Cervix cytology samples revealed increased methylation of the human markers FAM19A4/miR124-2 up to 8 years before adenocarcinoma.

INTRODUCTION: Methylation analysis of the promoter region of tumor-suppressor genes has previously shown high sensitivity for detection of high-grade cervical intraepithelial neoplasia (CIN) and cancer. HPV-testing has a high sensitivity to identify women at risk to develop cancer, and has been implemented in cervical screening programs in several countries. But in most HPV-positive women the infection will clear and they will not develop cancer. Testing for methylation could help to identify women who have potentially progressive cervical disease and need closer follow-up. The goal of the present study was to investigate the potential use of methylation as a triage test of HPV-positive women in the screening program. MATERIAL AND METHODS: A collection of liquid-based cytology (LBC) samples from 106 women, collected between 4 months and 8 years before histologically confirmed cervical cancer or CIN3, was analyzed for hypermethylation of the human genes FAM19A4 and miR124-2. RESULTS: Methylation was detected in 45% (33/73) of normal LBC samples from women who later developed CIN3+, compared with 10% (3/31) of normal LBC samples from women without subsequent dysplasia (P = 0.0006). Overall, methylation was detected in 39% (14/36), 51% (19/37), 61% (14/23) and 70% (7/10) of LBC samples from women who later developed CIN3, adenocarcinoma in situ (AIS), squamous cell carcinoma (SCC) and adenocarcinoma (ADC), respectively. Positive methylation analysis was not significantly more frequent than abnormal cytology of atypical squamous cells of unclear significance or worse (ASCUS+) in LBC samples collected 4 months to 8 years before SCC or AIS; however, prior to the development of ADC, methylation was observed in 7/10 LBC samples, despite normal cytology. Overall, LBC samples collected before invasive cancer (ADC and SCC) were more frequently positive in the methylation analysis than in cytological analysis of ASCUS+ (P = 0.048). For LBC samples collected more than 2 years before the development of AIS, SCC or ADC, methylation analysis showed a higher positivity rate than cytology did. CONCLUSIONS: Testing for methylation of FAM19A4/miR124-2 as a triage for HPV-positive women would be useful to identify women at risk of cancer development, especially adenocarcinoma. Further studies are needed to estimate the cost-effectiveness before introducing methylation testing in the screening program.

Cervical cancer in Region Skåne, Sweden 2017-2020 after the implementation of primary HPV screening: A quality assurance audit.

INTRODUCTION: Primary human papilloma virus (HPV) screening to detect cervical cancer and dysplastic lesions was implemented in Region Skåne 2017 for women aged 30-70. The aim of this study was to characterize the screening history of women diagnosed with cervical cancer to evaluate the performance of the screening program, as well as to assess the cancer treatments given and shortcomings in the follow-up of women with cervical dysplasia. MATERIAL AND METHODS: We performed a quality assurance audit. The data was collected from the National Cervical Cancer Prevention Registry, Region Skåne Labmedicin database and the Melior Journal system in 2017-2020. RESULTS: We identified 247 women diagnosed with invasive cervical cancer in Region Skåne in 2017-2020. Of these, 35 (14.2%) had a screening history over at least two screening rounds before diagnosis. There were 25 (10.1%) women diagnosed with cervical cancer in between screening intervals, i.e., interval cancer. The most common screening history in women with cervical cancer was irregular screening (143, 57.9%), followed by women being above screening age (44, 17.8%). HPV was detected in 96% of the cases, either in cervical cytology or in the tumor tissue. The screening program detected the disease in 96 (38.9%) of the patients, 149 (60.3%) were diagnosed through symptoms and two (0.80%) as a result of incidental findings. CONCLUSIONS: The most powerful tool in the prevention of cervical cancer is screening program attendance. Prolongation with HPV screening among elderly women will also reduce the incidence of cervical cancer. Today, such cancers are usually discovered when symptoms appear.

Circulating tumour HPV16 DNA quantification - A prognostic tool for progression-free survival in patients with HPV-related oropharyngeal carcinoma receiving curative chemoradiotherapy.

BACKGROUND AND PURPOSE: Circulating tumour (ct) human papillomavirus (HPV) DNA is detectable in HPV-related oropharyngeal carcinoma (OPSCC) patients and has the potential to become an important clinical tool. This study aimed to evaluate the prognostic significance of ctHPV16-DNA kinetics during treatment with chemoradiotherapy in HPV-related OPSCC. Patients with p16-positive OPSCC recruited to the ARTSCAN III trial, comparing radiotherapy plus cisplatin with radiotherapy plus cetuximab, constituted the study cohort. MATERIALS AND METHODS: Blood samples before start and at the end of treatment of 136 patients were analysed. ctHPV16-DNA was quantified by real-time (q)PCR. The correlation between ctHPV16-DNA levels and tumour burden was investigated with Pearson regression analysis. The prognostic value of ctHPV16-DNA levels at baseline and decline during treatment was evaluated by area-under-the-curve (AUC) calculations and analysed with univariable and multivariable Cox proportional hazards models. RESULTS: ctHPV16-DNA was detectable with qPCR in 108/136 patients before start of treatment and cleared in 74% of these patients at the end of treatment. Disease burden was significantly correlated with baseline ctHPV16-DNA levels (R = 0.39, p=<0.001). Both lower baseline levels and AUC-ctHPV16DNA were associated with improved progression-free survival (p = 0.01 and p < 0.001), overall survival (p = 0.013 and p = 0.002), but not local tumour control (p = 0.12 and p = 0.2, respectively), with a stronger association for AUC-ctHPV16DNA (likelihood ratio test 10.5 vs 6.5 in Cox regression analyses of progression-free survival). In multivariable analysis including tumour volume (GTV-T) and treatment allocation (cisplatin vs cetuximab), AUC-ctHPV16DNA remained a significant prognostic marker of progression-free survival. CONCLUSION: ctHPV16-DNA is an independent prognostic factor in HPV-related OPSCC.

Increased prevalence of human papillomavirus in fresh tissue from penile cancers compared to non-malignant penile samples: a case-control study.

BACKGROUND: HPV has been detected in approximately 50% of invasive penile cancers but with a large span between 24 and 89%, most likely due to different types of tumors and various methods for HPV analysis. Most studies of HPV in penile cancer have been performed using paraffin-embedded tissue, argued to be at risk for contaminated HPV analysis. Viral activity of HPV, by the use of HPV mRNA expression is well studied in cervical cancer, but seldom studied in penile cancer. The aim was to determine prevalence of HPV types in fresh tissue of penile cancers compared to non-malignant age-matched penile controls. Additional aims were to analyze the viral expression and copy numbers of HPV16-positive tumors and 10 mm adjacent to the tumor. METHODS: Fresh tissue from penile cancer cases was biopsied inside the tumor and 10 mm outside the tumor. Controls were males circumcised for non-malignant reasons, biopsied at surgery. PCR and Luminex assays were used for identification of HPV types. HPV16-positive samples were investigated for copy numbers and expression of HPV16-mRNA. RESULTS: Among tumors (n = 135) and age-matched controls (n = 105), HPV was detected in 38.5% (52/135) and 11.4% (12/105), respectively (p < 0.001), adjusted odds ratio 12.8 (95% confidence interval 4.9-33.6). High-risk HPV types were found in 35.6% (48/135) of tumors and 4.8% (5/105) of controls (p < 0.001). Among tumors and controls, HPV16 was present in 27.4% (37/135) and 1% (1/105), respectively (p < 0.001). Among HPV16-positive penile cancers, mean HPV16 viral copy/cell was 74.4 (range 0.00003-725.4) in the tumor and 1.6 (range 0.001-14.4) 10 mm adjacent from the tumor. HPV16-mRNA analysis of the tumors and 10 mm adjacent from the tumors demonstrated viral activity in 86.5% (32/37) and 21.7% (5/23), respectively. CONCLUSIONS: The prevalence of HPV was significantly higher in penile cancer (38.5%) than among age-matched non-malignant penile samples (11.4%). HPV16 predominates (27.4%) in penile tumors. HPV16 expression was more common in penile cancer than in adjacent healthy tissue, strongly suggesting an etiological role for HPV16 in the development of penile cancer.

Improving human papillomavirus (HPV) testing in the cervical cancer elimination era: The 2021 HPV LabNet international proficiency study.

BACKGROUND: Proficient Human Papillomavirus (HPV) genotyping services are essential to support HPV and cervical cancer elimination strategies, in particular to support HPV vaccine research. OBJECTIVES: To perform a global HPV genotyping proficiency study, with evaluation in relation to previous proficiency studies. STUDY DESIGN: The proficiency panel contained 44 coded samples (40 samples containing one or more purified HPV types (HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59/68a/68b) in human DNA, 1 human DNA control and 3 DNA extraction controls). Proficiency required detection of both single and multiple infections of 50 International Units of HPV 16/18, of 500 genome equivalents for other HPV types and no false positivity. RESULTS: One hundred and thirty-two laboratories submitted 211 datasets. Most assays used (182/211 datasets) were commercially available. An all-time high of 75% of the datasets were 100% proficient. One or more false positives were found in 17.5% of datasets. Among laboratories who participated in the 2019 proficiency study, full proficiency increased from 25% in 2019 to 60% in 2021. The high overall proficiency was mostly attributable to a large number of new laboratories, which used similar assays. CONCLUSIONS: The worldwide deterioration in comparability and reliability of HPV testing found in 2019 is now reversed and an overall increase in proficiency is found.

Incidence of penile intraepithelial neoplasia and treatment strategies in Sweden 2000-2019.

OBJECTIVES: To analyse the incidence, treatment strategies and complications associated with penile intraepithelial neoplasia (PeIN) in Sweden over a period of 20 years. MATERIALS AND METHODS: Data on PeIN from the Swedish National Penile Cancer Register were analysed regarding treatment in relation to age, size of the PeIN lesion, localization of the PeIN lesion and complications using chi-squared tests and logistic regression. The incidence of PeIN was calculated and age-standardized according to the European Standard population. RESULTS: Between 2000 and 2019 a total of 1113 PeIN cases were reported. The age-standardized incidence of PeIN was 1.40 per 100 000 men (95% confidence interval [CI] 1.32-1.49). An increase in incidence over time was seen, with a standardized incidence rate of 2.37 (95% CI 1.56-3.70) in 2019 compared to the baseline year, 2000. Surgical or topical treatments were given in 75.0% and 14.6% of cases, respectively. The complication rate was higher in laser surgery (12.1%, 7/58) compared to local surgery (4.6%, 16/348; P = 0.03) with an age-adjusted odds ratio (OR) of 2.82 (95% CI 1.10-7.19; P = 0.03). Local surgery was more common than laser surgery in the last 5 years compared to the first 5 years of the study period: OR 5.75 (95% CI 2.94-11.27). Treatments with imiquimod and topical 5-fluorouracil (5-FU) were more common than destructive methods such as photodynamic therapy, cryotherapy, curettage and electrocautery in the last 5 years compared to the first 5 years: OR 9.48 (95% CI 2.29-39.24). CONCLUSIONS: A twofold increase in the age-standardized incidence of PeIN was seen in Sweden over 20 years. Complications were three times more common in laser surgery compared to local surgery. Changes in treatment showed an increase of treatment strategies such as local surgery and treatment with imiquimod and topical 5-FU over time.

Co-testing in cervical screening among 40- to 42-year-old women is unreasonable.

INTRODUCTION: The screening program for cervical cancer in Sweden recommends the use of primary human papillomavirus (HPV) screening for women aged ≥30 to 65 years. Co-testing with both HPV analysis and cytology is recommended at the first screening after the age of 40 years. To fulfil co-testing, all screened women aged 40-42 years within the region of Skåne were co-tested. The aim of the audit was to investigate the proportion of severe dysplasia as diagnosed by cytology and histological follow-up among women with Aptima HPV-negative tests. We also calculated the cost of adding the cytology to the HPV primary screening program. MATERIAL AND METHODS: The local cytology registry was used to identify women aged 40-42 years who attended screening and were co-tested during the 4 years from January 2017 to December 2020. The Aptima HPV messenger RNA assay detects 14 HPV types. For Aptima HPV-negative women with high-grade cytology or histological high-grade squamous intraepithelial lesions (HSILs), we performed extended HPV typing for 40 HPV types with polymerase chain reaction using modified GP5+/6+ primers followed by a Luminex assay. To estimate the added cost of using cytology to identify each histologically confirmed cervical HSIL case among Aptima HPV-negative women, we used the current cost of €21.2 per cytology evaluation at our laboratory. RESULTS: Of 19 599 women, 5.8% (1137/19 599) had abnormal cytology. Among Aptima HPV-negative women, 0.11‰ (2/18 132) had histologically confirmed HSIL. One of the women was infected with HPV18 and the other with HPV73 at the diagnosis of HSIL. The calculated cost to find one HSIL, by adding cytology to HPV-negative cases, was approximately €200 000. CONCLUSIONS: The clinical benefit of a single cytology co-test added to an HPV-based screening program in women aged 40-42 years appears doubtful and economically unreasonable.

Tonsillar Cancer with High CD8+ T-Cell Infiltration Features Increased Levels of Dendritic Cells and Transcriptional Regulation Associated with an Inflamed Tumor Microenvironment.

Human papillomavirus (HPV) is the main causal agent of tonsillar cancer (TC) and HPV+ TC has a favorable prognosis compared to HPV- disease. In this study, we examined aspects of the tumor microenvironment of TC, focusing on T-cells, dendritic cells (DC), and macrophages. Fresh biopsies of TC and the contralateral healthy tonsil (HT) were obtained from 20 patients, analyzed by multiparameter flow cytometry, and assessed against a detailed HPV-status. Additionally, RNA-sequencing data from 38 TC samples available in the public database, The Cancer Genome Atlas (TCGA), were explored, focusing on the same leukocyte populations. HPV+ TC featured increased levels of CD8+ T-cells and antigen-presenting cells (cf. HPV- TC and HT, respectively). In HPV+ TC, CD8+ T-cell frequencies correlated to DC levels independently of tumor stage, HPV 16 copy number, and E7 oncogene expression as well as frequencies of other leukocytes. Similarly, RNA sequencing data were explored by dividing the HPV+ TCs according to predefined CD8+ T-cell scores in silico. Higher levels of genes expressed by antigen-presenting cells and effector T-cells, such as immune checkpoints and cytokines, were detected in the CD8HIGH HPV+ TC samples (cf. CD8LOW HPV+ TC). In conclusion, CD8HIGH HPV+ TC displays a unique inflammatory profile associated with increased effector T-cell functions and the presence of antigen-presenting cells in the tumor microenvironment. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions.

Immune delineation of laryngeal papilloma reveals enhanced neutrophil associated gene profile.

Laryngeal papilloma (LP) is a rare benign disease, caused by recurrent multisite papillomas that are referred to as recurrent respiratory papillomatosis (RRP). RRP is caused primarily by two types of human papillomavirus (HPV): HPV6 and HPV11. The immune dysregulation within the microenvironment of the lesions has been shown to likely play a role in the development of RRP. The present study aimed at analyzing the transcriptional profile of immune response genes and cancer-related genes in the LP microenvironment. We used the NanoString® nCounter® analysis system to study expression of 730 genes among seven paired samples of LP and healthy laryngeal (HL) tissue. qRT-PCR and flow cytometric analysis was performed to confirm identified transcripts and follow-up scores of infiltrating immune cells, respectively. In total, 113 differentially expressed transcripts were detected of which 37 showed increased expression levels and 76 decreased expression levels in the LP samples compared to the HL samples (fold change ≥ 2). Transcripts with increased expression levels included S100As (A7, A8, and A12), CEACAM1, neutrophil activation associated cytokines (IL8), chemokines (CXCL6), and IL receptors, e.g., IL4R. Transcripts with decreased expression in LP were associated with innate and adaptive immunity. Overall, HPV6 and 11 were present in 67% and 33% of the patients, respectively. There was a significant increase in neutrophils and a significant decrease in CD8+ T cells in LP. LP samples display an immune profile characterized by enhanced expression of neutrophilic markers and significantly reduced T cell-associated markers.

The 2019 HPV Labnet international proficiency study: Need of global Human Papillomavirus Proficiency Testing.

BACKGROUND: Accurate and internationally comparable human papillomavirus (HPV) testing services are essential for cervical cancer elimination programs. The WHO HPV Laboratory Network started issuing international HPV testing proficiency panels in 2008. OBJECTIVES: We report the results of the 2019 global proficiency study and evaluate the proficiency over time. STUDY DESIGN: The proficiency panel contained 40 coded samples containing mixes of purified HPV types (HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59/68a/68b) and 4 controls. Proficiency required detection of both single and multiple infections of 50 International Units of HPV 16/18, of 500 genome equivalents (10x higher concentration) for other HPV types, and no false positives (stricter requirement compared to previous panels). RESULTS: Seventy-eight laboratories submitted 110 datasets with 38 different assays. Most samples (38/44) were reported with 100% proficiency in most datasets. Mostly commercial assays were used (88/110 datasets). Overall, 47.3% of the datasets were 100% proficient. False positivity was detected in at least one sample in 30.1% of datasets. When analysing all datasets ever since 2008 using exactly the same proficiency criteria, there was a steady improvement up to 2017 (the proportion of datasets being completely proficient increased from 25% to 73%). However, in the 2019 proficiency testing the proportion of fully proficient datasets dropped to 50%. CONCLUSIONS: Although we initially documented a worldwide improvement in comparability and reliability of HPV testing services, the trend now appears to be reversed. In response, the International HPV Reference Center will provide support for improved quality of laboratory services, including issuing of global proficiency panels every year.

Equal prevalence of severe cervical dysplasia by HPV self-sampling and by midwife-collected samples for primary HPV screening: a randomised controlled trial.

OBJECTIVES: HPV self-sampling is an option for cervical screening. The aim of this randomised study was to investigate the compliance, prevalence of HPV, and prevalence of severe dysplasia in a vaginal self-sampling group in comparison to cervical samples collected by midwives (control arm). The hypothesis was that there would be no difference between vaginal self-sampling and cervical sampling to find high-grade cervical dysplasia or cancer. METHODS: Vaginal HPV self-sampling kits were sent by regular mail to 14 765 randomly selected women aged 30-64 years old in the screening programme. HPV-positive women were invited for a follow-up examination by their midwife in which they provided a cervical sample for cytological and HPV co-testing. The control arm consisted of 14 839 women who met the same inclusion criteria and were invited to have cervical sampling by midwives for primary HPV screening. All HPV samples were analysed by the Aptima HPV assay (Hologic Inc.). MAIN RESULTS: The participation rate was 33.5% in the self-sampling arm and 47.5% in the cervical sampling arm, (P < 0.0001). HPV was detected in 17.1% (95% confidence interval (CI), 16.1-18.23%) in the self-sampling arm and 4.5% (95% CI, 4.0-5.0%) in the cervical sampling arm. Histological, severe dysplasia was observed among 0.48% (95% CI, 0.3-0.72%) and 0.47% (95% CI, 0.3-0.66%) of the self-sampling and the cervical sampling groups, respectively. CONCLUSION: The self-sampling approach detects a similar proportion of severe dysplasia as regular screening. Thus, our study indicates that self-sampling could replace primary HPV screening of cervical samples.

Promotion of Cervical Screening among Long-term Non-attendees by Human Papillomavirus Self-sampling.

Cervical cancer is preventable through gynecological screening. To promote participation among non-attending women, self-collected vaginal samples for detection of high-risk human papillomavirus (hr-HPV) is an option. The aims of this study were to investigate the response of self-collected vaginal samples for hr-HPV testing among long-term non-attendees, to explore the attendance at follow-up among HPV-positive women, and to analyze the prevalence of hr-HPV and severe cervical dysplasia or cancer among the responders. A vaginal self-sampling kit was sent to 19,766 women aged 30-70 years who had not provided a cervical screening sample for ≥ 7 years in Skåne, Sweden. The self-sample was analyzed by the Aptima HPV mRNA assay (Hologic). Women testing positive for HPV were invited for follow-up. The response was 18.5% (3,646/19,757). The prevalence of HPV mRNA was 11.3% (412/3,636). Among HPV-positive women, 85.7% (353/412) attended follow-up, and of these, 44.8% (158/353) had HPV in the cervical sample. The HPV mRNA test of self-samples showed a positive predictive value of 9.3% ([33/353], 95% CI = 6.5-12.9) for detection of cytologically severe dysplasia. Histologically severe dysplasia or cancer was detected in 0.88% ([32/3,636], 95% CI = 0.6-1.2) among responders, including two cervical- and one vaginal cancer. In conclusion, almost one fifth of the long-term non-attendees participated in self-collected vaginal hr-HPV sampling. The prevalence of histologically confirmed high grade squamous intraepithelial lesion or cervical cancer was not increased significantly compared to regularly screened women in Sweden. The relatively high HPV prevalence among the self-samples indicates the importance of diagnostic follow-up with cervical HPV testing and reflex-cytology of HPV-positive cases.

HPV73 in cervical cancer and distribution of HPV73 variants in cervical dysplasia.

HPV73 is classified as possibly oncogenic and is not recognized by most commercial primary HPV screening platforms. The aim was to determine the prevalence of HPV73 among invasive cervical cancers, formalin-fixed paraffin embedded (FFPE) samples (N = 69), from southern Sweden during 2009 to 2010. Another aim was to determine proportions of HPV73 among Aptima HPV assay negative cervical cancers (N = 9, out of 206 cancers) and of high-grade cytological cervical diagnosis (N = 75, out of 5807 high grade lesions) in liquid-based cytology (LBC) samples collected between 2016 and 2019. We also investigated the distribution of HPV73 variants A1, A2 and B among HPV73-positive cases. HPV73 was detected by multiplex MGP-PCR and Luminex, and HPV73 variants were identified by sequencing PCR amplicons. HPV73 was detected in 2.9% (2/69, 95% CI: 0.18-9.9) of the FFPE cervical cancer series. Among the Aptima HPV-negative LBC samples, HPV73 was present in 55.5% (5/9) of the cancers and 29.3% (22/75) of the different grades of cervical diagnosis. The A1, A2 and B variants were present in 6.9% (2/29), 82.7% (24/29) and 10.3% (3/29) of the HPV73-positive women, respectively. Among the seven HPV73 cancer cases (two FFPE samples and five LBC samples), six A2 and one A1 isolate were detected. In summary, the A2 variant of HPV73 was most common in our region. In addition, the observed prevalence of HPV73 (2.9%) in cervical cancers and its relative high occurrence (55.5%) among Aptima HPV-negative cancers urge that detection of HPV73 should be included in future primary HPV screening programs.

Penile intraepithelial neoplasia, penile cancer precursors and human papillomavirus prevalence in symptomatic preputium: a cross-sectional study of 351 circumcised men in Sweden.

OBJECTIVES: To investigate the prevalence of pathological disease and spectrum of human papillomavirus (HPV) types among symptomatic foreskin tissue. PATIENTS AND METHODS: Consecutively excised symptomatic foreskins from 351 men were sent for histopathological evaluation. During the surgical procedure, a fresh biopsy was taken for HPV analysis by modified general primer polymerase chain reaction. A medical questionnaire regarding medication, smoking habits, number of lifetime sexual partners, former diseases and surgery performed on penis was completed by all participants. RESULTS: The most common clinical diagnosis and cause for circumcision was phimosis, seen in 85.2%. Histopathologically inflammatory dermatological conditions were present in 87% of the men. The most common histopathological diagnosis was lichen sclerosus (LS) observed among 58.7%. Notably, penile intraepithelial neoplasia (PeIN) was present in 2% without former clinical suspicion. Overall, HPV was detected in 17.1% of the men and 28 different HPV types were found. High-risk (HR) HPV types were identified in 9.1% and HPV16 was present in 2.3%. Current smoking increased the risk of HPV (crude odds ratio [OR] 2.8, confidence interval [CI] 1.4-5.6; P = 0.005). Having >15 lifetime sexual partners increased the risk of HPV (crude OR 2.6, 95% CI 1.4-5.1; P = 0.003) and when adjusted for current smoking the OR was substantially increased (OR 6.0, 95% CI CI 2.2-16.8; P < 0001). CONCLUSIONS: Histopathological evaluation of circumcised symptomatic foreskin revealed PeIN in 2% of the men without any clinical suspicion of malignancy and that treatable dermatological conditions were present in 87%, LS being the most common. HR-HPV types were present in 9%. Due to risk of malignant development both in PeIN and in inflammatory skin diseases we recommend sending all excised foreskins from patients with symptoms for histopathological evaluation as guidance for further clinical management.

Immune Phenotypes of Nasopharyngeal Cancer.

Nasopharyngeal cancer (NPC) features intralesional immune cells, but data are lacking on presence/distribution of T-cells and dendritic cells (DCs). Based on intralesional distribution of lymphocytes, a series of NPC biopsies (n = 48) were classified into "inflamed", "excluded", and "deserted" phenotypes. In addition, CD8+ T-cells and CD207+ DCs were quantified. The data were analyzed in relation to Epstein-Barr virus-encoded small RNA (EBER), Epstein-Barr virus (EBV) DNA, and survival. Separately, data on gene expression from a public database were analyzed. 61.7% of NPC lesions were "inflamed", 29.8% were "excluded", and 8.5% were "deserted". While CD8+ cells were present in cancer cell areas and in surrounding stroma, CD207+ cells were observed largely in cancer cell areas. High CD8+ T-cell presence was associated with EBV+ disease, but no such pattern was observed for CD207+ DCs. There was a difference in disease-free survival in favor of "inflamed" over "excluded" NPC. Gene expression analysis revealed differences between NPC and control tissue (e.g., with regard to interferon activity) as well as between subgroups of NPC based on CD8 expression (high vs. low). In conclusion, NPC lesions are heterogeneous with regard to distribution of CD8+ T-cells and CD207+ DCs. NPC can be classified into immune phenotypes that carry prognostic information. CD207+ DCs may represent a target for immunotherapy with potential to facilitate the antigen cross-presentation necessary to execute cytotoxic T-lymphocyte responses.

14-type HPV mRNA test in triage of HPV DNA-positive postmenopausal women with normal cytology.

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.

Detection of HPV mRNA in Self-collected Vaginal Samples Among Urban Ethiopian Women.

BACKGROUND/AIM: Cervical cancer is the most common cancer among women in Ethiopia. The objective was to evaluate the participation rate of a free of charge vaginal self-sample (Aptima multitest swab, Hologic) for the detection of human papillomavirus (HPV) in an Ethiopian cohort. PATIENTS AND METHODS: Specimens were collected from women employed by Ethiopian Airlines in Addis Abeba (N=5950). Samples were analysed for the presence of high-risk (HR) HPV mRNA by the Aptima HPV assay (Hologic) and HPV positive women were referred for cytology. Identification of HPV types among HPV positive samples was performed by Modified general primer-PCR and Luminex assay. RESULTS: Participation rate was 3.1% and the prevalence of HPV mRNA was 20.6% (37/180). CONCLUSION: Primary HPV mRNA screening with vaginal self-sampling may be an acceptable approach in Ethiopia. One out of five women harbor HPV in their vaginal self-sample in agreement with other similar studies from the region.

Cervical cancer prevention among long-term screening non-attendees by vaginal self-collected samples for hr-HPV mRNA detection.

BACKGROUND: The efficacy of cervical cancer screening programs is dependent on the participation rate. To increase participation among women not attending cervical cancer screening, self-collected samples for detection of high-risk human papillomavirus (hr-HPV) may be an option.The aims of this study were: to investigate the response rate to sending a self-collected vaginal sample for hr-HPV mRNA detection to long-term non-attendees; the compliance with follow-up among women positive for HPV in the self-sample; the prevalence of cervical dysplasia (high grade squamous intraepithelial lesion (HSIL), atypical squamous cells that cannot exclude HSIL (ASC-H) or adenocarcinoma in situ (AIS)) or cancer among the responders; as well as to explore reasons for not returning a self-sample. METHODS: A vaginal self-sampling kit was sent to 6023 women aged 30-70 years who had not provided a cervical screening sample for ≥7 years in the Region of Skåne, Sweden in November and December 2017. The self-sample was analyzed by Aptima HPV mRNA assay (Hologic). All vaginal self-samples returned no later than May 31, 2018 were included in the study. Follow-up of the results was registered until January 31, 2019 with a follow-up time varying between eight to 14 months. Women positive for hr-HPV mRNA were invited for a follow-up examination. This examination consisted of a cervical sample for cytological analysis and renewed Aptima HPV mRNA testing. Two hundred thirty-five women who had not returned the self-sample were randomly selected for telephone interviews, in order to explore their reasons. RESULTS: The response rate for the self-collected vaginal hr-HPV sample was 13.2% [(797/6023), 95% CI 12.4-14.1%] and 9.9% [(79/796), 95% CI 7.9-12.2%] were positive for hr-HPV mRNA. The prevalence of severe dysplasia or cancer in the whole group of responders was 1.3% [(10/796), 95% CI 0.6-2.3%], with a cervical cancer prevalence of 0.4% [(3/796), 95% CI 0.1-1.1%]. Only 27 women participated in the telephone interviews, no particular reason for not returning self-samples was observed. CONCLUSIONS: Self-collected vaginal hr-HPV samples increased participation in the cervical cancer screening among long-term non-attendees. The prevalence of cervical cancer was almost seven times higher for long-term non-attendees than in the organized screening population.

Detecting TP53 mutations in diagnostic and archival liquid-based Pap samples from ovarian cancer patients using an ultra-sensitive ddPCR method.

High-grade serous ovarian cancer (HGSOC) is the most common subtype of epithelial ovarian cancer and early detection is challenging. TP53 mutations are a hallmark of HGSOC and detection of these mutations in liquid-based Pap samples could provide a method for early diagnosis. Here we evaluate the use of IBSAFE, an ultra-sensitive droplet digital PCR (ddPCR) method, for detecting TP53 mutations in liquid-based Pap samples collected from fifteen women at the time of diagnosis (diagnostic samples) and/or up to seven years prior to diagnosis (archival samples). We analysed tumours for somatic TP53 mutations with next generation sequencing and were able to detect the corresponding mutations in diagnostic samples from six of eight women, while one patient harboured a germline mutation. We further detected a mutation in an archival sample obtained 20 months prior to the ovarian cancer diagnosis. The custom designed IBSAFE assays detected minor allele frequencies (MAFs) with very high assay sensitivity (MAF = 0.0068%) and were successful despite low DNA abundance (0.17-206.14 ng, median: 17.27 ng). These results provide support for further evaluation of archival liquid-based Pap samples for diagnostic purposes and demonstrate that ultra-sensitive ddPCR should be evaluated for ovarian cancer screening in high-risk groups or in the recurrent setting.

Intralesional EBV-DNA load as marker of prognosis for nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) is associated with the Epstein-Barr virus (EBV). The clinical presentation and prognosis of NPC is well described, but not in relation to intralesional EBV-DNA load. In a retrospective design, 48 patients with NPC were examined. Patient history was re-evaluated, and diagnostic biopsies were re-examined. Furthermore, intralesional EBV-DNA was quantitated and HPV status determined. Cancer stage, disease-free survival (DFS), and overall survival (OS) were assessed. Of the 48 patients, 36 (75%) patients featured lesions that were positive for EBER (Epstein-Barr virus-encoded small RNA) and 40 (83%) were positive for EBV-DNA. Seven patients (15%) were HPV positive. The levels of EBV-DNA ranged from 0.0005 to 94617 copies/cell. An EBV-DNA load of more than 70 copies/cell was associated with a prolonged DFS for EBV-DNA positive patients treated with curative intent (p = 0.046). In conclusion, the EBV-DNA load in NPC lesions appears to vary greatly. For patients with EBV-DNA positive NPC treated with curative intent, an EBV-DNA load of more than 70 copies/cell is associated with a better outcome in terms of 7-year DFS.