Assessment of the clinical and analytical performance of the Aptima SARS-CoV-2 assay using the VALCOR protocol.

BACKGROUND: The COVID-19 pandemic highlighted the importance of diagnostic testing against curbing the spread of SARS-CoV-2. The urgent need and scale for diagnostic tools resulted in manufacturers of SARS-CoV-2 assays receiving emergency authorization that lacked robust analytical or clinical evaluation. As it is highly likely that testing for SARS-CoV-2 will continue to play a central role in public health, the performance characteristics of assays should be evaluated to ensure reliable diagnostic outcomes are achieved. METHODS: VALCOR or "VALidation of SARS-CORona Virus-2 assays" is a study protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. Using clinical samples collated from VALCOR, the performance of Aptima SARS-CoV-2 assay was assessed against a standard comparator assay. Diagnostic test parameters such as sensitivity, specificity and overall per cent agreement were calculated for the clinical performance of Aptima SARS-CoV-2 assay. RESULTS: A total of 180 clinical samples were tested with an addition of 40 diluted clinical specimens to determine the limit of detection. When compared to the standard comparator assay Aptima had a sensitivity of 100.0% [95% CI 95.9-100.0] and specificity of 96.7% [95% CI 90.8-99.3]. The overall percent agreement was 98.3% with an excellent Cohen's coefficient of κ = 0.967 [95% CI 0.929-1.000]. For the limit of detection, Aptima was able to detect all of the diluted clinical samples. CONCLUSION: In conclusion. validation of Aptima SARS-CoV-2 assay using clinical samples collated through the VALCOR protocol showed excellent test performance. Additionally, Aptima demonstrated high analytical sensitivity by detecting all diluted clinical samples corresponding to a low limit of detection.

Accuracy of high-risk HPV DNA PCR, p16(INK4a) immunohistochemistry or the combination of both to diagnose HPV-driven oropharyngeal cancer.

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.

Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors.

BACKGROUND: Persistent infection with high-risk human papillomaviruses (hrHPV) types is causally linked with the development of cervical precancer and cancer. HPV types 16 and 18 cause approximately 70% of cervical cancers worldwide. OBJECTIVES: To evaluate the harms and protection of prophylactic human papillomaviruses (HPV) vaccines against cervical precancer and HPV16/18 infection in adolescent girls and women. SEARCH METHODS: We searched MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL) and Embase (June 2017) for reports on effects from trials. We searched trial registries and company results' registers to identify unpublished data for mortality and serious adverse events. SELECTION CRITERIA: Randomised controlled trials comparing efficacy and safety in females offered HPV vaccines with placebo (vaccine adjuvants or another control vaccine). DATA COLLECTION AND ANALYSIS: We used Cochrane methodology and GRADE to rate the certainty of evidence for protection against cervical precancer (cervical intraepithelial neoplasia grade 2 and above [CIN2+], CIN grade 3 and above [CIN3+], and adenocarcinoma-in-situ [AIS]), and for harms. We distinguished between the effects of vaccines by participants' baseline HPV DNA status. The outcomes were precancer associated with vaccine HPV types and precancer irrespective of HPV type. Results are presented as risks in control and vaccination groups and risk ratios (RR) with 95% confidence intervals in brackets. MAIN RESULTS: We included 26 trials (73,428 participants). Ten trials, with follow-up of 1.3 to 8 years, addressed protection against CIN/AIS. Vaccine safety was evaluated over a period of 6 months to 7 years in 23 studies. Studies were not large enough or of sufficient duration to evaluate cervical cancer outcomes. All but one of the trials was funded by the vaccine manufacturers. We judged most included trials to be at low risk of bias. Studies involved monovalent (N = 1), bivalent (N = 18), and quadrivalent vaccines (N = 7). Most women were under 26 years of age. Three trials recruited women aged 25 and over. We summarize the effects of vaccines in participants who had at least one immunisation.Efficacy endpoints by initial HPV DNA statushrHPV negativeHPV vaccines reduce CIN2+, CIN3+, AIS associated with HPV16/18 compared with placebo in adolescent girls and women aged 15 to 26. There is high-certainty evidence that vaccines lower CIN2+ from 164 to 2/10,000 (RR 0.01 (0 to 0.05)) and CIN3+ from 70 to 0/10,000 (RR 0.01 (0.00 to 0.10). There is moderate-certainty evidence that vaccines reduce the risk of AIS from 9 to 0/10,000 (RR 0.10 (0.01 to 0.82).HPV vaccines reduce the risk of any CIN2+ from 287 to 106/10,000 (RR 0.37 (0.25 to 0.55), high certainty) and probably reduce any AIS lesions from 10 to 0/10,000 (RR 0.1 (0.01 to 0.76), moderate certainty). The size of reduction in CIN3+ with vaccines differed between bivalent and quadrivalent vaccines (bivalent: RR 0.08 (0.03 to 0.23), high certainty; quadrivalent: RR 0.54 (0.36 to 0.82), moderate certainty). Data in older women were not available for this comparison.HPV16/18 negativeIn those aged 15 to 26 years, vaccines reduce CIN2+ associated with HPV16/18 from 113 to 6 /10,000 (RR 0.05 (0.03 to 0.10). In women 24 years or older the absolute and relative reduction in the risk of these lesions is smaller (from 45 to 14/10,000, (RR 0.30 (0.11 to 0.81), moderate certainty). HPV vaccines reduce the risk of CIN3+ and AIS associated with HPV16/18 in younger women (RR 0.05 (0.02 to 0.14), high certainty and RR 0.09 (0.01 to 0.72), moderate certainty, respectively). No trials in older women have measured these outcomes.Vaccines reduce any CIN2+ from 231 to 95/10,000, (RR 0.41 (0.32 to 0.52)) in younger women. No data are reported for more severe lesions.Regardless of HPV DNA statusIn younger women HPV vaccines reduce the risk of CIN2+ associated with HPV16/18 from 341 to 157/10,000 (RR 0.46 (0.37 to 0.57), high certainty). Similar reductions in risk were observed for CIN3+ associated with HPV16/18 (high certainty). The number of women with AIS associated with HPV16/18 is reduced from 14 to 5/10,000 with HPV vaccines (high certainty).HPV vaccines reduce any CIN2+ from 559 to 391/10,000 (RR 0.70 (0.58 to 0.85, high certainty) and any AIS from 17 to 5/10,000 (RR 0.32 (0.15 to 0.67), high certainty). The reduction in any CIN3+ differed by vaccine type (bivalent vaccine: RR 0.55 (0.43 to 0.71) and quadrivalent vaccine: RR 0.81 (0.69 to 0.96)).In women vaccinated at 24 to 45 years of age, there is moderate-certainty evidence that the risks of CIN2+ associated with HPV16/18 and any CIN2+ are similar between vaccinated and unvaccinated women (RR 0.74 (0.52 to 1.05) and RR 1.04 (0.83 to 1.30) respectively). No data are reported in this age group for CIN3+ or AIS.Adverse effectsThe risk of serious adverse events is similar between control and HPV vaccines in women of all ages (669 versus 656/10,000, RR 0.98 (0.92 to 1.05), high certainty). Mortality was 11/10,000 in control groups compared with 14/10,000 (9 to 22) with HPV vaccine (RR 1.29 [0.85 to 1.98]; low certainty). The number of deaths was low overall but there is a higher number of deaths in older women. No pattern in the cause or timing of death has been established.Pregnancy outcomesAmong those who became pregnant during the studies, we did not find an increased risk of miscarriage (1618 versus 1424/10,000, RR 0.88 (0.68 to 1.14), high certainty) or termination (931 versus 838/10,000 RR 0.90 (0.80 to 1.02), high certainty). The effects on congenital abnormalities and stillbirths are uncertain (RR 1.22 (0.88 to 1.69), moderate certainty and (RR 1.12 (0.68 to 1.83), moderate certainty, respectively). AUTHORS' CONCLUSIONS: There is high-certainty evidence that HPV vaccines protect against cervical precancer in adolescent girls and young women aged 15 to 26. The effect is higher for lesions associated with HPV16/18 than for lesions irrespective of HPV type. The effect is greater in those who are negative for hrHPV or HPV16/18 DNA at enrolment than those unselected for HPV DNA status. There is moderate-certainty evidence that HPV vaccines reduce CIN2+ in older women who are HPV16/18 negative, but not when they are unselected by HPV DNA status.We did not find an increased risk of serious adverse effects. Although the number of deaths is low overall, there were more deaths among women older than 25 years who received the vaccine. The deaths reported in the studies have been judged not to be related to the vaccine. Increased risk of adverse pregnancy outcomes after HPV vaccination cannot be excluded, although the risk of miscarriage and termination are similar between trial arms. Long-term of follow-up is needed to monitor the impact on cervical cancer, occurrence of rare harms and pregnancy outcomes.

Assessment of the clinical and analytical performance of three Seegene Allplex SARS-CoV-2 assays within the VALCOR framework.

The coronavirus disease 2019 (COVID-19) pandemic demonstrated the need for accurate diagnostic testing for the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pandemic has ended, accurate assays are still needed to monitor viral spread at national levels and beyond through population and wastewater surveillance. To enhance early detection, SARS-CoV-2 assays should have high diagnostic accuracy and should be validated to assure accurate results. Three distinct SARS-CoV-2 assays were evaluated with clinical samples using the VALCOR (VALidation of SARS-CORona Virus-2 assays) framework, with the TaqPath COVID-19 assay (ThermoFisher Scientific, USA) as a comparator. We evaluated clinical sensitivity, specificity, limit of detection (LOD), and overall concordance between comparator and three index Allplex SARS-CoV-2 assays (Seegene, South Korea): Allplex-SC2, Allplex-SC2Fast (Fast PCR), and Allplex-SC2FabR (SARS-CoV-2/FluA/FluB/respiratory syncytial virus). Analytical performance and LOD of index assays were assessed using a dilution series of three synthetic SARS-CoV-2 sequence reference materials (RMs). Ninety SARS-CoV-2 positives and 90 SARS-CoV-2 negatives were tested. All Allplex assays had 100.0% sensitivity (95%CI = 95.9%-100.0%). Allplex-SC2 and Allplex-SC2Fast assays had 97.8% specificity (95%CI = 92.3%-99.7%) and 98.9% overall concordance [κ = 0.978 (95%CI = 0.947-1.000)]. Allplex-SC2FabR assay showed 100.0% specificity (95%CI = 95.9%-100.0%) and 100.0% overall concordance [κ = 1.000 (95%CI = 1.000-1.000)]. LOD assessment of index assays revealed detection down to 2.61 × 102 copies/mL in clinical samples, while the analytical LOD was 9.00 × 102 copies/mL. In conclusion, the evaluation of the three Seegene Allplex SARS-CoV-2 assays showed high sensitivity and specificity and an overall good assay concordance with the comparator. The assays showed low analytical LOD using RM and even a slightly lower LOD in clinical samples. Non-overlapping target gene sequences between SARS-CoV-2 assays and RMs emphasize the need for aligning targeted sequences of diagnostic assays and RMs.IMPORTANCEThe coronavirus disease 2019 pandemic has a significant impact on global public health, economies, and societies. As shown through the first phases of the pandemic, accurate and timely diagnosis is crucial for disease control, prevention, and monitoring. Though the pandemic phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has concluded, diagnostic assays remain in demand to monitor SARS-CoV-2 at the individual patient level, regionally, and nationally, as well as to remain an infectious disease preparedness instrument to monitor any new SARS-CoV-2 dissemination across borders using population and wastewater surveillance. The anticipation by WHO and central health care policy entities such as the Center for Disease Control, EMA, and multiple national health authorities is that SARS-CoV-2 will reside as an endemic respiratory disease for years to come. The key strategic consideration is hence shifting from combating a pandemic situation with a high number of patients to instead allowing precise diagnostics of suspected patients with the intention of correct management in a low-prevalence setting.

Human papillomavirus testing versus repeat cytology for triage of minor cytological cervical lesions.

BACKGROUND: Atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intra-epithelial lesions (LSIL) are minor lesions of the cervical epithelium, detectable by cytological examination of cells collected from the surface of the cervix of a woman.Usually, women with ASCUS and LSIL do not have cervical (pre-) cancer, however a substantial proportion of them do have underlying high-grade cervical intra-epithelial neoplasia (CIN, grade 2 or 3) and so are at increased risk for developing cervical cancer. Therefore, accurate triage of women with ASCUS or LSIL is required to identify those who need further management.This review evaluates two ways to triage women with ASCUS or LSIL: repeating the cytological test, and DNA testing for high-risk types of the human papillomavirus (hrHPV) - the main causal factor of cervical cancer. OBJECTIVES: Main objective To compare the accuracy of hrHPV testing with the Hybrid Capture 2 (HC2) assay against that of repeat cytology for detection of underlying cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) or grade 3 or worse (CIN3+) in women with ASCUS or LSIL. For the HC2 assay, a positive result was defined as proposed by the manufacturer. For repeat cytology, different cut-offs were used to define positivity: Atypical squamous cells of undetermined significance or worse (ASCUS+), low-grade squamous intra-epithelial lesions or worse (LSIL+) or high-grade squamous intra-epithelial lesions or worse (HSIL+).Secondary objective To assess the accuracy of the HC2 assay to detect CIN2+ or CIN3+ in women with ASCUS or LSIL in a larger group of reports of studies that applied hrHPV testing and the reference standard (coloscopy and biopsy), irrespective whether or not repeat cytology was done. SEARCH METHODS: We made a comprehensive literature search that included the Cochrane Register of Diagnostic Test Accuracy Studies; the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE (through PubMed), and EMBASE (last search 6 January 2011). Selected journals likely to contain relevant papers were handsearched from 1992 to 2010 (December). We also searched CERVIX, the bibliographic database of the Unit of Cancer Epidemiology at the Scientific Institute of Public Health (Brussels, Belgium) which contains more than 20,000 references on cervical cancer.More recent searches, up to December 2012, targeted reports on the accuracy of triage of ASCUS or LSIL with other HPV DNA assays, or HPV RNA assays and other molecular markers. These searches will be used for new Cochrane reviews as well as for updates of the current review. SELECTION CRITERIA: Studies eligible for inclusion in the review had to include: women presenting with a cervical cytology result of ASCUS or LSIL, who had undergone both HC2 testing and repeat cytology, or HC2 testing alone, and were subsequently subjected to reference standard verification with colposcopy and colposcopy-directed biopsies for histologic verification. DATA COLLECTION AND ANALYSIS: The review authors independently extracted data from the selected studies, and obtained additional data from report authors.Two groups of meta-analyses were performed: group I concerned triage of women with ASCUS, group II concerned women with LSIL. The bivariate model (METADAS-macro in SAS) was used to assess the absolute accuracy of the triage tests in both groups as well as the differences in accuracy between the triage tests. MAIN RESULTS: The pooled sensitivity of HC2 was significantly higher than that of repeat cytology at cut-off ASCUS+ to detect CIN2+ in both triage of ASCUS and LSIL (relative sensitivity of 1.27 (95% CI 1.16 to 1.39; P value < 0.0001) and 1.23 (95% CI 1.06 to 1.4; P value 0.007), respectively. In ASCUS triage, the pooled specificity of the triage methods did not differ significantly from each other (relative specificity: 0.99 (95% CI 0.97 to 1.03; P value 0.98)). However, the specificity of HC2 was substantially, and significantly, lower than that of repeat cytology in the triage of LSIL (relative specificity: 0.66 (95% CI 0.58 to 0.75) P value < 0.0001). AUTHORS' CONCLUSIONS: HPV-triage with HC2 can be recommended to triage women with ASCUS because it has higher accuracy (significantly higher sensitivity, and similar specificity) than repeat cytology. When triaging women with LSIL, an HC2 test yields a significantly higher sensitivity, but a significantly lower specificity, compared to a repeat cytology. Therefore, practice recommendations for management of women with LSIL should be balanced, taking local circumstances into account.

HPV and head and neck cancers: Towards early diagnosis and prevention.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an increasing trend of its incidence. Alcohol consumption, smoking, and viral infections, such as the mucosal high-risk (HR) human papillomaviruses (HPVs) are major risk factors for HNSCC development. In particular, HR HPVs are mainly associated with a subset of oropharyngeal squamous cell carcinoma (OPSCC), while other head and neck sites are marginally affected by HPV infection. HPV16 is the most frequently HR HPV type associated with HNSCC. In contrast to the cervix, no screening programs or identifiable pre-malignant lesions have been characterized for HPV-related HNSCC. Therefore, identification of general diagnostic algorithms and HPV biomarkers that could facilitate the early diagnosis, disease evolution and recurrence for HPV-driven HNSCCs are urgently needed. We herein review the role of HPV in HNSCC with a focus on epidemiology, biology, applied diagnostic algorithms and available biomarkers in body fluids as early diagnostic tools in HPV-driven HNSCCs.

VALCOR: a protocol for the validation of SARS-corona virus-2 assays.

BACKGROUND: Testing for SARS-CoV-2, together with vaccination, is one of the most vital strategies in curbing the current COVID-19 pandemic. The pandemic has led to an unprecedented need for diagnostic testing and the rapid emergence of an abundance of commercial assays on the market. Due to the nature of the pandemic and in the interest of health protection, many of these assays received provisional authorisation for emergency use without thorough validation. To limit false negative and false positive results, it is key to define common criteria that SARS-CoV-2 assays need to fulfil. VALCOR or "VALidation of SARS-CORona Virus-2 assays" is a protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. OBJECTIVES: VALCOR is a study protocol for the validation of assays used for confirmation of the presence of SARS-CoV-2 in patients with COVID-19 disease or the screening of carriers of SARS-CoV-2 virus by the identification of viral RNA in oropharyngeal and/or nasopharyngeal specimens or other specimens from the human respiratory tract. METHODS: The VALCOR panel of samples will contain clinical human specimens and standardised artificial specimens. The collection of clinical specimens will include nasopharyngeal or oropharyngeal specimens or other specimens from the respiratory tract obtained from COVID-19 patients and healthy carriers of SARS-CoV-2 as well as specimens from subjects not carrying SARS-CoV-2. Artificial specimens include calibrated amounts of viral RNA of SARS-CoV-2 sequences provided by established competent agencies that produce reference materials for the assessment of the limit of detection of each assay. The panel of samples are sent from a central reference laboratory (having access to biobanks of clinical specimens tested already for SARS-CoV-2 with a reference comparator assay) to participating laboratories for testing with a SARS-CoV-2 index assay that requires evaluation. DISCUSSION: VALCOR provides a harmonised and standard framework to benchmark the testing performance of SARS-CoV-2 assays that are rapidly evolving. As the pandemic incited an urgent need for testing capacity, there is a gap in the comprehensive validation of SARS-CoV-2 assays. This study will generate comprehensive validation data for assays used for the diagnosis of SARS-CoV-2 and may serve as a basis for other validation protocols.

HPV DNA genotyping, HPV E6*I mRNA detection, and p16INK4a/Ki-67 staining in Belgian head and neck cancer patient specimens, collected within the HPV-AHEAD study.

BACKGROUND: The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries. METHODS: As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC-sites were tested for the presence of type-specific HPV RNA and p16INK4a over-expression. RESULTS: The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC. CONCLUSION: Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.

Prevaccination distribution of human papillomavirus types in women attending at cervical cancer screening in Belgium.

INTRODUCTION: Before the introduction of vaccination against human papillomaviruses (HPV) as a new strategy of combating cervical cancer, it is required to describe the baseline prevalence of HPV infection as well as the distribution of the different HPV types in the population and among women with cervical lesions. MATERIALS AND METHODS: Approximately 10,000 liquid cervical cell samples from women, resident of Flanders (North Belgium) and participating in cervical cancer screening, were assessed cytologically and virologically with a multiplex real-time PCR using primers targeting the E6/E7 genes of 16 HPV types. Correlations of HPV infection with age, geographic area, and occurrence of cytologic lesions were assessed. RESULTS: The prevalence of cytologic abnormalities was atypical squamous cells of undetermined significance (ASC-US), 1.6%; atypical glandular cells (AGC), 0.2%; low-grade squamous intraepithelial lesion (LSIL), 2.6%; atypical squamous cells, HSIL cannot be excluded (ASC-H), 0.3%; and high-grade squamous intraepithelial lesion (HSIL), 1.2%. The frequency of high-risk HPV infections was 11% in women without cytologic abnormalities, 77% in ASC-US, 32% in AGC, 85% in LSIL, and 93% in ASC-H and HSIL. The prevalence of high-risk HPV infection was highest in women of ages 20 to 24 years (29%) and decreased progressively with age. The percentage of women with HSIL in the entire study population attributable to infection with a particular type (AR(pop) %) was highest for HPV16 (32%), followed by HPV31 (22%), HPV39 (11%), and HPV52 (11%). HPV18 was responsible for 7% of the HSIL lesions. Elimination of HPV16 and HPV18 is expected to reduce the prevalence of ASCUS with 24%, AGC with 19%, LSIL with 29%, ASC-H with 31% and HSIL with 37%. DISCUSSION: Compared to other West European studies, the prevalence of HPV infection was considerably higher in cytologically negative women but similar in women with cervical lesions. These differences could be due to the use of a PCR with high analytic sensitivity. These data are relevant for estimating the expected and theoretical levels of vaccine protection offered as vaccinated girls gradually age into the groups from which our observations stem. Further periodic laboratory-based surveys, including genotyping of cervical cell samples and linkage with vaccine registries, are an important resource to address pending questions of the effect of HPV vaccination. Research is warranted to disentangle the causal role of individual HPV types in case of multiple infections.

Analysis of 13 million individual patient records pertaining to Pap smears, colposcopies, biopsies and surgery on the uterine cervix (Belgium, 1996-2000).

OBJECTIVE: Cervical cancer screening by surveys overestimate coverage because of selection and reporting biases. METHODS: The prepared Inter-Mutualistic Agency dataset has about 13 million records from Pap smears, colposcopies, cervical biopsies and surgery, performed in Belgium between 1996 and 2000. Cervical cancer screening coverage was defined as the proportion of the target population (women of 25-64 years) that has had a Pap smear taken within the last 3 years. Proportions and incidence rates were computed using official population data of the corresponding age group, area and calendar year. RESULTS: Cervical cancer screening coverage, in the period 1998-2000, was 59% at national level, for the target age group 25-64 years. Differences were small between the 3 regions. Variation ranged from 39% to 71%. Coverage was 64% for 25-29 year old women, 67% for those aged 30-39 years, 56% for those aged 50-54. The modal screening interval was 1 year. In the 3-year period 1998-2000, 3 million smears were taken from the 2.7 million women in the age group 25-64. Only 1.6 million women of the target group got one or more smears in that period and 1.1 million women had no smears, corresponding to an average of 1.88 smears per woman. CONCLUSION: Coverage reached only 59%, but the number of smears used was sufficient to cover more than 100% of the target population. Structural reduction of overuse and extension of coverage is warranted.

The Positive Predictive Value of High-Grade Squamous Intraepithelial Lesion on Cytology for the Histological Diagnosis of Cervical Intraepithelial Neoplasia 2 or Higher: A Systematic Review.

Cervical cancer is a major worldwide health problem. Therefore, regular cervical screening in order to make an early diagnosis can help to prevent cervical cancer, through identifying and treating preinvasive cervical lesions. The aim of this review is to evaluate the correlation between the cytological screening result and the final gold standard histological outcome in the diagnosis of cervical lesions. More specifically, the correlation between high-grade intraepithelial lesion (HSIL) on cytology and histological cervical intraepithelial neoplasia grade 2 or higher (CIN2+) was intended, by calculating the positive predictive value (PPV). PPV is an important value from a clinical point of view. An electronic search was carried out in the electronic databases MEDLINE (through PubMed) and the Cochrane Library (last searched beginning of December 2017), supplemented with the related article feature in PubMed and snowballing. Article selection (predefined inclusion and exclusion criteria) and data extraction were evaluated by two independent reviewers (N.K. and A.V.L.). After identifying 1,146 articles, 27 articles were finally included in this systematic review, representing 28,783 cytological HSIL diagnoses in total. The PPV of HSIL was 77.5% (range: 45.4-95.2%) for the histological diagnosis of CIN2+ and 55.4% (range: 36.4-67.6%) for the diagnosis of CIN3+. In this systematic review, 77.5% of the HSIL-positive women eventually had a CIN2+ diagnosis. The diagnostic value of a cytological HSIL result (conventional or liquid-based cytology) in the diagnosis of CIN2+ lesions is good, but a combination of tests could raise this value.

Efficacy of commercially available biological agents for the topical treatment of cervical intraepithelial neoplasia: a systematic review.

BACKGROUND: Cervical cancer is a major public health issue in the world, especially in developing countries. It can be prevented through vaccination against HPV (primary prevention) and through screening and treatment of cervical intraepithelial neoplasia (CIN) (secondary prevention). Surgical methods for treatment of CIN are linked to complications such as bleeding and adverse pregnancy outcomes. Furthermore, these methods are not generally available in resource-poor settings. Therefore, topical agents for local application on the cervix have been used since decades to overpass complications and limitations of the surgical methods. AIMS: Review of the literature on the efficacy of commercially available biological agents used for topical treatment of cervical intraepithelial neoplasia (CIN). METHODS: A systematic search through PubMed and the Cochrane database was performed up to December 2017, using the medical subheadings (MesH) for topical agent, treatment, and cervical intraepithelial neoplasia. Appropriate inclusion/exclusion criteria have been used for the selection of eligible clinical studies. Clinical studies containing a minimum of 20 women, aged 18-50 with a diagnosis of CIN 1-3, and at least a 4 weeks follow-up after the end of the topical treatment were included. RESULTS: The initial electronic database search resulted in a total of 849 articles. After screening titles and abstracts, 62 articles were selected as potential studies. Of these, six articles were included in the review after reading the full text: two were on 5-FluoroUracil, two on trans retinoic acid, one on Imiquimod, and one on Cidofovir. The reported regression/remission rates for CIN differed among studies. In CIN2 patients, the overall remission rate ranged between 43 and 93% for the active agents. CONCLUSION: Among the topical agents studied, 5-FluoroUracil showed good remission rates above 80%. Varying results seen in this review is due to the differences in quality of the design between studies. Large-scale and less biaised studies are needed to elucidate the true efficacy and safety of topical agents in the treatment of CIN.

Comparative study of the radiosensitizing and cell cycle effects of vinflunine and vinorelbine, in vitro.

BACKGROUND: Vinca alkaloids are an important class of anticancer agents and semisynthetic vinca alkaloids are developed to improve the therapeutic index of this class of drugs. In the present study, a direct comparison was made between vinflunine and vinorelbine regarding their radiosensitizing and cell cycle effects. METHODS: Four human tumour cell lines were tested under identical experimental conditions, using equitoxic concentrations of vinflunine and vinorelbine. RESULTS: Vinflunine and vinorelbine induced a comparable radiosensitizing effect (p-value never below 0.01) when cells were incubated for 24 h immediately prior to radiation. Regarding the cell cycle effects, a statistically significant concentration-dependent G2/M block was seen after 24 h incubation with vinorelbine in all tested cell lines. Similar results, with small cell line-related differences, were observed with vinflunine. CONCLUSION: The radiosensitizing effects of both semisynthetic vinca alkaloids were comparable (not statistically different) and nearly always cell line-specific and concentration-dependent. The cell cycle effects could be related to the observed radiosensitizing effects. Considering the more favourable toxicity profile of vinflunine, this agent might be more promising than vinorelbine for chemoradiation studies in the clinic.

Further mechanistic unravelling of the influence of the cell cycle effects on the radiosensitising mechanism of vinflunine, in vitro.

PURPOSE: Vinflunine is an innovative microtubule inhibitor belonging to the vinca alkaloid class that possesses radiosensitising properties, which could lead to promising activity in chemoradiation studies in the clinic. METHOD: In the current study, different incubation times with vinflunine, immediately before radiation and different time intervals between vinflunine treatment and radiation were investigated, in vitro, using four different human tumour cell lines differing in cell type and p53 status. Results were correlated with the cell cycle distribution at the moment of radiation, in order to elucidate the role of cell cycle perturbations caused by vinflunine on its radiosensitising effect. RESULTS: Radiosensitisation was observed in all cell lines, and maximal radiosensitisation was both cell line- and schedule-dependent. The cell cycle distributions were cell line-dependent also, and when correlated with the observed radiosensitising effects could explain many (but not all) of the radiosensitising properties of vinflunine. CONCLUSION: The cell cycle perturbations caused by vinflunine may definitely have an impact on its radiosensitising potential, but other factors must play a role because of some unaccountable differences between cell cycle distribution and the radiosensitising potential.

Efficacy of antiviral drug AV2 in the treatment of human papillomavirus-associated precancerous lesions of the uterine cervix: A randomized placebo-controlled clinical trial in Kinshasa, DR Congo. (KINVAV study).

BACKGROUND: Cervical Cancer (CC) is a major public health problem in DR Congo; the high incidence of CC is due to the inexistence of effective screening programs based on cytology and/or HPV detection followed by appropriate treatments. This situation highlights the need to implement efficacious and inexpensive treatment methods. This study aims at evaluating the efficacy of a topical antiviral drug named AV2® as a treatment for HPV-associated lesions of the cervix. METHODS: Women will undergo cytology sampling, HPV testing and Visual inspection of the cervix after application of 5% acetic acid (VIA). VIA-positive women will be randomized to one of two groups to receive treatment by either AV2®or placebo. They will undergo control examinations after two months and after six months. In case of persistent lesions on VIA, treatment by cryotherapy will be done. The primary outcomes will be the change of lesions, the clearance of HPV DNA, and the correlation of the two 2 months after treatment with AV2®. CONCLUSION: This study is the first large-scale study in Africa to evaluate systematically the efficacy and safety of a topical antiviral drug for the treatment of HPV- associated lesions of the cervix. Its findings will direct the planning of suitable algorithms for CC screening and treatment. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov - Unique identifier: NCT02346227, registered on November 8, 2014.

The influence of physical activity in the anti-tumor immune response in experimental breast tumor.

This study aimed to investigate the influence of physical activity in innate immunity to conduce to an effective antitumoral immune response analyzing the phenotype and activation status of infiltrating cells. We analysed the intracellular cytokines and the transcription factors of tumor infiltrating lymphocytes (TILS) and spleen leukocytes. The Nos2 gene expression was evaluated in spleen cells and futhermore the ROS production was measured and spleen cells; another cell evaluated was dendritic cells (TIDCs), their cytokines expression and membrane molecules; finally to understood the results obtained, we analysed the dendritic cells obtained from bone marrow. Were used female Balb/c mice divided into 4 groups: two controls without tumor, sedentary (GI) and trained (GII) and two groups with tumor, sedentary (GIII) or trained (GIV). The physical activity (PA) was realized acoording swimming protocol. Tumor was induced by injection of 4T1 cells. All experiments were performed in biological triplicate. After the experimental period, the tumor was removed and the cells were identified by flow cytometry with labeling to CD4, CD8, CD11c, CD11b, CD80, CD86 and Ia, and intracelular staining IL-10, IL-12, TNF-α, IFN-γ, IL-17, Tbet, GATA3, RORγt and FoxP3. The bone marrow of the animals was obtained to analyse the derivated DCs by flow cytometry and culture cells to obtain the supernatant to measure the cytokines. Our results demonstrated that the PA inhibit the tumoral growth although not to change the number of TILS, but reduced expression of GATA-3, ROR-γT, related with poor prognosis, and TNF-α intracellular; however occur one significantly reduction in TIDCS, but these cells expressed more co-stimulatory and presentation molecules. Furthermore, we observed that the induced PA stimulated the gene expression of Tbet and the production of inflammatory cytokines suggesting an increase of Th1 systemic response. The results evaluating the systemic influence in DCs showed that the PA improve significantly the number of those cells in bone marrow as well the number of co-stimulatory molecules. Therefore, we could conclude that PA influence the innate immunity by interfering to promote in process of maturation of DCs both in tumor and systemically, that by its turn promote a modification in acquired immune cells, representing by T helper to induce an important alteration transcription factors that are responsible to maintain a suppressive microenviroment, and thereby, allowing the latter cells can thus activate antitumor immune response. The PA was able improve the Th1 systemic response by enhance to Tbet gene expression, promote a slightly increased of Th1-type cytokines and decrease Gata3 and Foxp3 gene expression in which can inhibit the Th1 immune response.

Cell cycle effects of vinflunine, the most recent promising Vinca alkaloid, and its interaction with radiation, in vitro.

PURPOSE: Vinflunine (VFL) is a novel third generation Vinca alkaloid with superior antitumour activity in preclinical models and an anticipated more favourable toxicity profile compared to the other Vinca alkaloids. METHOD: We investigate the radiosensitising properties of VFL and its cell cycle effects in four human tumour cell lines (ECV304, MCF-7, H292, and CAL-27). The sulforhodamine B test was used to determine cell survival, and cell cycle analysis was performed by flow cytometry. Radiosensitisation (RS) was represented by dose enhancement factors (DEFs). RESULTS: Twenty-four hours treatment with VFL before radiation caused dose-dependent RS in all cell lines. This was most pronounced in ECV304 cells with RS already at VFL concentrations that reduced cell survival by 10% (IC10). DEFs ranged from 1.57 to 2.29 in the different cell lines. A concentration-dependent G2/M block was observed (starting at 4 h of incubation). After maximal G2/M blockade cells started cycling again, mainly by mitosis, while a small portion of cells started a polyploid cell cycle. Also drug removal immediately caused recycling of cells and induction of a polyploid cell population. The polyploid cell population was most impressively noticeable after prolonged incubation with VFL (48 h), in particular in CAL-27 and ECV304. This was never observed in a tested normal fibroblast cell line (Fi 360). The fate of these cells is of particular interest, but yet uncertain. CONCLUSION: VFL has radiosensitising potential. The exact role of the cell cycle effects of VFL in its radiosensitising mechanism is still not fully elucidated and requires further study.

Angiogenesis Markers in Gynecological Tumors and Patents for Anti-Angiogenic Approach: Review.

The formation of a tumor-associated vascular network is an important step in understanding the stages of tumor progression. This review aims to highlight the main markers of induction, proliferation and inhibition of angiogenesis, as well as the quantification of microvessel density, correlated with preclinical and clinical research in gynecologic cancers and also discussed related patents. Studies show that in the most advanced cases of gynecological cancers, biomarkers such as VEGF (Vascular Endothelial Growth Factor), MMP (Matrix Metalloproteinase), CD105 (Endoglin), TIMP (tissue inhibitors of metalloproteinases) and VASH (Vasohibin) are more expressed compared to healthy individuals. Continuous evaluation of these biomarkers in cancer cases could serve in the future as a basis for development of new therapeutic approaches, leading to a good response to cancer treatment, and thus increase survival of cancer patients.

Attendance at cervical cancer screening and use of diagnostic and therapeutic procedures on the uterine cervix assessed from individual health insurance data (Belgium, 2002-2006).

OBJECTIVE: To assess the coverage for cervical cancer screening as well as the use of cervical cytology, colposcopy and other diagnostic and therapeutic interventions on the uterine cervix in Belgium, using individual health insurance data. METHODS: The Intermutualistic Agency compiled a database containing 14 million records from reimbursement claims for Pap smears, colposcopies, cervical biopsies and surgery, performed between 2002 and 2006. Cervical cancer screening coverage was defined as the proportion of women aged 25-64 that had a Pap smear within the last 3 years. RESULTS: Cervical cancer screening coverage was 61% at national level, for the target population of women between 25 and 64 years old, in the period 2004-2006. Differences between the 3 regions were small, but varied more substantially between provinces. Coverage was 70% for 25-34 year old women, 67% for those aged 35-39 years, and decreased to 44% in the age group of 60-64 years. The median screening interval was 13 months. The screening coverage varied substantially by social category: 40% and 64%, in women categorised as beneficiary or not-beneficiary of increased reimbursement from social insurance, respectively. In the 3-year period 2004-2006, 3.2 million screen tests were done in the target group consisting of 2.8 million women. However, only 1.7 million women got one or more smears and 1.1 million women had no smears, corresponding to an average of 1.88 smears per woman in three years of time. Colposcopy was excessively used (number of Pap smears over colposcopies = 3.2). The proportion of women with a history of conisation or hysterectomy, before the age of 65, was 7% and 19%, respectively. CONCLUSION: The screening coverage increased slightly from 59% in 2000 to 61% in 2006. The screening intensity remained at a high level, and the number of cytological examinations was theoretically sufficient to cover more than the whole target population.