High-risk Human Papillomavirus mRNA Research Articles

High-risk HPV mRNA testing on self-samples offered to those who do not attend for organised cervical screening – real world data from the Dumfries and Galloway region in Scotland

BackgroundHPV self sampling can act as a tool to engage women in cervical screening and population based studies can inform optimal implementation of this approach. MethodsSelf sampling kits were mailed to women who had defaulted from routine screening resident in an entire territorial health board in Scotland. Kit return rates and compliance to colposcopy follow up in those who were HPV mRNA positive were assessed. Concordance of the self-sample with samples taken later at colposcopy was measured alongside PPV of an mRNA positive result for CIN2+. ResultsOf 4173 women invited to participate, 20.5 %, returned their kit and a greater return rate with increasing age was observed. HPV mRNA positivity was 12.0 %, and invalidity rate was approximately 3 %. Compliance to colposcopy follow up was 88.3 % and the PPV for an mRNA test for CIN2+ on a self sample was 25.6 %. hr-HPV concordance on the initial swab and the follow up swab and liquid based cytology (LBC) sample taken at colposcopy was 67.1 % and 30 % respectively. ConclusionsHPV self sampling using a “mail to all” approach is feasible in Scotland although only 1 in 5 actively responded to the offer. Future work to monitor screening behaviours in those who were invited but did not engage initially will help quantify any additional benefit(s) incurred.

High incidence of HPV infection in minors with oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma in minors is considered to be a distinct entity from OSCC in older patients, with an uncertain etiology. Human papillomavirus (HPV) infection may trigger the initiation and promote the progression of OSCC, but these roles have not been firmly established.We aimed to explore the correlation between HPV infection and the development of oral squamous cell carcinoma in minors and know the characteristics of OSCC in young patients more thoroughly.MethodFrom January 2013 to December 2022,6 cases of OSCC aged < 15 years were selected from the Department of Oral Pathology, Peking University School of Stomatology, Beijing, China. All cases underwent testing for high-risk HPV mRNA infection using the RNA scope technique, and immunohistochemical staining was performed to investigate the expression of p16, pan-cytokeratin (CK), CK5/6, CK7, CK8/18, epidermal growth factor receptor (EGFR), p53, and Ki-67. Furthermore, we reviewed the literature on OSCC in patients aged < 21 years.ConclusionsMinors OSCC is associated with HPV infection, and that p16 can serve as an immunohistochemical marker of HPV positivity.

HPV-Associated Squamous Cell Carcinoma of the Eyelid: Diagnostic Utility of p16 Immunohistochemistry and mRNA In Situ Hybridization.

High-risk (HR) Human papillomavirus (HPV) has been implicated in pathogenesis of squamous cell carcinomas (SCC) at several sites with mucocutaneous junctions, including the head and neck. SCC is the second most common eyelid malignancy. However, its association with transcriptionally active HR-HPV has not been adequately studied. Two index cases of eyelid HPV-associated SCC are described in detail. A retrospective cohort of eyelid SCC was examined for p16 immunoexpression. Cases demonstrating p16 positivity or equivocal staining were subjected to high-risk HPV mRNA in situ hybridization (ISH). Quantitative real-time PCR (qPCR) was performed in mRNA ISH-positive cases for HPV genotyping. The two index patients were older adult females, with upper eyelid tumours. On histology, both tumours were non-keratinizing SCC with trabecular and nested architecture reminiscent of oropharyngeal HPV-associated non-keratinizing SCC, prompting p16 immunohistochemistry, which was positive. HR-HPV mRNA ISH was positive, and qPCR detected HPV16 in both cases. Three of 20 (15%) archival cases showed p16 immunopositivity and two (10%) showed equivocal staining. However, mRNA ISH was negative. All cases showing p16 immunostaining and lacking HR-HPV were keratinizing SCCs. Thus, 9% of all eyelid SCC examined demonstrated HR-HPV. The prevalence of HR-HPV in eyelid SCC is low in Indian patients. HPV-associated SCC may mimic commoner eyelid carcinomas as it lacks overt keratinization. In basaloid-appearing eyelid carcinomas, p16 immunopositivity should be followed by reflex HR-HPV mRNA ISH, as p16 immunohistochemistry alone has low specificity. The prognostic role, if any, of HPV association needs further evaluation.

Sinonasal Adenosquamous Carcinoma - Morphology and Genetic Drivers Including Low- and High-Risk Human Papillomavirus mRNA, DEK::AFF2 Fusion, and MAML2 Rearrangement.

Sinonasal adenosquamous carcinoma is rare, and there are almost no studies detailing morphology or characterizing their genetic driver events. Further, many authors have termed sinonasal tumors with combined squamous carcinoma and glands as mucoepidermoid carcinoma but none have analyzed for the presence of MAML2 rearrangement. Cases from 2014 to 2020 were collected and diagnosed using World Health Organization criteria. They were tested for p16 expression by immunohistochemistry (70% cut-off), DEK::AFF2 fusion by fluorescence in situ hybridization (FISH) and AFF2 immunohistochemistry, MAML2 rearrangement by FISH, and low- and high-risk HPV by RNA ISH and reverse transcription PCR, respectively. Detailed morphology and clinical features were reviewed. There were 7 male (64%) and 4 female (36%) patients with a median age of 69 years, most Caucasian (10 of 11 or 91%). Most had tobacco exposure (8/11, 73%) and most presented with epistaxis, a visible nasal mass, and/or facial pain. Several had a precursor papillomas (3 of 11, 27%). The squamous component had variable keratinization, 5 of 11 (46%) of which would be described as keratinizing, 3 non-keratinizing, and 2 with mixed features. All had gland formation, by definition, and 2 of 11 (18%) had ciliated tumor cells. None of the 11 cases had MAML2 rearrangement and one had DEK::AFF2 fusion with associated positive nuclear AFF2 protein immunostaining. Most were p16 positive (7 of 11, 64%) and all 7 of these were hrHPV positive either by RNA ISH or RT-PCR. Two of the p16-negative tumors were positive for lrHPV by RNA ISH. Treatment included surgery alone (4 of 11, 36%), surgery with adjuvant radiation (5 of 11, 45%), and surgery with radiation and chemotherapy (2 of 11, 18%). Four of 11 patients (36%) suffered disease recurrence, two requiring re-operation and who were disease free at last follow-up, one receiving additional chemotherapy and who was alive with disease. The other elected to undergo palliative therapy and died of disease. Sinonasal adenosquamous carcinoma is a somewhat heterogeneous tumor not infrequently arising ex papilloma and having various drivers including high- and low-risk HPV and rarely DEK::AFF2 fusion. The prognosis appears favorable when proper treatment is possible.

Primary HPV-Associated Enteric-Type Adenocarcinoma of Vagina: A Case Report.

Primary enteric type adenocarcinomas of the vagina are extremely rare. We present a 63-year-old woman who had a polypoid mass localized to the distal vagina. The lesion was composed of a columnar glandular cell proliferation with focal cribriforming, reminiscent of tubular adenoma. Immunohistochemical stains were notable for expression of enteric markers (CDX2 and KRT20), as well as negativity for Mullerian markers (PAX8, ER, and PR), diffuse expression for p16, and positivity for high-risk HPV mRNA expression. Ultimately, a diagnosis of vaginal primary HPV-associated enteric type adenocarcinoma was rendered for this unusual lesion. To our knowledge, no prior cases of HPV-associated enteric type adenocarcinomas of the vagina have been described before.

Human Papillomavirus-Associated Oral Cavity Squamous Cell Carcinoma: An Entity with Distinct Morphologic and Clinical Features.

HPV-associated oral cavity squamous cell carcinoma(SCC) is not well-characterized in the literature, and also has a clinical significance that is poorly understood. We gathered a cohort of oral cavity (OC) SCC with nonkeratinizing morphology, either in the invasive or in situ carcinoma (or both), tested for p16 by immunohistochemistry and high risk HPV E6/E7 mRNA by RTPCR (reference standard for transcriptionally-active high risk HPV) and gathered detailed morphologic and clinicopathologic data. Thirteen patients from two institutions were proven to be HPV-associated by combined p16 and high risk HPV mRNA positivity. All 13 patients (100%) were males, all were heavy smokers (average 57 pack/year), and most were active drinkers (9/11 or 81.8%). All 13 (100%) involved the tongue and/or floor of mouth. All had nonkeratinizing features, but maturing squamous differentiation varied widely (0-90%; mean 37.3%). Nonkeratinizing areas had high N:C ratios and larger nests, frequently with pushing borders, and minimal (or no) stromal desmoplasia. The carcinoma in situ, when present, was Bowenoid/nonkeratinizing with cells with high N:C ratios, full thickness loss of maturation, and abundant apoptosis and mitosis. HPV was type 16 in 11 patients (84.6%) and type 33 in two (15.4%). Nine patients had treatment data available. These underwent primary surgical resection with tumors ranging from 1.6 to 5.2cm. Most had bone invasion (6/9-66.7% were T4a tumors), and most (6/9-66.7%) had extensive SCC in situ with all 6 of these patients having final margins positive for in situ carcinoma. HPV-associated OCSCC is an uncommon entity that shows certain distinct clinical and pathologic features. Recognition of these features may help pathologic diagnosis and could potentially help guide clinical management.

Accuracy and effectiveness of HPV mRNA testing in cervical cancer screening: a systematic review and meta-analysis.

Cervical cancer screening tests that identify DNA of the main causal agent, high-risk human papillomavirus (HPV) types, are more protective than cervical cytology. We systematically reviewed the literature to assess whether tests targeting high-risk HPV (hrHPV) mRNA are as accurate and effective as HPV DNA-based screening tests. We did a systematic review to assess the cross-sectional clinical accuracy to detect cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) or 3 or worse (CIN3+) of hrHPV mRNA versus DNA testing in primary cervical cancer screening; the longitudinal clinical performance of cervical cancer screening using hrHPV mRNA versus DNA assays; and the clinical accuracy of hrHPV mRNA testing on self-collected versus clinician-collected samples. We identified relevant studies published before Aug 1, 2021, through a search of Medline (PubMed), Embase, and CENTRAL. Eligible studies had to contain comparative data addressing one of our three clinical questions. Aggregated data were extracted from selected reports or requested from study authors if necessary. QUADAS and ROBINS-1 tools were used to assess the quality of diagnostic test accuracy studies and cohort studies. To assess cross-sectional clinical accuracy of mRNA testing versus DNA testing and clinical accuracy of hrHPV mRNA testing on self-collected versus clinician collected samples, we applied meta-analytical methods for comparison of diagnostic tests. To assess the longitudinal clinical performance of cervical cancer screening using hrHPV mRNA versus DNA assays, we compared the longitudinal sensitivity of mRNA tests and validated DNA tests for CIN3+ and the relative detection of CIN3+ among women who screened negative for hrHPV mRNA or DNA (both used as measures of safety) at baseline and pooled estimates by years of follow-up. A random-effect model for pooling ratios of proportions or risks was used to summarise longitudinal performance. For the hrHPV mRNA testing with APTIMA HPV Test (APTIMA), the cross-sectional accuracy could be compared with DNA assays on clinician-collected samples in eight studies; longitudinal performance was compared in four studies; and accuracy on self-samples was assessed in five studies. Few reports were retrieved for other mRNA assays, precluding their evaluation in meta-analyses. Compared with validated DNA assays, APTIMA was similarly sensitive (relative sensitivity 0·98 [95% CI 0·95-1·01]) and slightly more specific (1·03 [1·02-1·04]) for CIN2+. The relative sensitivity for CIN3+ was 0·98 (95% CI 0·95-1·01). The longitudinal relative sensitivity for CIN3+ of APTIMA compared with DNA assays assessed over 4-7 years ranged at the study level from 0·91 to 1·05 and in the pooled analysis between 0·95 and 0·98, depending on timepoint, with CIs including or close to unity. The detection rate ratios between 4 and 10 years after baseline negative mRNA versus negative DNA screening were imprecise and heterogeneous among studies, but summary ratios did not differ from unity. In self-collected samples, APTIMA was less sensitive for CIN2+ (relative cross-sectional sensitivity 0·84 [0·74-0·96]) but similarly specific (relative specificity 0·96 [0·91-1·01]) compared with clinician-collected samples. HrHPV RNA testing with APTIMA had similar cross-sectional sensitivity for CIN2+ and CIN3+ and slightly higher specificity than DNA tests. Four studies with 4-7 years of follow-up showed heterogeneous safety outcomes. One study with up to 10 years of follow-up showed no differences in cumulative detection of CIN3+ after negative mRNA versus DNA screening. APTIMA could be accepted for primary cervical cancer screening on clinician-collected cervical samples at intervals of around 5 years. APTIMA is less sensitive on self-collected samples than clinician-collected samples. Horizon 2020 Framework Programme for Research and Innovation of the European Commission, through the RISCC Network, WHO, Haute Autorité de la Santé, European Society of Gynaecological Oncology, and the National Institute of Public Health and the Environment.

A clinical analysis of oropharyngeal squamous cell carcinoma: a single-institution's experience.

We herein report the treatment outcome of oropharyngeal squamous cell carcinoma (OPSCC) at Kyushu University Hospital, the total number of OPSCC cases, and changes in the proportion of human papilloma virus (HPV)-related carcinomas over time. We performed a retrospective analysis of 237 cases treated for OPSCC at Kyushu University Hospital between 2013 and 2019. We performed HPV-mRNA in situ hybridization and p16 immunohistochemistry. This study included 197 males (82.1%) and 40 females (17.9%). The disease-specific, progression-free and overall survival (OS) were 69%, 62% and 61%, respectively, over the decade-long study period. p16-Immunohistochemistory and highrisk HPV mRNA in situ hybridization were positive in 114 (48.1%) and 105 (44.3%) cases, respectively. The number of HPV-related OPSCC cases increased according to an annual analysis. HPV+ cases had a significantly better prognosis than HPV- cases. In addition, p16+/HPV- cases had a significantly worse prognosis than p16+/HPV+ cases (OS: p = 0.0484). HPV+ OPSCC cases were associated with a younger age (< 60years old) (p = 0.0429), non-smoker (p = 0.0001), lateral tumor site (< 0.00001), lymphoid metastasis (< 0.0001) and low clinical stage (< 0.0001). The frequency of HPV-related OPSCC cases is increasing in Japan as well as worldwide, and such cases are characterized by no smoking habit, a young age, and a good prognosis. Even in p16+ OPSCC, HPV- cases had a poor prognosis, suggesting the importance of accurate HPV determination. To determine the intensity of treatment for HPV-related and non-related OPSCC, it is necessary to accumulate cases for the accurate HPV determination and comparison of treatment effects.

Raman Spectroscopy of Liquid-Based Cervical Smear Samples as a Triage to Stratify Women Who Are HPV-Positive on Screening.

Simple SummaryPersistent high-risk human papillomavirus (HPV) infection can lead to cervical precancer and cancer. Recently, HPV testing has been introduced for primary cervical screening, but the HPV DNA test cannot distinguish between transient and persistent HPV infection. Thus, there is an unmet clinical need to develop a new test to identify women with a high-risk persistent HPV infection. Raman spectra were recorded from cervical smear samples (n = 60) and, on the basis of HPV DNA and HPV mRNA test results, a classifier was developed to identify persistent HPV infection. A further blinded independent test set (n = 14) was used to validate the model, and sensitivity of 90% and specificity of 100% were achieved. Improved triage would allow women with a high-risk persistent HPV infection to be referred for immediate treatment, while women with a low-risk transient infection could avoid overtreatment.The role of persistent high-risk human papillomavirus (HPV) infection in the development of cervical precancer and cancer is now well accepted, and HPV testing has recently been introduced for primary cervical screening. However, the low specificity of HPV DNA testing can result in large numbers of women with an HPV-positive result, and additional triage approaches are needed to avoid over-referral to colposcopy and overtreatment. The aim of this study was to assess Raman spectroscopy as a potential triage test to discriminate between transient and persistent HPV infection. HPV DNA status and mRNA status were confirmed in ThinPrep® cervical samples (n = 60) using the Cobas 4800 and APTIMA HPV test, respectively. Raman spectra were recorded from single-cell nuclei and subjected to partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 14). Sensitivity of 85% and specificity of 92% were achieved for the classification of transient and persistent HPV infection, and this increased to 90% sensitivity and 100% specificity when mean sample spectra were used instead of individual cellular spectra. This study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify persistent HPV infection.

ROC analysis of p16 expression in cell blocks of metastatic head and neck squamous cell carcinoma

Oropharyngeal squamous cell carcinoma is associated with human papillomavirus (HPV) and often presents with early metastasis to cervical neck lymph nodes that are amenable to fine-needle aspiration (FNA). The most common method of HPV status determination is p16 immunohistochemistry (IHC). The literature suggests that a lower threshold is needed for p16 positivity on cell block. We examined and quantified p16 IHC staining on cell block and used receiver operating characteristics (ROC) curve analysis to determine an optimal cutoff value with high sensitivity and specificity. Thirty-six FNAs of metastatic squamous cell carcinoma from cervical lymph nodes with p16 IHC were evaluated. The p16 stain was quantified in 5% increments and high-risk HPV mRNA in situ hybridization was performed as a gold standard test. Statistical analysis was performed. Interobserver variability was evaluated and was shown to be low with an intraclass correlation coefficient of 0.857. ROC analysis was performed and showed that a cell block p16 IHC cutoff of 15% yielded the highest sensitivity (80%) and specificity (81.8%). Our data show that a threshold of 15% p16 staining in cell block maximizes sensitivity and specificity.

Oropharyngeal Squamous Cell Carcinoma With Discordant p16 and HPV mRNA Results: Incidence and Characterization in a Large, Contemporary United States Cohort.

Early studies estimate that 5% to 10% of oropharyngeal squamous cell carcinomas overexpress p16 but are unassociated with transcriptionally-active high-risk human papillomavirus (HPV). Patients with discordant HPV testing may experience clinical outcomes that differ from traditional expectations. To document the rate of p16 and HPV mRNA positivity, characterize patients with discordant testing, and identify features that may warrant selective use of HPV-specific testing after p16 IHC, a multi-institutional, retrospective review of oropharyngeal squamous cell carcinoma patients with p16 IHC and HPV mRNA testing by reverse transcriptase polymerase chain reaction was performed. Of the 467 patients, most had T1 or T2 tumors (71%), 82% were p16 positive, and 84% were HPV mRNA positive. Overall, most tumors were nonkeratinizing (378, 81%), which was strongly associated with p16 and HPV positivity (93% and 95%, respectively). Overall, 81% of patients were double positive, 14% double negative, and 4.9% discordant (3.4% p16 negative/HPV mRNA positive and 1.5% p16 positive/HPV mRNA negative). The survival rates of these discordant patient groups fell squarely between the 2 concordant groups, although in multivariate analysis for both disease-free survival and overall survival, discordant patients were not found to have statistically significantly different outcomes. Reclassifying patients by applying HPV mRNA testing when p16 results and morphology do not match, or when p16 results are equivocal, improved prognostication slightly over p16 or HPV mRNA testing alone. Patients with discordant testing demonstrate a borderline significant trend toward survival differences from those with concordant tests. When evaluated independently, patients who were p16 negative but HPV mRNA positive had a prognosis somewhat closer to double-positive patients, while those who were p16 positive, but HPV mRNA negative had a prognosis closer to that of double-negative patients. We suggest an algorithm whereby confirmatory HPV mRNA testing is performed in patients where p16 status is not consistent with tumor morphology. This captures a majority of discordant patients and improves, albeitmodestly, the prognostication.

In situ Hybridization for High-risk HPV RNA Identifies the Presence of High-risk HPV mRNA in Cervical Biopsies from Women with Prior High-risk HPV Negative ASC-H or HSIL Paps

In situ Hybridization for High-risk HPV RNA Identifies the Presence of High-risk HPV mRNA in Cervical Biopsies from Women with Prior High-risk HPV Negative ASC-H or HSIL Paps

The utility of high-risk human papillomavirus E6/E7 mRNA in situ hybridization in assessing HPV status on cell block

Assessment of human papillomavirus (HPV) status is critical to the treatment and prognosis of patients with oropharyngeal squamous cell carcinoma. Patients often present with enlarged cervical lymph nodes which are amenable to fine needle aspiration (FNA) and cell block creation. The most widely used method for assessing HPV status is the surrogate marker p16. Other HPV specific methods such as high-risk HPV E6/E7 mRNA in situ hybridization (ISH) have been shown to perform as well as p16 and are easier to interpret. Our study evaluates the utility of high-risk HPV mRNA ISH in cell block specimens. Thirty-six cases of metastatic squamous cell carcinoma in cervical neck lymph node FNAs were identified over a 3-year period. All cases had p16 immunohistochemistry (IHC) performed on cell block. HR HPV mRNA ISH was performed on the cell block and compared to the p16 results. Additionally, p16 and HR HPV mRNA ISH status was assessed in those cases with corresponding surgical resections. HR HPV mRNA ISH confirmed the p16 IHC (either positive or negative) in 24 of the 36 cases (66.7%). Six false negative cases were p16 negative/HR HPV mRNA ISH positive. HR HPV mRNA ISH was positive in 75% of the four p16 equivocal cases. Two cases were p16 positive/HR HPV mRNA ISH negative. HR HPV mRNA ISH is no more difficult to perform in the IHC lab and is easier to interpret than p16 IHC. HR HPV mRNA ISH is a useful alternative to p16 in cell block specimens.

Significance of transcriptionally-active high-risk human papillomavirus in sinonasal squamous cell carcinoma: Case series and a meta-analysis.

Sinonasal cancers represent a highly heterogeneous group of head and neck cancers, for which etiological and prognostic significance of high-risk human papillomavirus (HPV) infections has not yet been conclusively established. We investigated the presence of transcriptionally-active high-risk HPV in a series of 34 sinonasal squamous cell cancer (SNSCC) cases and evaluated the effect of transcriptionally-active HPV on the overall survival. In addition, we performed a meta-analysis of previously published studies, including this study, to summarize the prevalence of HPV positivity across histological subtypes of SNSCC. The presence of transcriptionally-active HPV was detected by HPV mRNA using the polymerase chain reaction (PCR) or in situ hybridization (ISH). p16 expression was evaluated as a surrogate marker for transcriptionally-active HPV infection by immunohistochemistry (IHC), the presence of high-risk HPV DNA was tested by PCR and the HPV genotypes were determined by sequencing of PCR amplicons. Transcriptionally-active HPV infections were found in ~25% of the SNSCC cases. The role of HPV infection in keratinizing SNSCC may be higher than previously reported (~32% in our study vs. ~0-6.3% in all other studies). Patients with transcriptionally-active HPV-positive SNSCCs were more likely to be diagnosed at earlier stages (p<0.05) and displayed better mean overall survival, although the difference between HPV-positive and HPV-negative groups was not statistically significant. In contrast to other non-oropharyngeal squamous cell carcinomas (non-OPSCCs) of the head and neck, in SNSCCs, p16/IHC and p16/IHC+HPV DNA displayed high specificity as surrogate markers of transcriptionally-active HPV infections. However, p16/IHC may have significantly lower sensitivity as a surrogate marker of transcriptionally-active HPV in SNSCCs compared to OPSCCs. Furthermore, in our group of SNSCCs, all cases positive for high-risk HPV DNA by PCR were also transcriptionally-active (causative) infections with positive HPV mRNA by ISH. Our results imply a possible different role of HPV-mediated carcinogenesis of squamous cell epithelium in oropharyngeal and sinonasal sites with the latter displaying a lower proportion of causative HPV infections; nevertheless, most cases positive for high-risk HPV DNA, p16/IHC or combination thereof were also found positive for transcriptionally-active HPV. The prognostic significance of HPV status in SNSCCs remains inconclusive and future studies should investigate the presence of transcriptionally-active HPV by direct HPV testing.

High-risk Human Papillomavirus Messenger RNA Testing in Wet and Dry Self-collected Specimens for High-grade Cervical Lesion Detection in Mombasa, Kenya.

Self-collection for high-risk human papillomavirus (hr-HPV) messenger RNA (mRNA) testing may improve cervical cancer screening. High-risk HPV mRNA with self-collected specimens stored dry could enhance feasibility and acceptance of specimen collection and storage; however, its performance is unknown. We compared the performance of hr-HPV mRNA testing with dry- as compared with wet-stored self-collected specimens for detecting high-grade squamous intraepithelial lesion or more severe (≥HSIL). A total of 400 female sex workers in Kenya participated (2013-2018), of which 50% were HIV positive based on enrollment procedures. Participants provided 2 self-collected specimens: one stored dry (sc-DRY) using a Viba brush (Rovers) and one stored wet (sc-WET) with Aptima media (Hologic) using an Evalyn brush (Rovers). Physician-collected specimens were collected for HPV mRNA testing (Aptima) and conventional cytology. We estimated test characteristics for each hr-HPV screening method using conventional cytology as the reference standard (≥HSIL detection). We also examined participant preference for sc-DRY and sc-WET collection. High-risk HPV mRNA positivity was higher in sc-WET (36.8%) than sc-DRY samples (31.8%). Prevalence of ≥HSIL was 6.9% (10.3% HIV positive, 4.0% HIV negative). Sensitivity of hr-HPV mRNA for detecting ≥HSIL was similar in sc-WET (85%; 95% confidence interval [CI], 66%-96%), sc-DRY specimens (78%; 95% CI, 58%-91%), and physician-collected specimens (93%; 95% CI, 76%-99%). Overall, the specificity of hr-HPV mRNA for ≥HSIL detection was similar when comparing sc-WET with physician collection. However, specificity was lower for sc-WET (66% [61%-71%]) than sc-DRY (71% [66%-76%]). Women preferred sc-DRY specimen collection (46.1%) compared with sc-WET (31.1%). However, more women preferred physician collection (63.9%) compared with self-collection (36.1%). Self-collected stored-dry specimens seemed to perform similarly to sc-WET for the detection of ≥HSIL.

A clinicopathological and molecular analysis of cervical carcinomas with basaloid features.

To investigate the relationship between adenoid basal carcinoma (ABC), adenoid cystic carcinoma (ACC) and squamous cell carcinoma (SCC) in the uterine cervix. We analysed the clinicopathological and molecular features in two pure ABCs, 15 SCCs with ABC-/ACC-like features and seven basaloid SCCs (BSCCs) by chart review, immunohistochemistry, human papillomavirus (HPV) RNA in-situ hybridisation and fluorescence in-situ hybridisation. All patients were alive with no evidence of disease, except for one patient with ACC-like features who died of disease at 18months post diagnosis. The mixed carcinomas comprised variable SCCs and ABC-/ACC-like components displaying vague transitional zones. All components consistently showed diffuse p16, p63 and SOX2, variable cytokeratin (CK)7 and CK17 and rare Ber-EP4 and MYB expression; there was a substantially lower Ki67 index in pure ABCs and the ABC-like components. The ACC-like components showed no myoepithelial differentiation (SMA, calponin and S100) and MYB gene fusions. CK7, CK17 and Ber-EP4 were characteristically stronger in BSCCs than in the mixed carcinomas (P<0.01). High-risk HPV (HR-HPV) E6/E7 mRNA was detected in 12 mixed carcinomas and seven BSCCs, but not in pure ABCs. The HR-HPV mRNA expression was higher in the SCC components and BSCCs than in the ABC-like components of mixed carcinomas (P<0.05). The ACC-like components in mixed carcinomas probably represent the morphological mimics of salivary ACCs. ABC-like components may be the potential precursor of the ACC-like and SCC components. HR-HPV oncogenes may play a role in the pathogenesis of SCCs with ABC-/ACC-like features.

Prevalence, correlates, and predictive value of high-risk human papillomavirus mRNA detection in a community-based cervical cancer screening program in western Uganda

BackgroundNew strategies are needed to combat the high incidence of cervical cancer in resource-limited settings such as sub-Saharan Africa. Screening for high-risk human papillomavirus (hrHPV) DNA is sensitive for pre-cancer, but its lack of specificity results in substantial overtreatment in low resource settings where additional testing (e.g., colposcopy) is rarely available. Testing for hrHPV E6/E7 mRNA may enhance specificity, but little is known about its performance characteristics in resource-limited settings.MethodsIn a series of community health fairs in rural Uganda, women aged 25 to 49 years provided self-collected vaginal samples, which were tested for hrHPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) E6/E7 mRNA with the Aptima® assay. Positive specimens underwent testing for HPV-16 and 18/45. After excluding pregnant women, all women testing positive for any hrHPV subsequently were offered cervical biopsy to determine pathology.ResultsA total of 1892 women provided a vaginal sample for hrHPV testing during 24 health fairs. The median age was 34 years, HIV prevalence was 10, and 95% had not been previously screened. Prevalence of any hrHPV E6/E7 mRNA was 21% (95% confidence interval (CI): 19 to 23%); the prevalence of HPV-16 was 2.6%, HPV-18/45 1.9%, and HPV 16 and 18/45 were jointly found in 0.1% of the study population. Younger age, pregnancy and HIV-positivity were independently associated with any hrHPV infection. Of the 255 evaluable cervical biopsies, the positive predictive value of detecting any hrHPV E6/E7 mRNA for presence of cervical intraepithelial neoplasia grade 2 or higher (“CIN 2+”) was 8.2% (95% CI: 5.1 to 12%). The positive predictive value associated with detection of HPV-16 mRNA (15%) or HPV-18/45 mRNA (15%) was only slightly higher.ConclusionAmong community-based women in Uganda, the prevalence of any hrHPV E6/E7 mRNA in vaginal samples was high, but the prevalence of the most oncogenic HPV types (16, 18, or 45) was substantially lower. Positive predictive value of hrHPV mRNA-positivity for CIN 2+ was also low, including when restricting to HPV 16/18/45-positivity. The findings emphasize the need to identify more specific screening approaches for cervical cancer.

Oral testing for high-risk human papillomavirus DNA and E6/E7 messenger RNA in healthy individuals at risk for oral infection.

Testing for oral high-risk human papillomavirus (HPV) DNA may be useful for identifying individuals at increased risk for HPV-driven oropharyngeal cancer (OPC). However, positivity for HPV DNA provides no information on the transforming potential of the infection. In contrast, the detection of high-risk HPV E6/E7 messenger RNA (mRNA) may help to identify clinically significant infections because of the indispensable role of E6/E7 viral oncoproteins in the carcinogenic process. Oral rinses were collected with a mouthwash from cancer-free individuals at increased risk for oral HPV infection. High-risk HPV DNA and mRNA were evaluated via the testing of the oral rinses with the Linear Array HPV genotyping test and the Aptima HPV assay, respectively. Overall, 310 subjects with no clinical evidence of lesions of the oral cavity and oropharynx were included in the study. Thirty-three (10.6%) harbored high-risk HPV DNA in their oral rinse. These cases, together with 10 random samples negative for high-risk HPV DNA, were tested with the Aptima assay. A valid result was obtained for 41 of the 43 specimens (95.3%). Among the 31 cases that were positive for high-risk HPV DNA and had a valid Aptima result, 4 (12.9%) were positive for HPV mRNA. HPV mRNA was not detected in any of the samples negative for high-risk HPV DNA. HPV mRNA is detectable in oral rinses of cancer-free subjects. Oral HPV mRNA testing may be useful in the screening and/or early detection of HPV-driven OPC by possibly identifying active and transforming oral infections. The testing of individuals at increased risk for HPV-related OPC via simply and noninvasively collected oral specimens is an attractive option for future screening strategies.

Strong SOX10 expression in human papillomavirus–related multiphenotypic sinonasal carcinoma: report of 6 new cases validated by high-risk human papillomavirus mRNA in situ hybridization test

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk HPV (HR-HPV) infection. Using HR-HPV messenger RNA (mRNA) in situ hybridization (ISH), we reported 6 new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma. Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified 6 HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium, and 1 had invasive squamous cell carcinoma (SCC). All HMSC cases were SOX10 positive, whereas the overlying atypical squamous epithelium and the SCC were SOX10 negative. One atypical HMSC-like case was also identified, which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, and SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals compared with HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium and the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

γH2Ax Expression as a Potential Biomarker Differentiating between Low and High Grade Cervical Squamous Intraepithelial Lesions (SIL) and High Risk HPV Related SIL.

BackgroundγH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas.MethodsImmunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed.ResultsGradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL.ConclusionsγH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state.