to CIN3+ detection, yet in combination a significantly higher sensitivity for CIN3+ (88.7%) was obtained at a specificity of 53.6% and a colposcopy referral rate of 53.6%. The combined strategy detected all six cervical cancers, whereas triage by cytology alone failed to detect two of them. CONCLUSIONS: Cytology and bi-marker CADM1/MAL-methylation analysis perform complementary for CIN2+/CIN3+ detection when used as triage tool on cervical scrapes of HPV positive women. This approach not only results in a higher CIN3+ sensitivity than cytology triage with an acceptable referral rate, but also seems to reduce the risk of missing cervical cancers and advanced high-grade lesions.

Acquisition and persistence of human papillomavirus 16 (HPV-16) and HPV-18 among men with high-HPV viral load infections in a circumcision trial in Kisumu, Kenya.

BACKGROUND: Circumcision and lower human papillomavirus (HPV) viral loads in men are possibly associated with a reduced risk of HPV transmission to women. However, the association between male circumcision and HPV viral load remains unclear. METHODS: Swab specimens from the glans and shaft of the penis were collected from men enrolled in a circumcision trial in Kisumu, Kenya. GP5+/6+ polymerase chain reaction (PCR) was used to identify HPV DNA types. HPV-16 and HPV-18 loads were measured with a LightCycler real-time PCR and classified as high (>250 copies/scrape) or low (≤250 copies/scrape). RESULTS: A total of 1159 men were randomly assigned to undergo immediate circumcision, and 1140 men were randomly assigned to the control arm (these individuals were asked to remain uncircumcised until the study ended). The hazard of acquisition of high-viral load infections in the glans was lower in the circumcision arm, compared with the control arm, for HPV-16 (hazard ratio [HR], 0.32 [95% confidence interval {CI}, .20-.49]) and HPV-18 (HR, 0.34 [95% CI, .21-.54]). The 6-month risk of HPV persistence among men with high-viral load infections in the glans at baseline was lower in the circumcision arm, compared with the control arm, for HPV-16 (risk ratio [RR], 0.36 [95% CI, .18-.72]) and HPV-18 (RR 0.34 [95% CI, .13-.86]). Weaker and less precise results were obtained for shaft samples. CONCLUSIONS: Male circumcision could potentially reduce the risk of HPV transmission to women by reducing the hazard of acquisition, and the risk of persistence of high-HPV viral load infections in the glans in men.

Methylation analysis of the FAM19A4 gene in cervical scrapes is highly efficient in detecting cervical carcinomas and advanced CIN2/3 lesions.

Primary testing for human papillomavirus (HPV) in cervical screening requires triage to differentiate women with transient infection from those with persistent infection who require more intensive management given their risk for cervical (pre)cancer. In this study, the clinical performance of a novel methylation marker FAM19A4 for the triage of high-risk (hr)HPV-positive women was evaluated. Using a training-validation set approach, we analyzed a FAM19A4 quantitative methylation-specific PCR (qMSP). The training set comprised hrHPV-positive cervical scrapes of 43 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and 135 women with ≤CIN1. The validation set comprised hrHPV-positive cervical scrapes of 52 women with CIN2+, including 33 CIN3+, 19 CIN2, and 166 women with ≤CIN1. The methylation threshold of FAM19A4 qMSP that gave rise to CIN3+ specificity of 70% in the training set was applied in the validation set. This resulted in CIN3+ sensitivity of 75.8% [95% confidence interval (CI), 61.1-90.4] at 67.0% (95% CI, 60.3-73.8) specificity. Next, the validated qMSP was applied to an independent series of hrHPV-positive cervical scrapes of 22 women with cervical cancer, 29 with advanced CIN2/3 [i.e., women with a known preceding hrHPV infection (PHI) lasting ≥5 years as proxy of longer duration of lesion existence], and 19 with early CIN2/3 (i.e., PHI <5 years). All carcinomas (22/22) and advanced CIN2/3 lesions (29/29) were FAM19A4 methylation-positive, compared with 42.1% (8/19; 95% CI, 19.9-64.3) of early CIN2/3 lesions. In conclusion, FAM19A4 is an attractive triage marker for hrHPV-positive women, with a high reassurance for the detection of cervical carcinoma and advanced CIN2/3 lesions.

CADM1, MAL and miR124-2 methylation analysis in cervical scrapes to detect cervical and endometrial cancer.

AIMS: Gene promoter hypermethylation is recognised as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer. METHODS: A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, cervical intraepithelial neoplasia grade 3 (CIN3) (n=16) or CIN2 (n=32), and women without evidence of CIN2 or worse (n=120) were assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR. RESULTS: All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI 58.0% to 94.4%) of women with endometrial cancer scored positive for DNA methylation for at least one of the three genes. In women without cancer, methylation frequencies increased significantly with severity of disease from 19.2% (23/120; 95% CI 12.1% to 26.2%) in women without CIN2 or worse to 37.5% (12/32; 95% CI 20.7% to 54.3%) and 68.8% (11/16; 95% CI 46.0% to 91.5%) in women with CIN2 and CIN3, respectively. Overall methylation positivity and the number of methylated genes increased proportionally to the lesion severity. CONCLUSIONS: DNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.

Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer.

OBJECTIVES: Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping. METHODS: 1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33-63 years, n=46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3+) of 50%, 60%, 70%, and 80%. RESULTS: The CIN3+ sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3+ sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3+ sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%). CONCLUSIONS: Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3+ sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.

Combined CADM1/MAL methylation and cytology testing for colposcopy triage of high-risk HPV-positive women.

Primary screening for high-risk human papillomavirus (hrHPV) requires a triage protocol. Repeat cytology testing at baseline and after 6 to 12 months has emerged as a reasonable triage approach, but carries the risk of loss to follow-up. Repeat cytology testing may be omitted if cytology is supplemented with another, complementary triage test at baseline. In this study, the performance of combined triage by cytology and DNA methylation analysis was assessed. In hrHPV-positive cervical scrapes (n = 250), cytology [threshold: atypical squamous cells of undetermined significance (ASCUS)], bi-marker CADM1/MAL methylation testing (at different assay thresholds), and combinations of both were evaluated for endpoints cervical intraepithelial neoplasia grade 2 or worse (CIN2(+)) and grade 3 or worse (CIN3(+)). At a predefined methylation threshold of 70% specificity for CIN3(+), combined triage revealed a CIN3(+) sensitivity of 86.8% [95% confidence interval (CI), 76.1-97.6] compared with 65.8% (95% CI, 50.7-80.9) for sole cytology triage testing. Corresponding CIN3(+) specificity was 64.8% (95% CI, 58.1-71.5) for combined triage and 78.6% (95% CI, 72.8-84.3) for sole cytology triage testing. For CIN2(+), the sensitivity of combined triage testing was 84.5% (95% CI, 75.2-93.8) compared with 65.5% (95% CI, 53.3-77.7) for sole cytology triage, with corresponding specificities of 69.9% (95% CI, 63.1-76.6) and 83.5% (95% CI, 78.0-89.0), respectively. In conclusion, combined triage reached substantially higher CIN2(+)/3(+) sensitivities compared with sole cytology at a slight drop in specificity. Therefore, it is an attractive triage strategy for colposcopy of hrHPV-positive women with a high reassurance for cervical cancer and advanced CIN lesions.

Triage by methylation-marker testing versus cytology in women who test HPV-positive on self-collected cervicovaginal specimens (PROHTECT-3): a randomised controlled non-inferiority trial.

BACKGROUND: Cytology is a widely used method of triaging women who test positive for human papillomavirus (HPV). However, self-sampled specimens, which can substantially increase participation in screening programmes, are not suitable for accurate cytological assessment. We investigated whether direct DNA methylation-based molecular triage on self-sampled cervicovaginal specimens was non-inferior to cytology triage on additional physician-collected cervical samples in the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or worse in women who did not attend cervical screening programmes. METHODS: In this randomised controlled non-inferiority trial, we invited women (aged 33-63 years) registered as non-attendees of cervical screening in the Netherlands in 2007 to submit a self-collected cervicovaginal sample for HPV testing. Using a computer-generated sequence, we randomly allocated women who tested positive for high-risk hrHPV on a self-sample to either triage by cytology on an additional physician-taken smear or direct triage on the self-sample by methylation analysis of MAL and miR-124-2 genes (1:1; stratified by age and region, with block sizes by age group). Triage-positive women in either group were referred for colposcopy. The primary endpoint was detection of CIN2 or worse, analysed by intention to treat. The non-inferiority margin was 0·80. This study is registered in the Primary Trial Register of the Netherlands, number NTR6026. FINDINGS: We invited 46,001 women to participate, 12,819 of whom returned self-sampled material; 1038 samples tested positive for high-risk HPV. Between Nov 1, 2010, and Dec 31, 2011, after exclusion of women who were ineligible, we enrolled and randomly allocated 515 women to methylation triage and 509 to cytology triage. The detection of CIN2 or worse with methylation triage was non-inferior to that with cytology triage (90 [17%] of 515 women vs 75 [15%] of 509 women; relative risk 1·19, 95% CI 0·90-1·57). Referral for colposcopy was more common in the molecular group (284 [55%] women) than in the cytology group (149 [29%] women; p<0·0001). Mean time to CIN2 or worse diagnosis was shorter in the molecular triage group (96 days, range 44-101) than in the cytology triage group (158 days, 71-222; p=0·00084). INTERPRETATION: DNA methylation analysis of MAL and miR-124-2 genes on HPV-test-positive self-samples is non-inferior to cytology triage in the detection of CIN2 or worse, opening the way to full molecular screening. FUNDING: Midden-West and Oost Screening Organisations and Stichting Achmea Gezondheidszorg.

Molecular characterization of p16-immunopositive but HPV DNA-negative oropharyngeal carcinomas.

Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (p16 IHC) but lack HPV DNA. The objective of this study was to characterize this group at the molecular level by performing sensitive HPV DNA- and RNA-based PCR methods and genetic profiling. All patients diagnosed with an OPSCC in the period 2000-2006 in two Dutch university medical centers were included (n = 841). The presence of HPV in a tumor sample was tested by p16 IHC followed by an HPV DNA GP5+/6+ PCR. p16 IHC scored positive in 195 samples, of which 161 were HPV DNA-positive and 34 (17%) HPV DNA-negative. In the latter group, a SPF10-LiPA25 assay, an HPV16 type-specific E7 PCR and an E6 mRNA RT-PCR were performed. Next, ten of these cases were further analyzed for loss of heterozygosity (LOH) of 15 microsatellite markers at chromosome arms 3p, 9p and 17p. Of the 34 p16-positive but PCR-negative OPSCCs, two samples tested positive by SPF10 assay, HPV16 E7 PCR and HPV16 E6 mRNA RT-PCR. Three samples tested positive by SPF10 assay but negative by the HPV16-specific assays. Nine of ten cases that were tested for LOH showed a genetic pattern comparable to that of HPV-negative tumors. This study categorizes p16-positive but HPV DNA-negative OPSCCs as HPV-negative tumors based on genetic profiling. This study highlights the importance of performing HPV testing in addition to p16 IHC for proper identification of HPV-associated OPSCCs.

A second generation cervico-vaginal lavage device shows similar performance as its preceding version with respect to DNA yield and HPV DNA results.

BACKGROUND: Attendance rates of cervical screening programs can be increased by offering HPV self-sampling to non-attendees. Acceptability, DNA yield, lavage volumes and choice of hrHPV test can influence effectiveness of the self-sampling procedures and could therefore play a role in recruiting non-attendees. To increase user-friendliness, a frequently used lavage sampler was modified. In this study, we compared this second generation lavage device with the first generation device within similar birth cohorts. METHODS: Within a large self-sampling cohort-study among non-responders of the Dutch cervical screening program, a subset of 2,644 women received a second generation self-sampling lavage device, while 11,977 women, matched for age and ZIP-code, received the first generation model. The second generation device was different in shape, color, lavage volume, and packaging, in comparison to its first generation model. The Cochran's test was used to compare both devices for hrHPV positivity rate and response rate. To correct for possible heterogeneity between age and ZIP codes in both groups the Breslow-Day test of homogeneity was used. A T-test was utilized to compare DNA yields of the obtained material in both groups. RESULTS: Median DNA yields were 90.4 µg/ml (95% CI 83.2-97.5) and 91.1 µg/ml (95% CI 77.8-104.4, p= 0.726) and hrHPV positivity rates were 8.2% and 6.9% (p= 0.419) per sample self-collected by the second - and the first generation of the device (p= 0.726), respectively. In addition, response rates were comparable for the two models (35.4% versus 34.4%, p= 0.654). CONCLUSIONS: Replacing the first generation self-sampling device by an ergonomically improved, second generation device resulted in equal DNA yields, comparable hrHPV positivity rates and similar response rates. Therefore, it can be concluded that the clinical performance of the first and second generation models are similar. Moreover, participation of non-attendees in cervical cancer screening is probably not predominantly determined by the type of self-collection device.

CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes increase proportional to degree and duration of underlying cervical disease.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with ≤ CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n = 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and ≥ 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically ≤ CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3- and 6.2-fold in CIN2/3, respectively, and 143.5- and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5- and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.