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OBJECTIVE: To evaluate the diagnostic accuracy of Electrical Impedance Spectroscopy (EIS)-assisted colposcopy in detecting CIN2+ Greek women towards standalone colposcopy, HPV mRNA testing, and p16/Ki67 immunostaining. METHODS: We conducted a cross-sectional observational study at the Cervical Pathology Clinic of the 2nd Obstetrics-Gynecology University Department of Hippokration Hospital Thessaloniki involving 316 patients from January 2022 to August 2023. All participants provided liquid-based cervical samples for cytology, HPV mRNA testing, and p16/Ki67 immunostaining. MAIN OUTCOME MEASURES: Subsequently, participants underwent both standalone colposcopy and EIS/ZedScan-assisted colposcopy, followed by cervical punch biopsies. RESULTS: The incorporation of EIS significantly enhanced the sensitivity of colposcopy, increasing it from 54.17% to 100%, equivalent to that of HPV mRNA testing and p16/Ki67 immunostaining, while achieving a high specificity (95.45%). The specificities observed with EIS/ZedScan-assisted and standalone colposcopy were notably superior to those of HPV-related biomarkers (HPV mRNA test and p16/Ki67 immunostaining). When compared to standalone colposcopy, HPV mRNA testing, and p16/Ki67 immunostaining, EIS/ZedScan-assisted colposcopy demonstrated the most favorable combination of Positive and Negative Predictive Values, at 90.57% and 100%, respectively. The inclusion of EIS/ZedScan in colposcopy led to the detection of 44 additional cases of true CIN2+ (100% of the total CIN2+ confirmed histologically) that were missed by standalone colposcopy. This discovery suggests a 45.83% increase in the detection of CIN2+ cases. CONCLUSIONS: The integration of EIS with colposcopy has demonstrated effectiveness in detecting cervical lesions, resulting in a significant detection increase of CIN2+ cases while offering optimal levels of sensitivity, specificity, and predictive values for CIN2+ detection.

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Cervical cancer remains a significant public health issue, particularly in regions with low screening uptake. This study evaluates the effectiveness of self-sampling and the 7-type HPV mRNA E6/E7 test in improving cervical cancer screening outcomes among a referral population in Mexico. A cohort of 418 Mexican women aged 25 to 65, referred for colposcopy and biopsy due to abnormal cytology results (ASC-US+), participated in this study. Self-samples were analyzed using both the 14-type HPV DNA test and the 7-type HPV mRNA E6/E7 test. The study assessed the sensitivity, specificity, positive predictive value (PPV), and the necessity of colposcopies to detect CIN3+ lesions. Participant acceptability of self-sampling was also evaluated through a questionnaire. The 7-type HPV mRNA E6/E7 test demonstrated equivalent sensitivity but significantly higher specificity (77.0%) and PPV for CIN3+ detection compared to the 14-type HPV DNA test (specificity: 45.8%, p < 0.001). The use of the HPV mRNA test as a triage tool reduced the number of colposcopies needed per CIN3+ case detected from 16.6 to 7.6 (p < 0.001). Self-sampling was highly accepted among participants, with the majority reporting confidence in performing the procedure, minimal discomfort, and willingness to undertake self-sampling at home. Self-sampling combined with the 7-type HPV mRNA E6/E7 testing offers a promising strategy to enhance cervical cancer screening by improving accessibility and ensuring precise diagnostics. Implementing these app roaches could lead to a significant reduction in cervical cancer morbidity and mortality, especially in underserved populations. Future research should focus on the long-term impact of integrating these methods into national screening programs and explore the cost-effectiveness of widespread implementation.

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OBJECTIVE: Self- collected specimens to detect high-risk (hr) HPV and high-grade cervical lesions (CIN2+) has been introduced aiming to increase cervical cancer screening coverage. The performance of self- collected specimen  compared to clinician collected specimen is one major concern. This study aimed to compare self-sampling HPV-DNA and clinician-sampling HPV-mRNA to detect hr-HPV and high-grade cervical lesions. METHODS: Women with abnormal cervical cytology and/ or positive hr-HPV who attended the colposcopy clinics in 10 tertiary hospitals in Bangkok were enrolled. Self-collected specimens were evaluated for  HPV DNA using Cobas® 4800 HPV test prior to the clinician-collected specimens which were tested for HPV mRNA with APTIMA® HPV Assay. Subsequent colposcopy with biopsy was performed. The detection rates of hr-HPV from both HPV tests and their performance to detect high-grade lesions pathology were compared. RESULTS: Data from 497 women's specimens were analyzed. Both samplings had 86.8% concordance rate in detecting hr-HPV (Kappa 0.670; 95% confidence interval [CI] 0.599-0.746, P value < 0.001). The sensitivity (95% CI) of self-collected specimen HPV DNA and clinician- collected specimen HPV-mRNA to detect high-grade lesions were 91.8% (85.4%-96.0%) and 90.2% (83.6%-94.9%) respectively. The corresponding negative predictive values (95% CI) were 91.9% (85.6%-96.0%) and 91.7% (86.0%-95.7%) respectively. CONCLUSION: HPV DNA testing from self-collected specimen to detect HR-HPV demonstrates high concordance with HPV mRNA testing from clinician-collected specimen. The sensitivity and negative predictive value of both tests to detect high-grade lesions are comparable.

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BACKGROUND: The study's purpose was to evaluate the performance of a five-type HPV mRNA test to predict cervical intraepithelial neoplasia grade 3 or worse (CIN3+) during up to 12 years of follow-up. METHODS: Overall, 19,153 women were recruited by gynecologists and general practitioners in different parts of Norway between 2003 and 2004. The study population comprised 9582 women of these women, aged 25-69 years with normal cytology and a valid five-type HPV mRNA test at baseline. Follow-up for CIN3+ through 2015 was conducted in the Norwegian Cervical Cancer Screening Programme. RESULTS: The cumulative incidence of CIN3+ by baseline status for HPV mRNA-positive and mRNA-negative women were 20.8% and 1.1%, respectively (p < 0.001). Age did not affect the long-term ability of the HPV mRNA test to predict CIN3+ during follow-up. CONCLUSION: The low long-term risk of CIN3+ among HPV mRNA-negative women and the high long-term risk among HPV mRNA-positive women strengthen the evidence that the five-type HPV mRNA test is an appropriate screening test for women of all ages. Our findings suggest that women with a negative result may extend the screening interval up to 10 years.

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BACKGROUND: A specific, cost-effective triage test for minor cytological abnormalities is essential for cervical cancer screening among younger women to reduce overmanagement and unnecessary healthcare utilization. We compared the triage performance of one 13-type human papillomavirus (HPV) DNA test and one 5-type HPV mRNA test. METHODS: We included 4115 women aged 25-33 years with a screening result of atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesions (LSIL) recorded in the Norwegian Cancer Registry during 2005-2010. According to Norwegian guidelines, these women went to triage (HPV testing and repeat cytology: 2556 were tested with the Hybrid Capture 2 HPV DNA test, which detects the HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68; and 1559 were tested with the PreTect HPV-Proofer HPV mRNA test, which detects HPV types 16, 18, 31, 33, and 45). Women were followed through December 2013. RESULTS: HPV positivity rates at triage were 52.8% and 23.3% among DNA- and mRNA-tested women (p < 0.001), respectively. Referral rates for colposcopy and biopsy and repeat testing (HPV + cytology) after triage were significantly higher among DNA-tested (24.9% and 27.9%) compared to mRNA-tested women (18.3% and 5.1%), as were cervical intraepithelial neoplasia grade 3 or worse (CIN3+) detection rates (13.1% vs. 8.3%; p < 0.001). Ten cancer cases were diagnosed during follow-up; eight were in DNA-tested women. CONCLUSION: We observed significantly higher referral rates and CIN3+ detection rates in young women with ASC-US/LSIL when the HPV DNA test was used at triage. The mRNA test was as functional in cancer prevention, with considerably less healthcare utilization.

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Identifying and reaching women at higher risk for cervical cancer is all-important for achieving the ambitious endpoints set in 2020 by the WHO for global cervical cancer control by 2030. HPV-based (vaginal) self-sampling (SS) represents a cost-effective screening strategy, which has been successfully implemented during the last decade both in affluent and constrained settings. Among other advantages, SS strategies offer convenience, diminished costs, flexibility to obtain a sample in the office or home, avoiding a pelvic exam and uncomfortable appointment with a healthcare professional, as well as social and cultural acceptability. SS implementation has been globally boosted during the COVID-19 pandemic. In pragmatic terms, social distancing, local lockdowns, discontinuation of clinics and reallocation of human and financial resources challenged established clinician-based screening; self-collection strategies apparently surpassed most obstacles, representing a viable and flexible alternative. With time, sufficient reassuring data has accumulated regarding specially designed SS devices, aspects of sample preparation, transport and storage and, importantly, optimization of validated PCR-based HPV testing platforms for self-collected specimens. Suboptimal rates of clinical follow-up post-SS screening, as well as overtreatment with reliance solely on molecular assays, have both been documented and remain concerning. Therefore, effective strategies are still required to ensure linkage to follow-up testing and management following positive SS results by trained health professionals with knowledge of HPV biology and management algorithms. Because of the prolonged SS screening intervals, implementation data are limited regarding subsequent screening rounds of SS-screened individuals; however, these are accumulating gradually. With further refinement of assays and validation of novel biomarkers in self-collected samples, there is a clear potential for increasing SS accuracy and PPV. The potential differentiation of self-collection protocols for vaccinated versus non-vaccinated individuals also represents an open issue. In conclusion, HPV-based self-collection techniques can effectively address limited uptake alongside other conventional cervical screening drawbacks; however, assays, logistics and infrastructures need further optimization to increase the efficacy, effectiveness and cost-effectiveness of SS approaches.

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AIM: To compare the diagnostic parameters of electrical impedance spectroscopy (EIS) via ZedScan, a device that measures spectra to differentiate between normal and abnormal cervical tissues, when used as an adjunct to colposcopies in the diagnosis of HSILs/CIN2+ in Greek women with abnormal referral cytology toward colposcopy alone and HPV mRNA-testing. METHODS: This study analyzed 86 women, patients of the Colposcopy and Cervical Pathology Clinic of 2nd Obstetrics and Gynecology Department, Aristotle University of Thessaloniki at Hippokration General Hospital, between January 2022 and September 2022. During the visits, women were subjected to cytology, colposcopy alone and then with EIS/ZedScan and histological sampling. RESULTS: Common use of colposcopies and EIS/ZedScan allowed detecting an additional 14 cases of CIN2+ (16.2%) that colposcopy alone failed to report. EIS enhanced the sensitivity of colposcopy from 80.65% to 100% equal with that of HPV-mRNA test while retaining a high specificity (94.74%) which is much higher than specificity of HPV mRNA-testing (65.45%). EIS-assisted colposcopy had the highest value combination of positive and negative predictive values (96.15% and 100%) compared to colposcopy alone (100% and 75%) and HPV mRNA-testing (72.46% and 100%). CONCLUSIONS: Colposcopies performed with EIS/ZedScan demonstrated effectiveness in the diagnosing of CIN2+ leading to a significant increase in the number of CIN2+ that would have been missed if only colposcopy was applied especially in women with LSIL referral cytology. EIS/ZedScan seems to possess the ideal diagnostic threshold for sensitivity, specificity, and predictive values for CIN2+ compared to colposcopy alone and HPV mRNA-testing.

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BACKGROUND: The incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODS: The accuracy of three possible test strategies, i.e. (a) hrHPV DNA PCR (DNA), (b) p16(INK4a) immunohistochemistry (IHC) (p16), and (c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTS: Ninety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16 + DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16 + DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONS: Single hrHPV DNA PCR and p16(INK4a) IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Disease prognostication can be encouraged based on this combined test result.

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BACKGROUND: Cervical cancer screening programs that use visual inspection with acetic acid to identify women with pre-cancerous lesions in Zimbabwe have had limited success due to challenges with human resource constraints and patient acceptability. Nucleic acid amplification tests for human papillomavirus (HPV) have been endorsed by the World Health Organization for cervical cancer screening, along with self-collection of samples. As evidence shows self-collected sampling to be acceptable and preferable, Zimbabwe's Ministry of Health and Child Care (MOHCC) required a comparative analysis on the agreement between self- and clinician-collected samples for testing with Hologic Aptima HPV mRNA assay, to determine if self-collected samples could be used as another method to increase coverage of cervical cancer screening programs. METHODS: In four public health facilities in Zimbabwe from July to August 2020, self- and clinician-collected HPV samples were obtained from HIV-positive women aged 30-49 years for HPV testing. RESULTS: A total of 280 self- and clinician-collected samples were tested and results were found to have good agreement (κ: 0.75, 95% CI: 0.66-0.82). HPV prevalence was 43.0% (95% CI: 37.0%-49.3%) for self-samples and 48.0% (95% CI: 41.0%-54.2%) for clinician-samples. CONCLUSIONS: Self-collected sampling had good agreement with clinician-collected and its inclusion in the national cervical cancer screening policy by Zimbabwe's MOHCC is expected to increase testing coverage, especially among underserved communities such as women living with HIV, as well as decentralize access to cervical cancer screening services for lower-level facilities and increase the geographical scope of where HPV testing can be offered through the country.

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BACKGROUND: In 2017, the South African National Department of Health (NDoH) Cervical Cancer Prevention and Control Policy was revised. Human papillomavirus (HPV) testing on self-collected samples may offer improved screening uptake. The objectives of the study were to compare the positivity of high-risk (hr)-HPV deoxyribonucleic acid (DNA) and hrHPV viral messenger ribonucleic acid (mRNA) between healthcare worker-collected cervical and self-collected vaginal samples and investigate the accuracy of the applicator-tampon-based self-collected samples in detecting hrHPV DNA and hrHPV mRNA. METHODS: A total of 527 women aged 18 years and older and seeking gynecology services at a tertiary hospital in Pretoria, South Africa, were enrolled. Vaginal samples were self-collected using SelfCerv applicator tampon, followed by cervical samples collected by a healthcare worker using a Cervex Brush® Combi. Both samples were tested with the Abbott m2000 analyzer for 14-hrHPV types and 285 paired samples were tested for hrHPV E6/E7 mRNA using the Aptima HR-HPV mRNA assay. The prevalence of hrHPV DNA and hrHPV E6/E7 mRNA was estimated and the positivity between the two collection methods was compared for the total group as well as per age group. RESULTS: HrHPV prevalence was 48.0% (95% CI 43.7-52.4) among healthcare worker collected samples and 47.6% (95% CI 43.3-52.0) among self-collected samples. There was no difference in positivity between healthcare worker collection (48.0%) and applicator-tampon-based self-collection, 47.6% (p-value = 0.90). The proportions of hrHPV were equal between the age groups as shown by the McNemar test (p = 0.9036) results for correlated proportions. The prevalence of hrHPV mRNA was 78.6% (95% CI 73.4-83.2) and 58.6% (95% CI 52.6-64.4) for healthcare worker- and self-collection, respectively. The McNemar test for correlated proportions was highly significant (p < 0.0001), indicating that the hrHPV mRNA proportions are not comparable, although this differed between age groups. CONCLUSIONS: Applicator-tampon-based self-collection has a comparable hrHPV DNA positivity rate as healthcare worker collection but different positivity rates for hrHPV mRNA. Self-sampling showed high concordance with healthcare worker-collected sampling for hrHPV DNA detection, especially regarding HPV 16/18 detection. HrHPV DNA was equally detected between the total group as well as per age group. Implementation of self-sampling using an applicator tampon as a primary screening tool may be considered.

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AIM: To evaluate the diagnostic performance of E6/E7 HPV-mRNA overexpression towards HPV-DNA testing and p16/Ki67 immunocytochemistry in a post-op population to verify if this biomarker can be effectively used as indicator of successful cervical intraepithelial neoplasia (CIN) treatment. METHODS: Our study retrospectively analyzed 197 patients of our Colposcopy Clinic between January 2013 and September 2020 coming with an abnormal Pap smear suggestive for colposcopy, and after a series of follow-ups including liquid-based cytology (LBC) and punch-biopsy sampling, there were surgically treated. LBC was used for cytology and molecular analysis of the three HPV-related biomarkers. RESULTS: Six months after treatment, 93% of the HPV-mRNA-positive women became negative while this applied to only 80.2% of the HPV-DNA-positive women. HPV persistence was 6.9% at 6-12 months after treatment. The comparison among cytology, colposcopy, HPV-DNA test, and HPV-mRNA test after treatment revealed that the last one is the only with a strong correlation with actual severity (histology during treatment) (ρ = 0.345, p = 0.006) implying that clinical cases with more severe CIN may have higher chances of unsuccessful treatment. HPV-mRNA test had higher sensitivity (100%), specificity (96.88%), and positive predictive value (45.45%) for CIN2+ recurrent lesions when compared with HPV-DNA testing (80%, 82.81%, 10.81% respectively) and p16/Ki67 immunocytochemistry (80%, 95.83%, 33.33% respectively) while their negative predictive values were similar. CONCLUSIONS: E6/E7 mRNA detection has higher diagnostic values for the prediction of treatment failure compared with HPV-DNA testing and p16/Ki67 immunocytochemistry, and as an outcome could be used as predictive indicator of CIN-treatment status.

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We describe the heretofore unreported case of an HPV-related carcinoma of the palatine tonsil with distinct areas of squamous cell- and adenoid cystic carcinoma-like differentiation in a 54-year old patient. The morphological, immunophenotypic and molecular findings of the tumor are illustrated. We discuss the parallels between the tumor and HPV-related multiphenotypic sinonasal carcinoma (HMSC) which is well-known to exhibit adenoid cystic carcinoma-like features. A review of the literature of high-risk HPV-associated non-squamous carcinomas of the oropharynx is presented.

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BACKGROUND: During 2013 and 2016 the region of Skåne, Sweden started to analyse human papillomavirus (HPV) and cytology in postmenopausal women 60-65 years of age. Our aim was to evaluate high-risk (HR) HPV mRNA testing for the triage of HPV DNA-positive postmenopausal women with normal cytology. METHODS: A total of 271 women, 60-65 years of age, underwent liquid-based cytology (LBC) and HPV testing by using the HR-HPV DNA MGP-PCR-Luminex assay. HR-HPV DNA-positive women with normal cytology underwent complimentary HPV mRNA testing (Aptima, Hologic Inc.). Over a period of 49 months (SD 11.0) the women received regular follow-ups at intervals of 12-18 months. Women with abnormal cytology and/or a positive HR-HPV DNA and/or mRNA result at two subsequent visits were scheduled for colposcopy and clinical examination. RESULTS: Over the surveillance period, 3.6% (10/271) of the HR-HPV DNA-positive women developed histologically confirmed high-grade squamous intraepithelial lesions (HSILs) or worse. The cumulative incidence rates (CIR) were 29.7% (CI 24.8-30.1) for HSIL or worse among HPV mRNA-positive women at enrolment (39.5% 107/271) and 0% among HPV mRNA-negative women (60.5%, 164/271), (p = 0.002). CONCLUSIONS: Postmenopausal women with normal cytology testing positive for HR-HPV mRNA are at increased risk for the development of high-grade cervical intraepithelial neoplasia (CIN), in contrast to women with a negative HR-HPV mRNA outcome. The HR-HPV mRNA APTIMA assay detecting 14 HR-HPV types may be a useful triage method among HPV DNA-positive postmenopausal women with normal cytology.

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The objective of this study was to investigate the hypothesis that HPV vaccination administered in patients with low-grade (LG) cytology shortly after an initial colposcopic assessment could prospectively alter HPV-related biomarkers. This was a prospective pilot observational study involving women attending a colposcopy clinic for evaluation of abnormal LG cytology that were advised to undergo HPV vaccination and proceeded accordingly. These women were compared with a matched unvaccinated group. Women requiring cervical biopsies or CIN treatment were excluded. INTERVENTION: A full three-dose HPV vaccination was undertaken with either the 2-valent or the 4-valent anti-HPV VLP vaccine. LBC samples were obtained prior and after the completion of the vaccination regimen and tested for HPV DNA genotyping (CLART-2 HPV test) and E6 and E7 mRNA (NASBA technique). RESULTS: Alterations of HPV-related biomarkers at a colposcopy reassessment appointment 12 months later. ANALYSIS: The p-values, relative risk (RR), absolute relative risk (ARR), number needed to treat (NNT) and 95% confidence intervals for each biomarker in each group were assessed. RESULTS: A total of 309 women were included in the analysis. One hundred fifty-two women received the vaccine. HPV vaccination reduced in a statistically significant manner (p < 0.05) HPV DNA positivity rates for genotypes 16, 18, and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18, and 31 genotypes, respectively, prior to vaccination. A less pronounced, statistically insignificant reduction was shown for women who tested positive for both HPV DNA and mRNA E6 and E7 expression for HPV16, 18, and 33 subtypes. Statistically significant reduction in HPV mRNA positivity was solely documented for genotype 31 (p = 0.0411). CONCLUSIONS: HPV vaccination appears to significantly affect the rates of HPV16, 18, and 31 DNA-positive infections in the population testing HPV DNA-positive for the aforementioned genotypes. The above findings deserve verification in larger cohorts.

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Aim of this study was to compare the 5-year risk of cervical intraepithelial neoplasia grade 2+ (CIN2+)/CIN3+ and the performance parameters at 3-year rescreening of a negative E6/E7 mRNA-human papillomavirus (HPV) test with those of a HPV-DNA-negative test. We studied a cohort of HPV-negative women tested with the Aptima HPV-mRNA Assay ("HPV-mRNA cohort") versus a cohort of HPV negatives tested with the Hybrid Capture 2 (HC2) DNA test living in neighboring areas. Both cohorts were rescreened after 3 years by a HPV-DNA test (HC2 or Cobas 4800 HPV test). HPV test positivity, referral to colposcopy and detection of CIN2+ at 3-year rescreening were computed. The Veneto Cancer Registry was checked to search for invasive cancers and CIN3 diagnosed up to 5 years from the negative baseline test. Some 22,338 HPV-mRNA and 68,695 HPV-DNA-negative women were invited to 3-year rescreening, and, respectively, 16,641 (74.5%) and 54,630 (79.6%) complied with the invitation. The proportion of HPV-positive tests, referral to colposcopy and detection of CIN2+ in the HPV-mRNA and HPV-DNA cohorts were, respectively. 4.0 and 3.9% (ratio 1.08; 95% confidence interval [CI] 0.99-1.17), 2.6 and 2.5% (ratio 1.06, 95% CI 0.95-1.18) and 1.4 and 1.7‰ (ratio 0.85, 95% CI 0.54-1.33). The relative 5-year cumulative risk of cancer and of CIN2+ in the HPV-mRNA and HPV-DNA cohorts were 4.5 and 8.7/100,000 (ratio 0.51; 95%CI 0.01-4.22) and 1.1 and 1.5/1,000 (ratio 0.74; 95%CI 0.45-1.16), respectively. A negative HPV-mRNA test confers a risk of invasive cervical carcinoma and of CIN2+ at 5 years comparable to that of a negative HPV-DNA test.

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HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3'UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3'UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif "AUUUA". Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3'UTR, indicating that the AU-rich elements of the 3'UTR are not contributing to RNA stability. Instead, the three "AUUUA" motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

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Primary HPV screening for cervical cancer by HPV mRNA testing (Aptima) was implemented in January 2017, for women ≥30 through 70 years, in the Region of Skåne, Sweden. HPV positive samples underwent cytology assessment, and women with any degree of abnormal cytology were referred for colposcopy. The aim was to audit the primary HPV screening program, by comparing the cytology results to those of corresponding women (aged ≥30 through 65 years) screened with conventional cytology during 2016. Overall, HPV was detected among 7.0% (4433/63,055) of the women ≥30-70 years in the primary HPV screening program. Among a co-tested (cytology and HPV) subgroup aged 40-42 years (N = 5039), HPV was detected in 100% (28/28) of high-grade squamous intraepithelial lesions (HSIL) and atypical squamous cells of undetermined significance (ASCUS) where HSIL could not be excluded (ASCH) (9/9), and in 80% (4/5) of cases of atypical glandular cells (AGC). Among women ≥30-65 years, the proportion ASCUS or worse (ASCUS+) was similar with cytology (3.52% [2016]) and primary HPV screening (3.70% [2017]). Only the proportion of ASC-H changed by the use of primary HPV screening, from 0.13% (2016) to 0.23% (2017) (p < 0.001). The colposcopy referral rate increased by 54% (3.70 vs 2.41%), when primary HPV screening was introduced. In conclusion, the implemented primary HPV screening approach demonstrated similar prevalence of ASCUS+ cytology as conventional screening. In addition, primary HPV screening decreased cytology assessments by 86% in our screening population of women 30 through 70 years taken into account the co-tested women.

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BACKGROUND: We recently reported a sensitivity of 85.5% to detect high-grade squamous intraepithelial lesions (HSIL)/adenocarcinoma in situ (AIS)/cancer by the use of self-collected vaginal samples analysed by the Aptima mRNA HPV assay (AHPV). OBJECTIVES: To increase detection of HPV among self-samples. STUDY DESIGN: We used a pre-heating step at 90 °C for 1 h on our previously AHPV-negative self-samples (N = 20) among women with AHPV-positive cervical samples. We also analysed AHPV results before and after the heating among a series of self-samples from women who had not attended cervical screening for > 7 years (N = 173). RESULTS: After heating, 55% (11/20) of the self-samples became AHPV-positive. By updating our original series 93.1% (121/130, 95% CI: 87.3-96.8) of the self-samples were AHPV-positive among women with AHPV-positive cervical samples, and among women with histologically confirmed cervical intraepithelial neoplasia or worse (CIN2+) now 95.3% (61/64, 95% CI: 86.9-99.0) of the self-samples were AHPV-positive. Among the 11 AHPV-positive self-samples we detected high-risk HPV types in 10 of the samples (HPV16 3 cases, HPV18 1, HPV31 1, HPV33 1, HPV 45 1, HPV51 2, HPV 56 and 58 1, HPV42 and 90 1 [low risk]) by multiplex PCR and Luminex assay. Among the self-samples from the non-attenders 16% (27/170) and 5.3% (8/152) were AHPV-positive after and before the heating step, respectively (P = 0.0022). Concerning validity of AHPV-results, 99% (170/172) were valid after the heating step compared to 88% (152/172) before the heating step (P < 0.0001). CONCLUSIONS: A pre-heating step on vaginal self-samples increased HPV detection by the AHPV assay.

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Because of its non-invasive nature urine testing may enable increased screening for HPV in women who avoid cervical sampling. Comparisons have shown fewer HPV positives in urine. The objectives were to compare first-void urine (FVU) treated with proteinase K (PK) to untreated FVU and cervical samples collected from women attending a colposcopy clinic using an Aptima HPV mRNA assay, and comparing the HPV rates to cytology and pathology results. Female FVU (n = 433) was treated with Aptima Transfer Solution (ATS) containing PK within 24 h or after months of storage. Untreated female FVU samples were HPV-positive in 20.8-27.6% compared to 34.4-45.6% of ATS-treated FVU and 44.9-48.4% of PreservCyt samples. Good overall agreement for HR-HPV detection between ATS-FVU and PreservCyt was observed (81.1%; k 0.63). Validation of ATS treatment was performed on 356 male FVU, detecting 6.7% HPV positive compared to 3.4% of untreated samples (p = 0.059). Although HPV presence in ATS FVU and PreservCyt samples were similar, significantly more women with abnormal cervical cytology and histopathology were HPV-positive in cervical specimens than in ATS-treated FVU.

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Randomized clinical trials using human papillomavirus (HPV) DNA testing have found increased protection against cervical cancer and HPV-based screening is globally recommended for women ≥30 years of age. HPV-mRNA is a promising alternative target for cervical screening tests, but assessing equivalence requires longitudinal evaluation over at least the length of a screening interval. Our aim is to analyze the longitudinal sensitivity of HPV-mRNA and HPV-DNA in cervical samples taken up to 7 years before severe cervical intraepithelial neoplasia or worse (CIN3+). From a population-based cohort of 95,023 women in Sweden, cervical samples were frozen at -80°C between May 2007 and January 2012. Registry linkages identified that 1,204 of these women had CIN3+ 4 months to 7 years after enrolment. Baseline samples were analyzed for HPV-mRNA (Aptima, Hologic) and for HPV-DNA (Cobas 4800, Roche) and results from both tests obtained for 1,172 women. For both women <30 and ≥ 30 years, HPV-mRNA had similar sensitivity for CIN3+ as HPV-DNA (p = 0.0217 and p = 0.0123 in noninferiority testing for at least 90% relative sensitivity, respectively). Among women ≥30 years, the longitudinal sensitivities for CIN3+ occurring 5-7 years later were comparable [76.3% (95% CI: 65.8%-84.3%) and 82.5% (95% CI: 72.6%-89.4%)] as were the longitudinal negative predictive values for absence of CIN3+ [99.97% (95% CI: 99.95-99.98) and 99.98% (95% CI: 99.96-99.99)], for the HPV-mRNA and HPV-DNA test. In conclusion, HPV-mRNA testing has similar longitudinal sensitivity as HPV-DNA, implying that HPV-mRNA testing can safely be used for cervical screening.

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