Alpha, Beta, and Gamma Human Papillomaviruses in Genital Lichen Sclerosus: A Retrospective Cross-Sectional Study.

BACKGROUND: Lichen sclerosus (LS) is an inflammatory disease mostly arising at the genital level. It is unclear whether human papillomaviruses (HPVs) have an etiological significance in LS, and data on their prevalence in patients with LS are controversial. OBJECTIVES: The authors assessed alpha, beta, and gamma HPV prevalence in patients with genital LS. The association of HPV positivity with demographic and clinical factors was also investigated. METHODS: One hundred thirty-two formalin-fixed, paraffin-embedded LS samples (2016-2020) were retrieved from the archives of a pathology department. Alpha HPVs were genotyped with the INNO-LiPA HPV Genotyping Extra II kit. Beta and gamma HPVs were searched by multiplex Polymerase Chain Reaction. Immunostaining for p16 INK4a was performed on high-risk HPV-positive samples. RESULTS: Patients had a median age of 61 years, were mostly women ( n = 73, 55.3%), and with an early disease stage ( n = 79, 59.8%). Alpha HPVs were detected in 12/132 cases (9.1%). Among the 5 high-risk HPV-positive cases, only 2 displayed a strong and diffuse p16 INK4a staining. Beta genus was the most prevalent (35/132, 26.5%) and HPV5 was the most frequent beta genotype (25/132, 18.9%). There were 3 gamma HPV-positive cases among those with a valid result (3/131, 2.3%). Multiple infections with genotypes belonging to different genera were infrequent (3/131, 2.3%). No significant differences in the prevalence of the individual genera were observed according to sex and disease stage. CONCLUSIONS: Of the 3 HPV genera, beta genus showed the highest prevalence. Further research is needed to clarify whether the presence of beta HPVs in genital LS has a clinical significance.

Accuracy of different triage strategies for human papillomavirus positivity in an Italian screening population.

How to manage human papillomavirus (HPV)-positive women in cervical cancer screening remains debated. Our study compared different strategies to triage HPV positivity in a large cohort of women participating in a population HPV-based screening program. Women were tested for HPV (Cobas 4800; Roche), and those positive were triaged with cytology; cytology-positives were referred to colposcopy, while negatives were referred to 1-year HPV retesting. All HPV-positive women were also evaluated with p16/ki67 dual staining (Roche). All lesions found within 24 months of follow-up were included in the analyses. Of the 70 146 women tested, 4757 (6.8%) were HPV-positive. Of these, 1090 were cytology-positive and were referred to colposcopy. Of the 2958 HPV-positive/cytology-negative women who presented at 1-year retesting, 1752 (59.9%) still tested positive. Cumulatively, 532 CIN2+ (including 294 CIN3+) were found. The sensitivity of cytology, HPV16/18 and p16/ki67 as triage test for CIN3+ was 67.9%, 56.0% and 85.0%, respectively. The positive predictive value (PPV) for immediate colposcopy referral was 21.0%, 15.8% and 22.9%, respectively. Combining cytology with typing increased sensitivity to 83.9% and lowered PPV to 14.8%, while combining p16/ki67 and typing increased sensitivity to 91.1%, lowering the PPV to 15.9%. Women negative to p16/ki67 triage presented a cumulative 1-year CIN3+ risk of about 1%. In conclusion, when triaging HPV positivity, p16/ki67 performed better than cytology with or without HPV16/18 genotyping. The strategies that included dual staining achieved sensitivity and low 1-year risk for CIN3+ sufficiently high enough to permit considering extending the surveillance interval to 2 to 3 years for HPV-positive/triage-negative women.

Performance of HPV E6/E7 mRNA assay as primary screening test: Results from the NTCC2 trial.

As the primary screening test, E6/E7 mRNA has shown similar sensitivity for CIN3+ and lower positivity rate than the HPV DNA test. Nevertheless, the overall mRNA positivity is too high for immediate colposcopy, making a triage test necessary. The aim was to estimate the mRNA performance as a primary test with different triage strategies. All HPV DNA-positives were tested for mRNA, cytology and p16/ki67. A sample of HPV DNA-negatives was also tested for mRNA to estimate test specificity. We included all CIN3+ histologically diagnosed within 24 months since recruitment. Of the 41 127 participants, 7.7% were HPV DNA-positive, of which 66.4% were mRNA-positive. Among the HPV DNA-negatives, 10/1108 (0.9%) were mRNA-positive. Overall, 97 CIN3+ were found. If mRNA was used as the primary test, it would miss about 3% of all CIN3+ with a 22% reduction of positivity compared with HPV DNA. The weighted specificity estimate for

Evaluating programmed death-ligand 1 (PD-L1) in head and neck squamous cell carcinoma: concordance between the 22C3 PharmDx assay and the SP263 assay on whole sections from a multicentre study.

AIMS: The introduction of immunotherapy for patients with head and neck squamous cell carcinoma (HNSCC) raises the need for harmonisation between different types of antibody and immunohistochemistry platform for evaluating the expression of PD-L1 by use of the combined positive score (CPS) in this tumour. The aim of this study was to compare the expression of PD-L1 as determined with the CPS and two widely used assays (the 22C3 PharmDx assay and the SP263 assay) in a cohort of HNSCCs. METHODS AND RESULTS: We analysed 43 whole sections of HNSCC with two different anti-PD-L1 antibodies, 22C3 and SP263. The results, expressed as the CPS, were evaluated by 10 trained pathologists and statistical analyses were performed. We found a very similar results for PD-L1 expression between the 22C3 PharmDx assay and the SP263 assay in our cohort, and a strong and significant correlation between the two assays for all specimens (P < 0.0001). The interobserver reliability among pathologists for the continuous scores of CPS with the intraclass correlation coefficient and the correlation between the two assays were both good. Moreover, the rate of agreement between assays was high at all cut-offs and was best for the most relevant cut-off of CPS ≥ 1, and the kappa values were always in the range of almost perfect. CONCLUSIONS: Two different assays (the 22C3 PharmDx assay and SP263 assay) for PD-L1 in HNSCC showed high agreement. These data suggest that these two assays are interchangeable in the selection of patients with HNSCC for immunotherapy.

Oral human papillomavirus infection in HIV-infected and HIV-uninfected MSM: the OHMAR prospective cohort study.

OBJECTIVE: We aimed to assess incidence and clearance of oral human papillomavirus (HPV) infection and the respective risk factors in HIV-infected and uninfected men who have sex with men (MSM). METHODS: Oral rinse and gargles were collected semiannually from 244 MSM (103, 42.2% HIV-infected). HPV-DNA testing was performed with the Linear Array HPV Genotyping test. A Markov model was used for estimation of incidence, clearance and risk factor analysis. RESULTS: Incidence rates for any HPV were 21.2 and 15.0×1000 person-months in HIV-infected and uninfected MSM, respectively. The respective figures for high-risk HPVs were 10.7 and 6.5×1000 person-months. The clearance rate was 4-12 times higher than the respective incidence rate. HIV-infected MSM with >95 lifetime oral sex partners showed increased incidence of any HPV (adjusted HR, aHR: 8.46, 95% CI 1.89 to 37.92). Condomless oral sex appeared the strongest predictor for incident infection by high-risk HPVs in this group (aHR: 13.40, 95% CI 2.55 to 70.53). Those aged >46 years (aHR: 0.30, 95% CI 0.12 to 0.74) and those with nadir CD4+ T count of <200 cells/mm3 (aHR: 0.14, 95% CI 0.03 to 0.75) displayed a significantly reduced clearance of any and high-risk HPVs, respectively. HIV-uninfected MSM aged >46 years had increased risk of acquiring any HPV (aHR: 3.70, 95% CI 1.30 to 10.52) and high-risk HPV (aHR: 5.33, 95% CI 1.06 to 26.68). Any HPV clearance declined in those with more than six recent oral sex partners (aHR: 0.18, 95% CI 0.05 to 0.65). CONCLUSIONS: Acquisition of oral HPV infection in MSM seems to occur rarely, whereas clearance seems to be a frequent event. Oral HPV natural history in these at-risk subjects is differently influenced by age and sex behaviour, depending on HIV status.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Determinants of Viral Oncogene E6-E7 mRNA Overexpression in a Population-Based Large Sample of Women Infected by High-Risk Human Papillomavirus Types.

Cervical cancer screening by human papillomavirus (HPV) DNA testing with cytology triage is more effective than cytology testing. Compared to cytology, the HPV DNA test's higher sensitivity, which allows better protection with longer intervals, makes it necessary to triage the women with a positive result to compensate its lower specificity. We are conducting a large randomized clinical trial (New Technologies for Cervical Cancer 2 [NTCC2]) within organized population-based screening programs in Italy using HPV DNA as the primary screening test to evaluate, by the Aptima HPV assay (Hologic), the use of HPV E6-E7 mRNA in a triage test in comparison to cytology. By the end of June 2016, data were available for 35,877 of 38,535 enrolled women, 2,651 (7.4%) of whom were HPV DNA positive. Among the samples obtained, 2,453 samples were tested also by Aptima, and 1,649 (67.2%) gave a positive result. The proportion of mRNA positivity was slightly higher among samples tested for HPV DNA by the Cobas 4800 HPV assay (Roche) than by the Hybrid Capture 2 (HC2) assay (Qiagen). In our setting, the observed E6-E7 mRNA positivity rate, if used as a triage test, would bring a rate of immediate referral to colposcopy of about 4 to 5%. This value is higher than that observed with cytology triage for both immediate and delayed referrals to colposcopy. By showing only a very high sensitivity and thus allowing a longer interval for HPV DNA-positive/HPV mRNA-negative women, a triage by this test might be more efficient than by cytology.

Altered peritumoral microRNA expression predicts head and neck cancer patients with a high risk of recurrence.

Head and neck squamous cell carcinoma is typically characterized by a high incidence of local recurrences. It has been extensively shown that mucosa from head and neck squamous cell carcinoma patients carries both genetic and gene expression alterations, which are mostly attributable to major etiologic agents of head and neck squamous cell carcinoma. We previously identified a signature of microRNAs (miRNAs) whose high expression in tumors is predictive of recurrence. Here, we investigated whether the deregulation of miRNA expression in the tumor-surrounding mucosa is correlated to disease recurrence. Specifically, comparing the miRNA expression in matched tumoral, peritumoral, and normal tissues collected from head and neck squamous cell carcinoma patients, we identified 35 miRNAs that are deregulated in both tumoral and peritumoral tissues as compared with normal matched samples. Four of these composed a miRNA signature that predicts head and neck squamous cell carcinoma local recurrence independently from prognostic clinical variables. The predictive power of the miRNA signature increased when using the expression levels derived from both the peritumoral and the tumoral tissues. The expression signal of the miRNAs composing the predictive signature correlated with the transcriptional levels of genes mostly associated with proliferation. Our results show that expression of miRNAs in tumor-surrounding mucosa may strongly contribute to the identification of head and neck squamous cell carcinoma patients at high risk of local recurrence.

Cytology and human papillomavirus testing on cytobrushing samples from patients with head and neck squamous cell carcinoma.

BACKGROUND: The increasing incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) highlights the need for simple and effective tools to evaluate head and neck lesions and their HPV status. The main objective of the current study was to investigate the association between abnormal cytology and HPV infection, assessed on cytobrushing samples, and histologically confirmed HNSCC. Second, the authors attempted to investigate whether HPV status on cytobrushing samples reflected that of the tumoral tissue. METHODS: A total of 164 samples from HNSCC, nonmalignant lesions, or healthy mucosae of the oral cavity and oropharynx were collected by cytobrushing in PreservCyt solution and evaluated by liquid-based cytology and Linear Array HPV genotyping test. All the findings from the cytologic samples were compared with those from the corresponding histologic samples. RESULTS: Patients with abnormal cytology had a significantly higher risk of having an HNSCC (odds ratio [OR], 9.18; 95% confidence inteval [95% CI], 3.27-26.49). The association was stronger for oral cancer (OR, 10.86; 95% CI, 2.51-51.06) than oropharyngeal cancer (OR, 8.45; 95% CI, 1.62-49.82). HPV positivity in the oropharyngeal cytobrushing was associated with a nearly 5-fold higher risk of having abnormal cytology (OR, 4.57; 95% CI, 1.57-13.57) as well as histologically proven oropharyngeal cancer (OR, 5.09; 95% CI, 1.09-31.61). Comparing the HPV status on cytologic and corresponding histologic samples from patients with HNSCC, we found that 90.4% of the cases were concordant (kappa, 0.796). CONCLUSIONS: Abnormal brushing cytology is strongly associated with a diagnosis of HNSCC, whereas HPV positivity on cytobrushing samples is only associated with oropharyngeal cancer. HPV testing on cytobrushing samples represents a valid option for the assessment of HPV infection in patients with oropharyngeal cancer.

Interobserver agreement in the interpretation of anal cytology.

BACKGROUND: Anal cytology represents a tool for anal cancer screening in high-risk populations. In addition to accuracy, the reproducibility of the interpretation is of key importance. The authors evaluated the agreement of anal cytologic interpretation between two cytopathologists. METHODS: Liquid-based cytologic slides from human immunodeficiency virus (HIV)-negative men who have sex with men (MSM) were evaluated by two readers with at least 10 years of expertise in cervical cytology. Cases with a discordant interpretation were reviewed, and a consensus was reached. Human papillomavirus (HPV) genotyping was performed using a proprietary HPV genotyping test. Unweighted and weighted Cohen kappa and 95% confidence interval (CI) values were calculated. RESULTS: Overall, 713 slides that were adequate for interpretation were evaluated (MSM: median age, 33 years). An HPV test was performed on 620 samples (87.0%). Considering a dichotomous interpretation (negative for intraepithelial lesion or malignancy vs. atypical squamous cells of undetermined significance or worse), the crude agreement between the two readers was 93.3% (kappa = 0.82; 95% CI, 0.77-0.87). Once a consensus for discordant cases was reached, the best agreement was found for the negative for intraepithelial lesion or malignancy category (511 of 528 samples; 96.8%), whereas the atypical squamous cells of undetermined significance category showed the lowest agreement (90 of 117 samples, 76.9%). Considering the individual cytologic categories, overall agreement was 92.1% (kappa = 0.85; 95% CI, 0.81-0.89). The discordant interpretations were not associated with high-risk HPV infection, HPV16 infection, or MSM age. CONCLUSIONS: The results indicating excellent interobserver agreement in this study substantiate the use of anal cytology in the setting of human immunodeficiency virus-negative MSM.

Circulating tumor HPV DNA in the management of HPV+ oropharyngeal cancer and its correlation with MRI.

BACKGROUND: First aim was to compare ddPCR assays of ctHPVDNA with p16 IHC and qualitative HPV PCR. Second aim was to carry out longitudinal blood sampling to test for association of ctHPVDNA with histological confirmed recurrence. Third aim was to perform a multidimensional assessment which included: (1) clinical features; (2) ctHPVDNA; (3) MRI-based tumor size measurements of primary tumor (PT) and cervical lymph node metastases (CLNM). METHODS: Plasma samples were collected before treatment and during follow-up, and ddPCR assay comprising E6 of HPV16 and HPV 33 and HPV 35 was used. RESULTS: Present study was conducted at diagnosis in 117 patients and revealed a ctHPVDNA sensitivity of 100% (95% CI 95.5-100) and a specificity of 94.4 (95% CI 81.3-99.3), positive predictive value (PPV) of 94.4 (95% CI 81.3-99.3), and negative predictive value (NPP) of 100% (95% CI 89.7-100). During follow-up ctHPVDNA had a sensitivity of 100% (95% CI 72.1-100)% and specificity of 98.4% (95% CI 91.7-100)%, PPV% of 90.9% (95% CI 62.3-98.4) and NPV% of 100% (95% CI 94.3-100) for ability to detect recurrence. Correlation between both the CLNM volume and the sum of PT and CLNM volume was observed. CONCLUSIONS: ctHPVDNA was superior to p16 in identification of HPV-OPSCC at diagnosis. Introduction of ctHPVDNA, beyond diagnostic setting, represents a great opportunity to improve follow-up protocol of OPSCC patients.

Combined TP53 status in tumor-free resection margins and circulating microRNA profiling predicts the risk of locoregional recurrence in head and neck cancer.

Locoregional recurrences represent a frequently unexpected problem in head and neck squamous cell carcinoma (HNSCC). Relapse often (10-30%) occurs in patients with histologically negative resection margins (RMs), probably due to residual tumor cells or hidden pre-cancerous lesions in normal mucosa, both missed by histopathological examination. Therefore, definition of a 'clean' or tumor-negative RM is controversial, demanding for novel approaches to be accurately explored. Here, we evaluated next generation sequencing (NGS) and digital PCR (dPCR) as tools to profile TP53 mutational status and circulating microRNA expression aiming at scoring the locoregional risk of recurrence by means of molecular analyses. Serial monitoring of these biomarkers allowed identifying patients at high risk, laying the ground for accurate tracking of disease evolution and potential intensification of post-operative treatments. Additionally, our pipeline demonstrated its applicability into the clinical routine, being cost-effective and feasible in terms of patient sampling, holding promise to accurately (re)-stage RMs in the era of precision medicine.

Comparison of HPV-positive triage strategies combining extended genotyping with cytology or p16/ki67 dual staining in the Italian NTCC2 study.

BACKGROUND: Each high-risk HPV genotype has different oncogenic potential, and the risk of CIN3+ varies according to genotype. We evaluated the performance of different strategies of HPV-positivity triage combining cytology, p16/ki67 dual staining (DS), and extended genotyping. METHODS: Samples from 3180 consecutive women from the NTCC2 study (NCT01837693) positive for HPV DNA at primary screening, were retrospectively analyzed by the BD Onclarity HPV Assay, which allows extended genotyping. Genotypes were divided into three groups based on the risk of CIN3+. HPV DNA-positive women were followed up for 24 months or to clearance. FINDINGS: Combining the three groups of genotypes with cytology or DS results we identify a group of women who need immediate colposcopy (PPV for CIN3+ from 7.8 to 20.1%), a group that can be referred to 1-year HPV retesting (PPV in those HPV-positive at retesting from 2.2 to 3.8), and a group with a very low 24-month CIN3+ risk, i.e. 0.4%, composed by women cytology or DS negative and positive for HPV 56/59/66 or 35/39/68 or negative with the Onclarity test, who can be referred to 3-year retesting. INTERPRETATION: Among the baseline HPV DNA positive/cytology or DS negative women, the extended genotyping allows to stratify for risk of CIN3+, and to identify a group of women with a risk of CIN3+ so low in the next 24 months that they could be referred to a new screening round after 3 years. FUNDING: Italian Ministry of Health (grant number RF-2009-1536040). Hologic-Genprobe, Roche Diagnostics, and Becton & Dickinson provided financial and non-financial support.

T and NK cells: two sides of tumor immunoevasion.

Natural Killer (NK) cells are known to reject several experimental murine tumors, but their antineoplastic activity in humans is not generally agreed upon, as exemplified by an interesting correspondence recently appeared in Cancer Research. In the present commentary, we join the discussion and bring to the attention of the readers of the Journal of Translational Medicine a set of recent, related reports. These studies demonstrate that effectors of the adaptive and innate immunity need to actively cooperate in order to reject tumors and, conversely, tumors protect themselves by dampening both T and NK cell responses. The recently reported ability of indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) expressed by melanoma cells to down-regulate activating NK receptors is yet another piece of evidence supporting combined and highly effective T/NK cell disabling. Major Histocompatibility Complex class I (MHC-I) molecules, including Human Leukocyte Antigen E (HLA-E), represent another class of shared activating/inhibitory ligands. Ongoing clinical trials with small molecules interfering with IDO and PGE2 may be exploiting an immune bonus to control cancer. Conversely, failure to simultaneously engage effectors of both the innate and the adaptive immunity may contribute to explain the limited clinical efficacy of T cell-only vaccination trials. Shared (T/NK cells) natural immunosuppressants and activating/inhibitory ligands expressed by tumor cells may provide mechanistic insight into impaired gathering and function of immune effectors at the tumor site.

Melanoma molecular classes and prognosis in the postgenomic era.

Gene expression profiling is a powerful method to classify human tumours on the basis of biological aggressiveness, response to therapy, and outcome for the patient, but its application in melanoma lags behind that of other cancers. From more than 100 articles available on the topic, we selected 14 focusing on patients' outcome. We review and briefly discuss salient findings, and list ten reasons why melanoma molecular classes are not yet used in clinical diagnosis and prognosis. The available evidence suggests that we are on the verge of creating a framework for the use of melanoma molecular classes in prognosis, but so far there is little consensus to put together informative diagnostic and prognostic gene sets.

Incidence of abnormal anal cytology in HIV-infected and HIV-uninfected men who have sex with men.

INTRODUCTION: Anal cytology is used in the prevention of anal cancer, which disproportionally affects men who have sex with men (MSM). Data on the incidence of cytologic abnormalities in these individuals are scant. METHODS: MSM with baseline negative anal cytology and at least one further adequate cytology were included. Incidence rate for positive atypical squamous cells of undetermined significance (ASC-US+) was calculated. Kaplan-Meier curves were compared by log-rank test according to HIV status, baseline high-risk human papillomavirus (HPV) (high-risk HPV-negative, HPV16-positive, other high-risk HPV-positive [non-HPV16]) and high-risk HPV persistence (positive from baseline to the first ASC-US+ or last visit for those who remained cytologically negative). Cox univariate and multivariate analyses were performed. RESULTS: A total of 250 MSM were included: 52/153 (34.0%) HIV-uninfected MSM had an ASC-US+ report at follow-up (incidence: 13.1 × 100 person-years; 95% CI, 9.8-17.2); 48/97 (49.5%) HIV-infected MSM developed cytologic abnormalities (incidence: 16.0 × 100 person-years; 95% CI, 11.8-21.2). ASC-US+ incidence in HIV-uninfected and HIV-infected MSM did not differ significantly (p = .32). Kaplan-Meier curves did not differ significantly according to baseline high-risk HPV. Differences were significant between those with and without persistent high-risk HPVs, both among HIV-uninfected (p = .03) and HIV-infected MSM (p = .008). Age (adjusted hazard ratio [aHR], 0.98; 95% CI, 0.96-0.99), high-risk HPV persistence (aHR, 1.57; 95% CI, 1.02-2.39), and condomless receptive anal sex (aHR, 1.99; 95% CI, 1.31-3.03) were predictors for incident ASC-US+. CONCLUSIONS: Despite the limited number of subjects, in our study HIV-uninfected and HIV-infected MSM have a similar ASC-US+ incidence. Occurrence of ASC-US+ was significantly affected by age, high-risk HPV persistence, and condomless receptive anal sex. The assessment of HPV persistence might identify those MSM at higher risk for anal lesions.

Claspin as a biomarker of human papillomavirus-related high grade lesions of uterine cervix.

BACKGROUND: Claspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint. Claspin expression is significantly high in several human solid tumors. Furthermore, high levels of claspin have been found in cervical cancer cell lines. Nevertheless, no data are available regarding claspin expression in cervical tissues. METHODS: In order to investigate whether claspin immunoreactivity is related to the lesion severity and High-Risk (HR) HPV infection, we analyzed claspin expression by immunohistochemistry in a series of cervical biopsies which represent the steps occurring during cervical carcinogenesis (normal tissues, Cervical Intraepithelial Neoplasias 1, 2 and 3, Squamous Cell Carcinomas). All patients also had a cervico-vaginal sample for HPV testing, collected immediately before the colposcopy-guided biopsy. The HR-HPV DNA detection was performed by the HR-HPV Hybrid Capture 2 test. HPV genotyping was performed using the Linear Array HPV Genotyping Test. RESULTS: Our results evidenced a constant and significant increase of the rate of claspin positivity from the normal tissues to carcinomas (pχ2(trend) < 0.0001). In fact, the normal tissues displayed either no or faint claspin immunoreactivity, whereas a moderate/high positivity was observed in 16% of the CIN1, 76% of the CIN2, 87.5% of the CIN3 and 93.3% of the cancers. Moreover, we found a statistically significant correlation between claspin expression and HR-HPV infection (pχ2 < 0.0001), irrespective of the genotype. Finally, we demonstrated the feasibility of claspin immunostaining in cervical cytology. CONCLUSIONS: Our findings indicate that in vivo claspin expression is significantly related to HR-HPV infection and lesion grade both in histological and cytological samples. Therefore, the analysis of claspin expression could be clinically relevant in the diagnosis of HPV-related cervical lesions, in particular when applied to cervico-vaginal cytology. Moreover, giving information on the proliferation rate of each lesion, claspin immunostaining may contribute to the evaluation of progression risk, thus being helpful in patient management. Nevertheless, only large prospective studies may clarify the true clinical usefulness of claspin expression in distinguishing lesions with different progression potential.

Anal cytological abnormalities and epidemiological correlates among men who have sex with men at risk for HIV-1 infection.

BACKGROUND: The incidence of anal cancer, a Human Papillomavirus (HPV)-related neoplasia, has been increasing in recent decades, mainly in men who have sex with men (MSM). Cytological changes of the anal epithelium induced by HPV can be detected through an anal pap smear. This study aimed to evaluate the prevalence and epidemiological correlates of anal cytological abnormalities among relatively young MSM at risk for HIV-1 infection, to help clarify whether or not this population deserves further investigation to assess the presence of anal cancer precursor lesions. METHODS: MSM were recruited among attendees of a large STI clinic for a HIV-1 screening program. Anal samples, collected with a Dracon swab in PreservCyt, were used both for liquid-based cytology and HPV testing by the Linear Array HPV Genotyping Test. Data regarding socio-demographic characteristics and sexual behavior were collected in face-to-face interviews. RESULTS: A total of 346 MSM were recruited (median age 32 years). Overall, 72.5% of the individuals had an anal HPV infection, with 56.1% of them being infected by oncogenic HPV genotypes. Anal cytological abnormalities were found in 29.8% of the cases (16.7% ASC-US and 13.1% L-SIL). Presence of ASC-US+ was strongly associated with infection by any HPV type (OR = 4.21, 95% CI: 1.97-9.23), and particularly by HPV 16 and/or 18 (OR = 5.62, 95% CI: 2.33-13.81). A higher proportion of ASC-US+ was found in older MSM, in those with a higher number of lifetime partners and in those with a history of ano-genital warts. However, none of these variables or the others analyzed showed any significant association with abnormal cytological findings. CONCLUSIONS: The presence of anal cytological abnormalities in about one third of the recruited MSM and their strong association with HPV infection, in particular that caused by HPV 16 and/or 18, might provide a further complement to the data that now support the introduction of HPV vaccination among MSM to protect them from the development of HPV-associated diseases. Additional studies are needed to determine whether and how screening for anal cancer precursor lesions should be performed in younger MSM.

p16/Ki-67 dual staining in cervico-vaginal cytology: correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy.

OBJECTIVES: To evaluate the CINtec PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16(INK4a) and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities. METHODS: 140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec PLUS kit and the morphology assessment. RESULTS: Cytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84-31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39-47.77 vs COR=4.20, 95% CI: 0.99-20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16-29.12). CONCLUSIONS: This preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion.

Incidence and clearance of anal high-risk Human Papillomavirus infection and their risk factors in men who have sex with men living with HIV.

HIV-infected men who have sex with men (MSM) display the highest prevalence of anal infection by high-risk Human Papillomaviruses (hrHPVs) and incidence of anal carcinoma. Anal specimens were genotyped by the Linear Array. Incidence and clearance of anal infection by hrHPVs, hrHPVs other than HPV16, low-risk HPVs, and four individual types (6,11,16,18) were estimated using a two-state Markov model. Determinants for incidence and clearance were assessed by logistic regression. Overall, 204 individuals were included (median age 42 years, IQR = 34-49). For hrHPVs, incidence and clearance rates were 36.1 × 1000 person-months (p-m) (95% CI 23.3-56.5) and 15.6 × 1000 p-m (95% CI 10.7-23.3), respectively. HPV16 showed a higher incidence than HPV18 (10.2 vs. 7.2 × 1000 p-m). Its clearance was more than twofold lower than that of HPV18 (30.1 vs. 78.2 × 1000 p-m). MSM receiving cART displayed a 68% to 88% decrease in risk of acquiring hrHPVs, hrHPVs other than HPV16, HPV16, and HPV18 (adjusted Hazard Ratio [aHR] 0.13, 95% CI 0.02-0.67; aHR 0.22, 95% CI 0.06-0.78; aHR 0.32, 95% CI 0.12-0.90; aHR 0.12, 95% CI 0.04-0.31, respectively) than patients not treated. A nadir CD4 + count < 200 cells/mm3 significantly reduced the clearance of hrHPVs other than HPV16 (aHR 0.39, 95% CI 0.17-0.90). cART use reduces the risk of acquiring anal infection by hrHPVs.