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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 forCIN2 was 94.5% (95% CI = 93.9%-94.9%) and sensitivity for CIN3+ was 96.9% (95% CI = 91.3%-99.1%). If all the weighted estimated 6.0% mRNA-positive women had been referred to colposcopy, PPV for CIN3+ would have been 4.2%. Cytology or p16/ki67 triage would decrease immediate referral to 1.7% and 2.0%, increasing PPV to 11.2% and 11.7%, respectively; total colposcopy referral would be 4.0% and 3.9%, respectively. As the primary screening test, the mRNA assay showed a positivity rate lower than that of HPV DNA, with a small number of CIN3+ missed. Triage with cytology or p16/ki67 would only marginally decrease overall colposcopy referral.

Purpose: Men at risk of missed prostate cancer on a negative biopsy often undergo a rebiopsy. We evaluated whether global hypomethylation, measured through LINE-1 methylation, and GSTP1 hypermethylation on a negative biopsy are associated with subsequent prostate cancer diagnosis.Experimental Design: We performed a case–control study nested in an unselected series of 737 men who received at least two prostate biopsies at least three months apart at the Molinette Hospital (Turin, Italy). Two pathology wards were included for replication purposes. The study included 67 cases and 62 controls in Ward 1 and 62 cases and 66 controls in Ward 2. We used pyrosequencing to analyze LINE-1 and GSTP1 methylation in the negative biopsies. Odds ratios (OR) of prostate cancer diagnosis were estimated using conditional logistic regression.Results: After mutual adjustment, GSTP1 hypermethylation was associated with an OR of prostate cancer diagnosis of 5.1 (95% confidence interval: 1.7–14.9) in Ward 1 and 2.0 (0.8–5.3) in Ward 2, whereas an association was suggested only for low LINE-1 methylation levels (P = 0.007).Conclusions: GSTP1 hypermethylation on a negative biopsy is associated with the risk of prostate cancer on a rebiopsy, especially of high-grade prostate cancer. Consistent results were found only for extremely low LINE-1 methylation levels. Clin Cancer Res; 22(4); 984–92. ©2015 AACR.

Kernel density estimate of LINE-1 methylation distribution in biopsies vs. transurethral resection of the prostate (TURP) in the Ward 1 and Ward 2 combined. Cases and controls are presented separately.

BACKGROUND Epigenetics may play a role in wheezing and asthma development. We aimed to examine infant saliva DNA methylation in association with early childhood wheezing. METHODS A case-control study was nested within the NINFEA birth cohort with 68 cases matched to 68 controls by sex, age (between 6 and 18 months, median: 10.3 months) and season at saliva sampling. Using a bumphunting region-based approach, we examined associations between saliva methylome measured using Illumina Infinium HumanMethylation450k array and wheezing between 6 and 18 months of age. We tested our main findings in independent publicly available data sets of childhood respiratory allergy and atopic asthma, with DNA methylation measured in different tissues and at different ages. RESULTS We identified one wheezing-associated differentially methylated region (DMR) spanning ten sequential CpG sites in the promoter-regulatory region of PM20D1 gene (family-wise error rate

Human papilloma virus (HPV) testing is more sensitive but less specific than cytology. We evaluated stand-alone genotyping as a possible triage method. During a multicentre randomised controlled trial comparing HPV testing to conventional cytology, HPV-positive women were referred to colposcopy and followed up if no high-grade lesion was detected. HPV-positive samples were genotyped by GP5+/GP6+ primed polymerase chain reaction followed by reverse line blot. Genotypes were hierarchically ordered by positive predictive value (PPV) for CIN grade 2 or more (CIN2+), and grouped by cluster analysis into three groups (A, B and C in decreasing order). Receiver operating characteristic curves were computed. Among 2,255 HPV-positive women with genotyping, 239 CIN2+ (including 113 CIN3+) were detected at baseline or during a 3-year follow-up. HPV33 had the highest PPV with CIN2+ and CIN3+ as the endpoint and when considering lesions detected at baseline or also during follow-up. HPV16 and HPV35 were the second and third, respectively. Cross-sectional sensitivity for CIN2+ at baseline was 67.3% (95% CI 59.7-74.2), 91.8% (95% CI 86.6-95.5) and 94.7% (95% CI 90.2-97.6), respectively, when considering as "positive" any of the HPV types in group A (33, 16 and 35), A or B (31, 52, 18, 59 and 58) and A or B or C (39, 51, 56, 45 and 68). The corresponding cross-sectional PPVs for CIN2+ were 15.8% 95% (CI 13.2-18.7), 12.0% (95% CI 10.3-13.9) and 9.6% (95% CI 8.2-11.1), respectively. HPV33, 16 and 35 confer a high probability of CIN2+ but this rapidly decreases when adding other genotypes.

Background The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis. Methods Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided. Results 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both). Conclusions p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

The present study aimed to evaluate the association between altered methylation and histologically confirmed high grade cervical intraepithelial neoplasia (hgCIN).Methylation levels in selected host (CADM1, MAL, DAPK1) and HPV (L1_I, L1_II, L2) genes were measured by pyrosequencing in DNA samples obtained from 543 women recruited in Curitiba (Brazil), 249 with hgCIN and 294 without cervical lesions. Association of methylation status with hgCIN was estimated by Odds Ratio (OR) with 95% confidence interval (CI).The mean methylation level increased with severity of the lesion in the host and viral genes (p-trend 0.05), with the exception of L1_II region (p-trend = 0.075). Positive association was found between methylation levels for host genes and CIN2 and CIN3 lesions respectively [CADM1: OR 4.17 (95%CI 2.03-8.56) and OR 9.54 (95%CI 4.80-18.97); MAL: OR 5.98 (95%CI 2.26-15.78) and OR 22.66 (95%CI 9.21-55.76); DAPK1: OR 3.37 (95%CI 0.93-12.13) and OR 6.74 (95%CI 1.92-23.64)]. Stronger risk estimates were found for viral genes [L1_I: OR 10.74 (95%CI 2.66-43.31) and OR 15.00 (95%CI 3.00-74.98); L1_II: OR 73.18 (95%CI 4.07-1315.94) and OR 32.50 (95%CI 3.86-273.65); L2: OR 4.73 (95%CI 1.55-14.44) and OR 10.62 (95%CI 2.60-43.39)]. The cumulative effect of the increasing number of host and viral methylated genes was associated with the risk of CIN2 and CIN3 lesions (p-trend 0.001).Our results, empowered by a wide cervical sample series with a large number of hgCIN, supported the role of methylation as marker of aggressiveness.

Background Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for accurate detection of high-grade cervical lesions and cancer. Methylation status has been widely investigated for HPV16, sporadically for other HPV types. Methods Objective of this methodological study was to set up molecular methods to test the methylation levels in the twelve oncogenic HPV types by pyrosequencing, minimizing the number of HPV type-specific PCR protocols. Target CpGs were selected on the HPV L1 (two regions, L1 I and L1 II) and L2 genes. Study samples included DNA stored at Turin, Italy, purified by cervical cells collected in Standard Transport Medium or PreservCyt from women who participated in two studies (N = 126 and 140) nested within the regional organized screening programme. PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types. Results Generation of consensus primers was successful for L1 I and II regions, unsuccessful for L2 region, for which HPV type-specific primers remained necessary. The difference between replicated tests on the same sample was ≤4% in 88%, 77% and 91% of cases when targeting the L1 I, L1 II and L2 regions, respectively. The corresponding intra-class correlation coefficients (ICC) were 0.94, 0.87 and 0.97 respectively. When comparing methylation measures based on consensus and type-specific primers, ICC was 0.97 for the L1 I region and 0.99 the for L1 II region. Conclusions The proposed protocols, applying consensus primers suitable to amplify the oncogenic HPV types and minimize the number of PCR reactions, represent a promising tool to quantify viral methylation in women positive for any high risk HPV type. Impact Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.

BACKGROUND The triage of human papillomavirus (HPV)-positive women is needed to avoid overreferral to colposcopy. p16INK4a immunostaining is an efficient triage method. p16INK4a/Ki-67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand-alone p16INK4a staining. METHODS Within a pilot project, HPV-positive women were referred to colposcopy if cytology was abnormal or unsatisfactory or HPV testing was still positive after 1 year. For 500 consecutive women, a slide obtained during colposcopy was immunostained for p16INK4a/Ki-67 and independently interpreted by 7 readers without previous experience with dual staining. Four of these readers were experts in cervical pathology and 3 were not. Kappa values for multiple raters, sensitivity, and specificity for cervical intraepithelial neoplasia type 2-positive histology were computed. Because women with normal cytology were underrepresented, estimates for all HPV-positive women were obtained as weighted means of cytology-specific estimates. RESULTS The overall kappa for HPV-positive women was 0.70 (95% confidence interval [95% CI], 0.60-0.77). Kappa values were not found to be significantly different between expert and nonexpert readers with regard to cervical cytology but were significantly increased (P =. 0066) after consensus discussion. The overall specificity estimate for HPV-positive women was 64.0% (95% CI, 57.4%-70.2%): 66.7% (95% CI, 59.8%-73.0%) for experts and 60.5% (95% CI, 59.8%-73.0%) for nonexperts. Among women with abnormal cytology, the sensitivity was 85.5% (95% CI, 77.9%-90.8%): 85.8% (95% CI, 77.9%-91.2%) for experts and 85.1% (95% CI, 76.6%-90.9%) for nonexperts. CONCLUSIONS p16INK4a/Ki-67 immunostaining demonstrated good reproducibility and specificity when triaging HPV-positive women. Dual-staining interpretation can be performed, after short training, even by staff who are not experts in cervical cytology. This allows HPV-based screening with triage to be performed in settings in which such expert staff is not available. Cancer (Cancer Cytopathol) 2015;123:212–218. © 2014 American Cancer Society.

Human papilloma virus (HPV) testing is more sensitive but less specific than cytology. We evaluated stand‐alone genotyping as a possible triage method. During a multicentre randomised controlled trial comparing HPV testing to conventional cytology, HPV‐positive women were referred to colposcopy and followed up if no high‐grade lesion was detected. HPV‐positive samples were genotyped by GP5+/GP6+ primed polymerase chain reaction followed by reverse line blot. Genotypes were hierarchically ordered by positive predictive value (PPV) for CIN grade 2 or more (CIN2+), and grouped by cluster analysis into three groups (A, B and C in decreasing order). Receiver operating characteristic curves were computed. Among 2,255 HPV‐positive women with genotyping, 239 CIN2+ (including 113 CIN3+) were detected at baseline or during a 3‐year follow‐up. HPV33 had the highest PPV with CIN2+ and CIN3+ as the endpoint and when considering lesions detected at baseline or also during follow‐up. HPV16 and HPV35 were the second and third, respectively. Cross‐sectional sensitivity for CIN2+ at baseline was 67.3% (95% CI 59.7–74.2), 91.8% (95% CI 86.6–95.5) and 94.7% (95% CI 90.2–97.6), respectively, when considering as “positive” any of the HPV types in group A (33, 16 and 35), A or B (31, 52, 18, 59 and 58) and A or B or C (39, 51, 56, 45 and 68). The corresponding cross‐sectional PPVs for CIN2+ were 15.8% 95% (CI 13.2–18.7), 12.0% (95% CI 10.3–13.9) and 9.6% (95% CI 8.2–11.1), respectively. HPV33, 16 and 35 confer a high probability of CIN2+ but this rapidly decreases when adding other genotypes., What's new? The Human papilloma virus (HPV) infection genotype is an early predictor of cervical lesions. The current focus lies on high‐risk genotypes 16 and 18, frequently found in cancers. Here the authors studied the predictive value of all carcinogenic types and identified infections with HPV16, but also HPV33 and 35, as conferring very high risk for the development of high‐grade cervical intraepithelial neoplasia (CIN2+). However, HPV genotyping alone did not reliably triage women, and combinations with other methods are recommended.

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.

The role of global DNA methylation in prostate cancer (PCa) remains largely unknown. Our aim was to summarize evidence on the role of global DNA hypomethylation in PCa development and progression.We searched PubMed through December 2013 for all studies containing information on global methylation levels in PCa tissue and at least one non-tumor comparison tissue and/or studies reporting association between global methylation levels in PCa tissue and survival, disease recurrence or at least one clinicopathological prognostic factor. We summarized results using non-parametric comparisons and P-value summary methods.We included 15 studies in the review: 6 studies with both diagnostic and prognostic information, 5 studies with only diagnostic information and 4 studies with only prognostic information. Quantitative meta-analysis was not possible because of the large heterogeneity in molecular techniques, types of tissues analyzed, aims and study designs. Summary statistical tests showed association of DNA hypomethylation with PCa diagnosis (P0.006) and prognosis (P0.001). Restriction to studies assessing 5-methylcytosine or long interspersed nucleotide element-1 revealed results in the same direction. Analyses restricted to specific clinicopathological features showed association with the presence of metastasis and tumor stage in all tests with P0.03, and no association with Gleason score (all tests P0.1 except for the weighted Z-test, P=0.05).DNA hypomethylation was associated with PCa development and progression. However, due to the heterogeneity and small sample sizes of the included studies, along with the possibility of publication bias, this association requires additional assessment.

Adult height is associated with testicular cancer risk. We studied to what extent this association is explained by parental height, childhood height and age at puberty. We conducted a case-control study on germ-cell testicular cancer patients diagnosed in 1997-2008 and resident in the Province of Turin. Information was collected using mailed questionnaires in 2008-2011. Specifically, we asked for adult height (in cm), height at age 9 and 13 (compared to peers) and age at puberty (compared to peers). We also asked for paternal and maternal height (in cm) as indicators of genetic components of adult height. The analysis included 255 cases and 459 controls. Odds ratios (ORs) of testicular cancer were estimated for the different anthropometric variables. Adult height was associated with testicular cancer risk [OR: 1.16, 95% confidence interval (CI): 1.03-1.31 per 5-cm increase]. The risk of testicular cancer was only slightly increased for being taller vs. shorter than peers at age 9 (OR: 1.55, 95% CI: 0.91-2.64) or age 13 (OR: 1.26, 95% CI: 0.78-2.01), and parental height was not associated with testicular cancer risk. The OR for adult height was 1.32 (95% CI: 1.12-1.56) after adjustment for parental height. Among participants with small average parental height ( 180 cm) vs. short (

OBJECTIVE In many African Sub-Saharan countries, human papilloma virus (HPV) prevalence data are not available. The current study estimated the prevalence of HPV virus in the female population of Djibouti. METHODS Approximately 1000 asymptomatic women 16 to 64 years old were enrolled from 3 of the main health structures of Djibouti in 2014 and 2015; 998 cervical samples were tested for HPV-DNA of high risk types, 499 during the first year, and 499 during the second. Positive samples were typed with an HPV genotyping kit. RESULTS The women were an average age of 38.8 years (SD, 10.2); 54 women tested positive for HPV (prevalence rate, 5.4% [95% confidence interval, 4.0-6.8]). The highest prevalence was observed among the women younger than 35 years. HPV66 was the most prevalent (15.4% of the infections), followed by HPV31 and HPV52 (10.8% both) and HPV16 (9.2%). All 54 women who tested HPV-positive underwent a Pap test, which was positive in 8 cases (14.8%): 2 high-grade squamous intraepithelial lesion (HSIL) and 6 low-grade (LSIL). CONCLUSIONS The HPV prevalence shows a curve by age similar to that of other African countries. The proportion of HPV16 is among the lowest ever seen in similar studies. The findings suggest to Djibouti the choice of a strategy of screening that includes forms of cytological triage, thus limiting recourse to colposcopy.

Background Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Objective Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. Methods We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. Results We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. Conclusion The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

Purpose: Men at risk of missed prostate cancer on a negative biopsy often undergo a rebiopsy. We evaluated whether global hypomethylation, measured through LINE-1 methylation, and GSTP1 hypermethylation on a negative biopsy are associated with subsequent prostate cancer diagnosis. Experimental Design: We performed a case–control study nested in an unselected series of 737 men who received at least two prostate biopsies at least three months apart at the Molinette Hospital (Turin, Italy). Two pathology wards were included for replication purposes. The study included 67 cases and 62 controls in Ward 1 and 62 cases and 66 controls in Ward 2. We used pyrosequencing to analyze LINE-1 and GSTP1 methylation in the negative biopsies. Odds ratios (OR) of prostate cancer diagnosis were estimated using conditional logistic regression. Results: After mutual adjustment, GSTP1 hypermethylation was associated with an OR of prostate cancer diagnosis of 5.1 (95% confidence interval: 1.7–14.9) in Ward 1 and 2.0 (0.8–5.3) in Ward 2, whereas an association was suggested only for low LINE-1 methylation levels ( Conclusions: GSTP1 hypermethylation on a negative biopsy is associated with the risk of prostate cancer on a rebiopsy, especially of high-grade prostate cancer. Consistent results were found only for extremely low LINE-1 methylation levels. Clin Cancer Res; 22(4); 984–92. ©2015 AACR.

Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982–1988 and 1993–1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95–2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03–21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.

Introduction We investigated clustering patterns of human papillomavirus (HPV) in a large study, the New Technologies in Cervical Cancer (NTCC) screening study. Materials and Methods Women aged 25–60 years who attended cervical screening in eight different areas in Northern and Central Italy were tested for HPV infection with Hybrid Capture 2 (HC2). Genotyping of the HC2-positive samples was performed for 13 HPV types targeted by GP5+/GP6+ PCR, followed by Reverse Line Blot. Logistic regression was used to model type-specific HPV positivity, adjusted for age, study area, and specific HPV type prevalence. Subject-level random effects were added to represent unobservable risk factors common to all HPV types. Results A total of 36,877 women were included. Of 2833 HC2-positive women, 2108 were confirmed to be positive for any of the 13 specific HPV infections using the PCR assay, and amongst them 430 (20.4% of all PCR-positive women) were infected with multiple types. The observed-to-expected ratio for infection with ⩾2 HPV types was 1.21 (95% Credible Interval: 1.13–1.30). Amongst the 78 combinations of specific HPV types, none of the pairs reached the chosen level of significance, p -value Conclusions Multiple HPV infections occurred more frequently than predicted by chance. The excess of multiple infections was small, though not completely absent, after controlling for all sources of common correlation between HPV types. The present analysis of the NTCC screening study showed no evidence that specific HPV types have the tendency to be found more or less often than others in coinfections.

Oral Diseases17, 572–576 Background: Fanconi’s anemia (FA) is a rare recessive genetic disorder characterized by bone marrow failure, developmental and congenital abnormalities, which frequently evolves to aplastic anemia and neoplasias, primarily acute leukemia and head-neck carcinomas. Risk of malignancies increases after hematopoietic stem cell transplantation (HSCT), and the role of human papillomavirus (HPV) in FA carcinogenesis have been proposed. Objective: To investigate prevalence of oral HPV in FA patients without oral malignant lesions. Materials and methods: After oral examination, 76 subjects without detectable oral malignant lesions were included and classified in four groups: 20 FA submitted to HSCT (I), 22 FA not submitted to HSCT (II), 18 severe aplastic anemia (SAA) submitted to HSCT (III) and 16 healthy subjects (IV). Liquid-based cytology sampling, HPV screening by polymerase chain reaction and genotyping by reverse hybridization were performed. Results: The HPV detection rates were: group I 35%, group II 27.3%, group III 38% and group IV 6.25%. Prevalence of high risk HPV types, mainly HPV16, was detected. Compared with control group, suggestions for increased likelihood of being HPV infected in SAA (OR = 9.55, 95% CI: 1.01–125.41) and FA patients submitted to HSCT (OR = 8.08, 0.83–72.29) emerged. Conclusion: Patients without oral malignant lesions submitted to HSCT, have high prevalence of oral HPV. HPV screening and close follow up should be considered in these patients.

Summary Background Human papillomavirus (HPV) testing is known to be more sensitive, but less specific than cytology for detecting cervical intraepithelial neoplasia (CIN). We assessed the efficacy of cervical-cancer screening policies that are based on HPV testing. Methods Between March, 2004, and December, 2004, in two separate recruitment phases, women aged 25–60 years were randomly assigned to conventional cytology or to HPV testing in combination with liquid-based cytology (first phase) or alone (second phase). Randomisation was done by computer in two screening centres and by sequential opening of numbered sealed envelopes in the remaining seven centres. During phase one, women who were HPV-positive and aged 35–60 years were referred to colposcopy, whereas women aged 25–34 years were referred to colposcopy only if cytology was also abnormal or HPV testing was persistently positive. During phase two, women in the HPV group were referred for colposcopy if the HPV test was positive. Two rounds of screening occurred in each phase, and all women had cytology testing only at the second round. The primary endpoint was the detection of grade 2 and 3 CIN, and of invasive cervical cancers during the first and second screening rounds. Analysis was done by intention to screen. This trial is registered, number ISRCTN81678807. Findings In total for both phases, 47 001 women were randomly assigned to the cytology group and 47 369 to HPV testing. 33 851 women from the cytology group and 32 998 from the HPV-testing group had a second round of screening. We also retrieved the histological diagnoses from screening done elsewhere. The detection of invasive cervical cancers was similar for the two groups in the first round of screening (nine in the cytology group vs seven in the HPV group, p=0·62); no cases were detected in the HPV group during round two, compared with nine in the cytology group (p=0·004). Overall, in the two rounds of screening, 18 invasive cancers were detected in the cytology group versus seven in the HPV group (p=0·028). Among women aged 35–60 years, at round one the relative detection (HPV vs cytology) was 2·00 (95% CI 1·44–2·77) for CIN2, 2·08 (1·47–2·95) for CIN3, and 2·03 (1·60–2·57) for CIN2 and 3 together. At round two the relative detection was 0·54 (0·23–1·28) for CIN2, 0·48 (0·21–1·11) for CIN3, and 0·51 (0·28–0·93) for CIN2 and 3 together. Among women aged 25–34 years, there was significant heterogeneity between phases in the relative detection of CIN3. At round one the relative detection was 0·93 (0·52–1·64) in phase one and 3·91 (2·02–7·57) in phase two. At round two the relative detection was 1·34 (0·46–3·84) in phase one and 0·20 (0·04–0·93) in phase two. Pooling both phases, the detection ratio of CIN2 for women aged 25–34 years was 4·09 (2·24–7·48) at round one and 0·64 (0·23–1·27) at round two. Interpretation HPV-based screening is more effective than cytology in preventing invasive cervical cancer, by detecting persistent high-grade lesions earlier and providing a longer low-risk period. However, in younger women, HPV screening leads to over-diagnosis of regressive CIN2. Funding European Union, Italian Ministry of Health, Regional Health Administrations of Piemonte, Tuscany, Veneto and Emilia-Romagna, and Public Health Agency of Lazio.

Summary Background Human-papillomavirus (HPV) testing is more sensitive, but less specific, than conventional cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We assessed whether HPV testing with triage by p16-INK4A overexpression can increase specificity while maintaining sensitivity. Methods HPV-positive women were enrolled between June 10, 2003, and Dec 31, 2004 in a multicentre randomised controlled trial, which compared stand-alone HPV testing by Hybrid Capture 2 (experimental group) with conventional cytology, were referred for colposcopy. In seven of nine centres, cytospin preparations from these women were tested for p16-INK4A overexpression by immunostaining. The sensitivity and specificity for CIN grade 2 or more, determined at blind review of histology, were calculated for these women. We also estimated the relative sensitivity and relative referral to colposcopy that would have been obtained by HPV testing with p16-INK4A triage compared with conventional cytology. This trial is registered as a Standard Randomised Controlled Trial, number ISRCTN81678807. Findings 24 661 women were randomly assigned to the experimental group. 1137 women (74% of those undergoing colposcopy in relevant centres), including 50 with CIN2 and 42 with CIN3 or cancer, had valid p16-INK4A immunostaining. For the endpoint of CIN2+, sensitivity and specificity of p16-INK4A (deemed positive with any number of stained cells—except endocervical, metaplastic, and atrophic cells if morphologically normal) in HPV-positive women of any age were 88% (81 of 92; 95% CI 80–94) and 61% (633 of 1045; 57–64), respectively. In the 35–60-year age group, the relative sensitivity of HPV testing and p16-INK4A triage versus conventional cytology for CIN2+ was 1·53 (95% CI 1·15–2·02) and relative referral was 1·08 (0·96–1·21). In the 25–34-year age group, relative sensitivity was 3·01 (1·82–5·17) and relative referral was 1·15 (0·96–1·37). In the latter age group, when 5% or more stained cells were deemed positive, the corresponding values were 2·06 (1·20–3·68) and 0·58 (0·46–0·73), respectively. Interpretation HPV testing with p16-INK4A triage produces a significant increase in sensitivity compared with conventional cytology, with no substantial increase in referral to colposcopy. Funding European Union, Italian Ministry of Health, Regional Health Administrations of Piemonte, Tuscany, Veneto, and Emilia-Romagna, and Public Health Agency of Lazio Region.

Purpose: Surgical margin status is reported to be a relevant prognostic factor in head and neck squamous cell carcinoma (HNSCC), associated with a high risk of local recurrence. This study examines whether gene-promoter hypermethylation could be detected in HNSCC surgical margins with no histologic evidence of malignancy, and if so, whether it reflects epigenetic events of primary tumors. Experimental Design: Promoter methylation status of MGMT, p16, and DAP-K genes was evaluated by methylation-specific PCR in 20 primary HNSCC tumors. Histopathologically negative surgical margins of hypermethylated tumors were collected, and their methylation status compared with the primary tumor status. Results: Promoter hypermethylation in at least one of the three tested genes was detected in 65% (13 of 20) of tumors. MGMT was hypermethylated in 50% (10 of 20), DAP-K in 45% (9 of 20), and p16 in 20% (4 of 20) of tumors. Methylation status was analyzed in 35 margins from 11 of 13 patients showing promoter hypermethylation in the tumor tissue. Identical methylation events were seen for at least one gene in primary tumor and surgical margins in 9 of 11 cases (82%). Association was found for gene-specific hypermethylation status in tumors and paired surgical margins, and gene-specific concordance was 63% for MGMT (κ = 0.24), 90% for DAP-K (κ = 0.74), and 90% for p16 (κ = 0.79). Conclusions: Our results support the hypothesis that detection of gene promoter hypermethylation in HNSCC tumor cells–free surgical margins may be a helpful biomarker to identify molecularly altered fields in areas adjacent to the tumor.

The triage of human papillomavirus (HPV)-positive women is needed to avoid overreferral to colposcopy. p16(INK4a) immunostaining is an efficient triage method. p16(INK4a) /Ki-67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand-alone p16(INK4a) staining.Within a pilot project, HPV-positive women were referred to colposcopy if cytology was abnormal or unsatisfactory or HPV testing was still positive after 1 year. For 500 consecutive women, a slide obtained during colposcopy was immunostained for p16(INK4a) /Ki-67 and independently interpreted by 7 readers without previous experience with dual staining. Four of these readers were experts in cervical pathology and 3 were not. Kappa values for multiple raters, sensitivity, and specificity for cervical intraepithelial neoplasia type 2-positive histology were computed. Because women with normal cytology were underrepresented, estimates for all HPV-positive women were obtained as weighted means of cytology-specific estimates.The overall kappa for HPV-positive women was 0.70 (95% confidence interval [95% CI], 0.60-0.77). Kappa values were not found to be significantly different between expert and nonexpert readers with regard to cervical cytology but were significantly increased (P =. 0066) after consensus discussion. The overall specificity estimate for HPV-positive women was 64.0% (95% CI, 57.4%-70.2%): 66.7% (95% CI, 59.8%-73.0%) for experts and 60.5% (95% CI, 59.8%-73.0%) for nonexperts. Among women with abnormal cytology, the sensitivity was 85.5% (95% CI, 77.9%-90.8%): 85.8% (95% CI, 77.9%-91.2%) for experts and 85.1% (95% CI, 76.6%-90.9%) for nonexperts.p16(INK4a) /Ki-67 immunostaining demonstrated good reproducibility and specificity when triaging HPV-positive women. Dual-staining interpretation can be performed, after short training, even by staff who are not experts in cervical cytology. This allows HPV-based screening with triage to be performed in settings in which such expert staff is not available.

Within a large Italian randomized trial on new technologies for cervical cancer screening involving 7 laboratories with different levels of experience, an intralaboratory and interlaboratory quality control program for human papillomavirus (HPV) DNA testing by Hybrid Capture 2 (HC2; Digene, Gaithersburg, MD) was implemented. To monitor the hybridization and detection steps, target samples containing purified, concentration-defined, HPV DNA were introduced in each test run. Only 3 of 1,024 showed a mistake in a positive vs negative classification with a 1 relative light unit (RLU)/positive control specimen (PC) ratio cutoff. To monitor the preanalytic steps (particularly denaturation), blinded specimens (33 collected in PreservCyt [Cytyc, Boxborough, MA] and 36 in Specimen Transport Medium [STM, Digene]) were centrally prepared, divided into aliquots, and sent to each laboratory. The multiple-rater κ scores for negative (

Background: Age- and type-specific high-risk human papillomavirus (hrHPV) incidence estimates in screen-eligible women are relevant from a public health perspective because they provide an indication of the effect of vaccination on the occurrence of screen-positives in HPV-based screening. However, limited data from women over 25 years of age are available. Methods: In 24,105 hrHPV-negative women participating in Dutch (Population-Based Screening Study Amsterdam: POBASCAM) and Italian (New Technologies for Cervical Cancer: NTCC) population-based randomized controlled screening trials the age- and type-specific distribution of incident hrHPV infections detected at the next screening round was assessed. HPV types were grouped into vaccine (bivalent: HPV16/18; polyvalent HPV16/18/31/33/45/52/58) and nonvaccine types. Results: The incidence of screen-detected hrHPV among women ages 29 to 56 years was 2.54% (95% confidence interval, 2.30–2.78) in POBASCAM and 2.77% (2.36–3.19) in NTCC. In both studies, the incidence of bivalent, polyvalent, and nonpolyvalent infections decreased with age (P < 0.0001). Among women with incident infection(s), vaccine-type positivity changed quadratically with age, in particular for the polyvalent vaccine (P values: POBASCAM: bivalent 0.264, polyvalent 0.038; NTCC bivalent 0.039, polyvalent 0.005). However, more than 20% and 50% of women with incident hrHPV were positive for bivalent and polyvalent vaccine types, respectively, in all ages in both studies. Conclusions: We observed decreasing age trends of hrHPV vaccine and nonvaccine type incidences and age-related differences in the vaccine-type positivity among women with incident infections. Most importantly, hrHPV infections continued to be detected in all ages and the contribution of vaccine types remained substantial. Impact: Our results indicate a considerable reduction of new hrHPV infections in vaccinated cohorts, ensuing revision of screening guidelines. Cancer Epidemiol Biomarkers Prev; 24(1); 111–8. ©2014 AACR.

Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays a role in cellular response to alkylating agents. In the present study aimed to: (i) evaluate the concordance between MGMT promoter methylation status in tumor tissue and plasma; (ii) monitor MGMT promoter methylation status in plasma taken before and during temozolomide treatment; (iii) explore the value of MGMT promoter methylation status in plasma as a prognostic/predictive biomarker in glioma patients. We enrolled 58 patients with histologically confirmed glioma at different grades of malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-embedded tumor tissue was available for 48 patients. Blood samples were collected from all patients before temozolomide treatment (baseline) and at each MRI examination for a 12-month period. MGMT promoter methylation status was assessed in both sample types by real time PCR with a specific probe. The frequency of MGMT promoter methylation was 60.4 % in tumor tissue and 41.38 % in plasma. MGMT promoter methylation status was concordant in the two sample types (Kappa = 0.75, 95 % confidence interval (CI) 0.57–0.93; p value

The prevalence of infections with human papillomavirus (HPV) specific genotypes differs by age and areas. Knowledge of these differences will help predicting how prophylactic HPV vaccination and screening program could best be integrated.To investigate variations in the HPV distribution between areas and ages in Italy and the impact of vaccination on HPV prevalence.37,367 women aged 25-60 years who attended cervical screening in eight different areas in Northern and Central Italy were tested for HPV infection with the high-risk hybrid capture (hr-HC2) assay. hr-HC2 positive samples were genotyped by an intensive integrated strategy.hr-HPV types were detected in 79.1% of HC2 positive women. HPV16 was the most frequent type, followed by HPV31, HPV18 and HPV56. A statistically significant variability in HPV type distribution between centres (overall χ84df(2)=195.86p0.001) was observed. No significant overall difference in the HPV type distribution was observed in the age groups 25-34, 35-44 and 45-60 years. Considering cross-protection, overall 57.6% (95%CI 56.0-59.3) of all infections by hr-HPV types was preventable by vaccination with the bivalent vaccine and 49% (95%CI 46.9-51.1) with the quadrivalent vaccine. The variability between centres was statistically significant with both bivalent (χ7df(2)=43.8, p0.0001) and quadrivalent vaccine (χ7df(2)=32.9, p0.0001).We observed differences in HPV genotype distribution according to centres but not to age. Results suggest that the higher proportion of HPV16/18 related high grade CIN in younger women could be the result of faster progression and not of earlier infection by these types.

Background/objectives It has been suggested that subfertility and testicular cancer share genetic and environmental risk factors. We studied both subfertility and the strongest known testicular cancer susceptibility gene, the c-KIT ligand (KITLG), whose pathway is involved in spermatogenesis. Methods The EPSAM case-control study is comprised of testicular cancer patients from the Province of Turin, Italy, diagnosed between 1997 and 2008. The present analysis included 245 cases and 436 controls from EPSAM, who were aged 20 years or older at diagnosis/recruitment. The EPSAM questionnaire collected information on factors such as number of children, age at first attempt to conceive, duration of attempt to conceive, use of assisted reproduction techniques, physician-assigned diagnosis of infertility, number of siblings, and self-reported cryptorchidism. Genotyping of the KITLG single nucleotide polymorphism (SNP) rs995030 was performed on the saliva samples of 202 cases and 329 controls. Results Testicular cancer was associated with the number of children fathered 5 years before diagnosis (odds ratio (OR) per additional child: 0.78, 95% confidence interval (CI): 0.58–1.04) and sibship size (OR per additional sibling: 0.76, 95% CI: 0.66–0.88). When considering the reproductive history until 1 year before diagnosis, attempting to conceive for at least 12 months or fathering a child using assisted reproduction techniques was not associated with the risk of testicular cancer, nor was age at first attempt to conceive or physician-assigned diagnosis of infertility. The SNP rs995030 was strongly associated with risk of testicular cancer (per allele OR: 1.83; 95%CI: 1.26–2.64), but it did not modify the association between number of children and the risk of testicular cancer. Conclusion This study supports the repeatedly reported inverse association between number of children and risk of testicular cancer, but it does not find evidence of an association for other indicators of subfertility.

To explore the feasibility of designing vaccination protocols in acute leukemia patients with cytokine gene-transduced leukemic cells, we studied in vitro the growth potential of human leukemic cells transduced with the interleukin-2 (IL-2), IL-7, or IL-7 plus IL-2 genes, as well as the capacity of generating both autologous and allogeneic cytotoxic lymphocytes directed against the parental cells. A lymphoblastic T-cell line, ST4, obtained from a patient in long-lasting complete remission, was retrovirally engineered with the IL-2, IL-7, and IL-7 plus IL-2 genes; in addition, clones releasing different amounts of the cytokines were obtained by limiting dilution. Mixed lymphocyte-tumor cultures (MLTCs) were set up with parental or transduced leukemic cells as stimulators and with autologous or allogeneic lymphocytes as responders. When nonirradiated ST4 parental cells or clones producing50 international units (IU)/mL/10(6) cells/72 hours of IL-2 were used as stimulators, leukemic overgrowth was observed in MLTCs within 16 days of culture. When clones producing80 IU/mL/10(6) cells/72 hours of IL-2 were used as stimulators, the proliferation of leukemic cells was blocked and the transduced leukemic cells were completely cleared from the cultures by day 16; repeated restimulations with IL-2-producing leukemic cells were required to sustain long-term lymphocyte survival. On the contrary, when IL-7- or IL-7-IL-2-producing cells were used as stimulators, only a delay in leukemic cell overgrowth was observed, and lymphocytes were completely cleared from the cultures after day 60. IL-7 production by the different clones ranged between 11 and 36 ng/mL/10(6) cells/72 hours, whereas the highest IL-2-producing IL-7-IL-2 clone released 50 IU/mL/10(6) cells/72 hours of IL-2. When the stimulator efficacy of the highest IL-2-producing clone (ST4/IL-2#A7) was compared with that of exogenous IL-2 plus parental cells, a 7-fold higher amount of exogenous IL-2 was required to achieve the same results obtained with IL-2-producing leukemic cells. Autologous and allogeneic long-term MLTCs (up to 35 days) with ST4/IL-2#A7 as the stimulator were capable of generating cytotoxic effectors equally endowed with both major histocompatibility complex (MHC) class I-unrestricted and -restricted activity against parental ST4 cells. By day 18 of both autologous and allogeneic cultures, a substantial proportion of CD56+ cells was consistently recorded; this was coupled to a predominantly MHC-unrestricted cytotoxic activity directed against parental ST4 cells. CD56+ cells decreased considerably at the end of the different MLTCs, together with the unrestricted cytotoxic activity. At this time,50% of the cells were CD8+, and 55% of the activity could be blocked by an anti-MHC class I monoclonal antibody. The results of this study demonstrate that IL-2 gene-transduced human acute leukemia cells cocultured with both autologous and allogeneic lymphocytes are capable of inducing a strong MHC-unrestricted anti-leukemic activity and subsequently "educating" MHC class I-restricted anti-leukemic effectors. The evidence that the immunogenic potential of human leukemic blasts can be boosted after transfer of the IL-2 gene suggests that the possibility of using leukemic cells engineered to release IL-2 as a therapeutic vaccine needs to be explored further.

The Hybrid Capture 2 (HC2) test targets 13 human papillomavirus (HPV) types. Here, cross-reactivity with non-HC2-targeted HPV types is described. We aimed to define the proportion of HC2-positive women who had negative results with HC2-targeted HPV types and estimate its determinants and impact on women's health management. The New Technologies for Cervical Cancer (NTCC) trial was followed in two predetermined phases. Women in the experimental arm were tested for the presence of HPV DNA by HC2 following a sample collection in PreservCyt (first phase) or Digene specimen transport medium (STM) (second phase). HPV genotyping was performed on DNA samples from HC2-positive women by PCR with GP5 + /GP6 + primers and reverse line blot (RLB) hybridization. Untyped samples were submitted to direct sequencing or restriction fragment length polymorphism. Multivariate logistic regression analysis estimated the adjusted odds ratios (ORs) between the presence of HC2-targeted types and age, viral load, and type of transport medium. Out of 2,920 HC2-positive samples, 2,310 (79.1%) were positive on RLB for HC2-targeted types, 396 were positive (13.6%) for only non-HC2-targeted types (mostly represented by HPV-53, HPV-66, and HPV-70), and in 214 (7.33%) samples, no HPV types were detected. The probability of detecting HC2-targeted types increased with increasing viral load expressed as the relative light unit/positive-control specimen ratio (RLU/PC) (OR for unitary increase of log RLU/PC, 1.35; 95% confidence interval [CI], 1.30 to 1.42) and with STM versus PreservCyt (OR, 1.56; 95% CI, 1.25 to 1.84). If only the samples containing HC2-targeted types tested positive, the positive predictive value (PPV) would have increased from 7.0% (95% CI, 6.1% to 8.0%) to 8.4% (95% CI, 7.3 to 9.6), although 4.9% (95% CI, 2.4% to 8.8%) of cervical intraepithelial neoplasia grade 2 + (CIN2 + ) cases would have been missed. In conclusion, STM use and an increased cutoff would reduce the HC2 analytical false-positive rate and increase the positive predictive value for high-grade CIN. The gain in clinical sensitivity by detecting non-HC2-targeted HPV types is limited.

Background Although among women a decreasing prevalence of human papillomavirus (HPV) infection with increasing age has been consistently observed in high-resource countries, different age profiles have been reported elsewhere. Methods We compared the age profile of high-risk (HR)-HPV prevalence in nine different areas of Northern and Central Italy by studying the women recruited in the intervention arm of the New Technologies in Cervical Cancer study and tested by Hybrid Capture 2. Differences in the age-distribution of HPV infection were investigated in each centre by the joinpoint approach in a logistic model. 46,900 women aged 25 to 60 years were included in the analysis. Results The HR-HPV age-standardised (on Italian population) prevalence ranged from 5.7% (Trento) to 10.3% (Ravenna). HR-HPV prevalence decreased as a logistic function of increasing age in 6 of 9 centres (Trento, Verona, Florence, Bologna, Imola, and Viterbo). The effect of age on HR-HPV prevalence slopes did not differ significantly among these 6 centres, whereas significant heterogeneity in intercepts (p

Summary Background Immunostaining for p16-INK4A (henceforth p16) is a sensitive and specific method for detection of high-grade cervical intraepithelial neoplasia (CIN) in women infected with human papillomavirus (HPV), but longitudinal data have not been obtained. We investigated the relation between p16 status and risk of CIN during 3 years of follow-up. Methods Women aged 25–60 years were enrolled between June 10, 2003, and Dec 31, 2004, in a multicentre randomised trial comparing HPV testing with cytology. HPV-positive women were referred for colposcopy and, in seven of nine centres, were tested for p16 overexpression by immunostaining. If no CIN was detected, these women were followed up at yearly intervals until clearance of HPV infection. The primary endpoint was histologically confirmed CIN of grade 2 or worse (CIN of grade 2 [CIN2], CIN of grade 3 [CIN3], or invasive cervical cancer) at recruitment or during follow-up. We calculated the absolute and relative risks by p16 status at recruitment. We also calculated the longitudinal sensitivity of p16 testing. Additionally, we assessed the relative sensitivity of an alternative strategy (referral to colposcopy and follow-up of only HPV-positive, p16-positive women) versus conventional cytology in two age groups. Percentages were weighted by the inverse of the tested fraction. The trial in which this study is nested is registered, number ISRCTN81678807. Findings Of 1042 HPV-positive women who were tested for p16 with no CIN detected during the first round of screening, 944 (91%) had further HPV tests. 793 (84%) of these 944 were followed up until detection of CIN2 or worse, HPV infection clearance, or for at least 3 years. CIN2 or worse was detected during follow-up in more p16-positive women (31 of 365, 8·8% [95% CI 5·8–11·8]) than in p16-negative women (17 of 579, 3·7% [1·9–5·4]; relative risk [RR] 2·61 [95% CI 1·49–4·59]). RR was higher in women aged 35–60 years at recruitment (3·37 [1·39–8·15]) than in those aged 25–34 years (2·15 [1·00–4·61]), but age was not a significant modifier. CIN3 or worse was detected during follow-up in more p16-positive women (16 of 365, 4·4% [2·3–6·6]) than in p16-negative women (six of 579, 1·3% [0·2–2·3]; RR 3·90 [95% CI 1·57–9·68]). Longitudinal sensitivity of p16 testing for detection of CIN3 or worse during follow-up at all ages was 77·8% (95% CI 63·9–91·6). The relative sensitivity of the alternative strategy compared with conventional cytology was 2·08 (1·13–3·56) in women aged 35–60 years and 2·86 (1·28–5·36) in those aged 25–34 years. HPV-positive, p16-negative women aged 35–60 years had a higher cumulative risk of CIN3 or worse during recruitment or follow-up (2·0%, 95% CI 0·3–3·7) than did HPV-negative women (0·01%, 0–0·04) or those who were cytologically normal (0·04%, 0·02–0·09) at recruitment. Interpretation p16 overexpression is a marker for CIN2 or worse or for development of CIN2 or worse within 3 years in HPV-positive women, especially those aged 35–60 years. HPV-positive, p16-positive women need immediate colposcopy and, if the assessment is negative, annual follow-up. Immediate colposcopy can be avoided in HPV-positive, p16-negative women, who can be safely managed with repeat screening after 2–3 year intervals. Funding European Union; Italian Ministry of Health; Regional Health Administrations of Piemonte, Tuscany, Veneto and Emilia Romagna; and Public Health Agency of Lazio Region.

Cervical cancer (CC) is caused by oncogenic human papillomaviruses (HR-HPVs) infection and exhibits well-defined clinical stages associated with different steps of tumorigenesis. Screening for CC based on HPV is more effective than cytology, but it may increase costs and over treatment of spontaneously regressive high-grade lesions (hgCIN). Thus triage of HPV positive women is needed. Several microRNAs (miRNAs) are dysregulated in CC and hgCIN. However, the studies so far are based on miRNA candidate genes or array approach, mainly in tumor/healthy tissues. The aim of this study is to investigate, for the first time, miRNA profiles by next-generation sequencing in exfoliated cervical cells of HPV-positive women in relation to the presence of hgCIN lesions or as predictors of persistence/progression of CIN lesions. The study is nested in a large Italian multi-centre randomised controlled trial recruiting women in population-based screening programs that actively invite women aged 25-64 years. The study population includes HPV-positive women with CIN2 and CIN3 or without hgCIN lesion. For the discovery phase libraries of 100 samples of exfoliated cervical cells have been set up for miRNA sequencing. For the validation an additional set of 250 samples has already been selected. Preliminary results shows that, after correction for multiple testing, 44 miRNAs resulted dysregulated in women with vs. those without prevalent hgCIN. This set includes some miRNAs previously reported in the literature, such as miR-100 and miR-126, and other additional miRNAs not included in arrays before. The association of miRNAs with occurrence of hgCIN and clearance of infection during follow up reveals other significantly differentially expressed miRNAs under investigation for their biological roles. The high stability of miRNAs, in contrast to the fast degradation of mRNA and proteins, allows their accurate determination also in samples of exfoliated cervical collected during screening. Validation of the results, associations with other investigated markers and with HPV genotypes will be performed. Study supported by Italian Association on Cancer Research (AIRC, IG2013 N.14119). Citation Format: Alessio Naccarati, Daniela De Maria, Francesca Cordero, Barbara Pardini, Stavrula Gouzounis, Maddalena Arigoni, Raffaella Rizzolo, Laura De Marco, Anna Gillio-Tos, Paolo Vineis, Guglielmo Ronco. miRNA as markers of CIN risk and persistence for optimization of HPV-based cervical cancer screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-366.

OBJECTIVE OF THE PROJECT: The introduction of the HPV test as a primary screening test will cause important changes in the screening system based on cytology. The purposes of this report are: to define the best screening policies with HPV-based screening on the basis of the resulting efficacy and of undesired effects; comparing them to cytology-based screening; to identify their best conditions of application; to evaluate economic cost, feasibility and impact on the organisation of services of such policy in the Italian situation.This report contains a section on efficacy and undesired effects based on a systematic review of literature conducted in strict coordination with the preparation of a supplement to the European Guidelines for quality assurance in cervical cancer screening. This chapter corresponds to a preliminary version of the chapter of the European Guidelines on primary screening with HPV. The sections on costs, impact on organisation, and social, ethical and legal impact reflect the Italian situation; they are based on a review of the available Italian data (including unpublished data, mainly from on-going pilot projects) and on a structured analysis of what will result if the proposed protocol is applied to the Italian situation.Efficacy and undesired effects. There is clear scientific evidence that a screening based on validated tests for the DNA of oncogenic HPV as primary test and applying an appropriate protocol is more effective than screening based on cytology in preventing invasive cancers of the uterine cervix. In addition, it entails a limited--if any--increase of the undesired effects both in terms of unneeded referral to diagnostic work-up and in terms of over-diagnosis and consequent overtreatment of spontaneously regressive lesions. The crucial elements of such protocol are the followings: HPV-positive women are not to be directly referred to colposcopy, but the use of triage systems is essential. The currently recommendable method is based on performing cytology in HPV positive women. If the result of this test is abnormal, the woman is immediately referred to colposcopy; if cytology is normal, the woman is invited to repeat a new HPV test after one year. In case such a test is still positive, the woman is referred to colposcopy; in case of negative result, the woman will be re-invited for a new screening round at the regular interval. In organised population-based screening programmes the interval after a negative primary HPV test should be at least 5 years. There is evidence that the 5-year cumulative risk of high-grade CIN after a negative HPV test is lower than the 3-year risk after a normal cytology. On the other hand, the probability of unneeded colposcopies and treatments would plausibly be relevant with 3-year intervals after a negative HPV test. HPV-based screening should not start before 30-35 years. There is evidence that below 30 years HPV-based screening leads to an increased overdiagnosis of CIN2 that would regress spontaneously, with consequent overtreatment. Some increase in overdiagnosis is plausible also between 30 and 34 years. Below such ages, cytological screening is the recommended test. Only tests for the DNA of oncogenic HPV, validated according to the European guidelines as for sensitivity and specificity for high-grade lesions, should be applied. There is no evidence that double testing with cytology and HPV is more protective than stand-alone HPV as primary test, although it entails a small and not relevant increase in sensitivity vs stand-alone HPV. On the contrary, there is evidence that double testing causes a substantial increase in referral to colposcopy and a decrease in its PPV. For this reason, if HPV is used as primary screening test, it is recommended not to add cytology in parallel. Cost and economic evaluation. It is estimated that, if the protocol described is applied, in the current Italian situation the overall costs of HPV-based screening are lower than those of conventional cytological screening applied at the current 3-year intervals, although the cost of each screening round is higher. Impact on organization. For reasons of quality and cost, both the interpretation of cytology and HPV testing require a centralisation. This need is particularly strong, in terms of costs, for HPV test execution. It is therefore recommended to perform the HPV test in a limited number of reference laboratories of large size. This also makes monitoring and evaluating the spontaneous activity easier. HPV-based screening entails problems of organisation related to the need of triage, to complex protocols and to reconversion of the activities of cytological interpretation. Social, ethical and legal impact. The communication of the result of the HPV test to women, particularly if positive, is a further crucial aspect in order to reduce not only the emotional impact, but also the possible risks that women are inappropriately managed or lost to follow-up. Great efforts must be put in the education of healthcare professionals, both directly involved in organised programmes or not, particularly private gynaecologists and general practitioners.In conclusion, the crucial requirement to introduce HPV-based screening programmes is the capacity to guarantee the application of appropriate screening protocols. If protocols do not respect the criteria described above they can cause relevant increase of undesired effects and costs compared to cytology-based screening. Therefore they should be avoided, except in studies able to provide clear evidence about human and economic costs. For this purpose, correct education and information both to healthcare professionals and to the population is needed. In the Italian situation, where organised screening and a relevant spontaneous activity coexist, their interaction is crucial. Actions directed to integrate them and to guarantee as more uniformity of interventions as possible are needed, in particular through the integration of registries and thorough monitoring and a progressive homogenization of protocols. In order to grant the safety of transition, it is needed that the HPV-based organised screening activities are strictly monitored and that the National Centre for Screening Monitoring (ONS) ensures coordination. Knowledge about HPV based screening is still rapidly evolving. It is possible that currently on-going researches suggest changes to the optimal protocols in the next few years, particularly as for the management of HPV positive women. In addition, studies on the validation of new assays were recently published and others are expected. It is suggested to exploit the organised screening activity to produce scientific evidence, in order to clarify the still uncertain aspects of optimal protocols. Different protocols in terms of screening intervals, age of application and management of HPV positive women should be studied in the frame of controlled implementation, through multicentre projects coordinated by ONS. Finally, it is suggested the creation of a National working group to promptly update the recommendations for screening and the list of assays to be considered as validated. On the bases of the results obtained in the first vaccinated cohorts reaching the screening age, for the future, it will be crucial to deliver specific recommendations to the population vaccinated against HPV during adolescence.

causal relation between high risk Papilloma virus and cervical carcinoma is definitely ascertained. HPV test is suggested both as primary screening test and as triage test for selecting women who should undergo colposcopy examination. Evidence is clear for triage after ASC-US cytology. A recent study suggested the implementation after LSIL cytology in women 35 or older but the issue is controversial.We present a pilot study on the implementation of HPV test triage in the framework of cervical cancer screening. The study was conducted in respect to: participation, predictive value, and cost analysis, separately for ASC-US and LSIL cytology.HPV test was offered to women with Pap test result ASC-US or LSIL (35 and over), as an add-on to the "Prevenzione serena Screening Program" protocol. All samples were analyzed in the same specialized laboratory. HPV positive women were invited to colposcopy, negative will be invited at the scheduled interval for negative screening tests.Piedmont (NW Italy), LHAs of Novara and Verbano. In the 1-year study period 15,614 Pap tests were performed: 153 women were eligible for the triage.Participation at HPV test, HPV test results, costs.ninety two percent of women invited to HPV test actually participated: 66.9% of them were positive (52.8% after ASC-US and 82.8% after LSIL). Regarding colposcopy and histological results, CIN2+ were 9.4% of positive HPV tests in ASC-US group and 17.1% in LSIL group. Cost analysis showed limited differences between triage (offered after ASC-US and LSIL cytology) and traditional protocol. Triage is economically convenient when limited to women with ASC-US cytology.the pilot study demonstrated the feasibility of adding a triage phase with HPV test in cervical cancer screening protocol. Additional cost is balanced by the saving due to the reduction in the number of colposcopy exams: the process is economically convenient when limited to women with ASC-US cytology. When extended to LSIL cytology the marginal cost increases because of the higher prevalence of HPV positive results and total cost of triage with HPV test is close to the cost of immediate colposcopy referral.

This study compares colposcopy referrals of 2 management strategies: oncogenic human papillomavirus (HPV)-DNA testing (Hybrid Capture 2 assay, Qiagen, Germantown, MD) and repeat cytology. In the New Technology in Cervical Cancer Trial, 22,708 subjects were randomly assigned to undergo both HPV and liquid-based cytologic testing. Women aged 35 to 60 years old with unsatisfactory cytologic findings were directly referred for colposcopy if the HPV test result was positive, and were referred for repeat cytologic examination if the HPV test result was negative; women aged 25 to 35 years old were referred for repeat cytologic examination independent of HPV test results. A positive or a second unsatisfactory cytologic examination referred women for colposcopy. Five hundred sixty women had unsatisfactory cytologic findings. Colposcopy referral was not significant and slightly higher with HPV testing than repeat cytologic test (9.8% vs 6.8%, P = .11). When cytologic testing was repeated 36.8% were unavailable for follow-up and most of the colposcopies were performed in HPV-negative women. For unsatisfactory cytologic findings, HPV triage is a more logical and efficient management strategy than a repeat cytologic test.

Aberrant DNA methylation plays a role in prostate cancer progression. We studied the relationships among DNA methyltransferase (DNMT) genotype, DNA methylation, Gleason score, and mortality in two cohorts of prostate cancer patients, previously reported with associations between DNA methylation in GSTP1, APC, and RUNX3 and prostate cancer mortality. Herein, we considered possible causal relationships between the studied variables, assuming that (1) DNMT activity affects tumor tissue methylation, (2) methylation status affects tumor morphology, and thus the Gleason score, and (3) DNA methylation affects mortality via Gleason score.The cohorts comprised 438 patients diagnosed at one Italian pathology ward before 1997, with DNA obtained from paraffin-embedded tumor tissues. The polymorphism rs406193 in the DNMT3b gene was assessed by allele discrimination in real-time PCR. According to the assumed causal model, we analyzed the effects of rs406193 (T carriers vs others) on the Gleason score without adjusting for gene methylation, and the effects of rs406193 on gene methylation and prostate cancer mortality without adjusting for Gleason score.We found no evidence of association between T carriers and the number of methylated genes. However, T carriers had reduced risk of a Gleason score 8+ (odds ratio = 0.57, 95 % CI 0.39-0.85), and a hazard ratio of 0.81 (0.61-1.09) of dying from prostate cancer, which would have been erroneously estimated of 0.93 if adjusted for Gleason score.These findings provide clues on the role of a DNMT3b SNP in prostate cancer progression and illustrate the importance of considering possible causal relationships in the analyses.

Hepatitis C virus (HCV) and Epstein-Barr virus (EBV) have been repeatedly associated with risk of non-Hodgkin's lymphoma (NHL) in studies focusing on serological evidence of infection. We investigated NHL risk in association with detection of HCV-RNA or EBV-DNA in the peripheral blood mononuclear cells (PBMC). The study involved 91 NHL cases and 182 controls nested in the Italian branch of the EPIC (European Prospective Investigation of Cancer and nutrition) cohort, which obtained blood samples from 47,749 healthy volunteers between 1993 and 1998 in 5 Italian cities. NHL cases were identified until June 2005 through linkage with records of the Cancer, Mortality, and Hospital Discharge Registries. For all study subjects, we performed viral genome analyses on DNA and RNA extracted from buffy-coats and analysed EBV and HCV antibodies. The odds ratios (ORs) of NHL were 1.2 (95% confidence intervals: 0.4-3.8; 5 exposed cases) for PBMC HCV infection and 1.2 (0.7-2.3; 24 exposed cases) for PBMC EBV infection. Similar OR estimates were found for detection of EBV and HCV antibodies. These null results, although based on a relatively small sample size, suggest that persistent EBV and HCV infection in the PBMC is not a stronger predictor of NHL risk than serological evidence of infection.

Background Occult Hepatitis C virus (HCV) infection is a new pathological entity characterized by presence of liver disease and absence or very low levels of detectable HCV-RNA in serum. Abnormal values of liver enzymes and presence of replicative HCV-RNA in peripheral blood mononuclear cells are also observed. Aim of the study was to evaluate occult HCV occurrence in a population unselected for hepatic disease. Methodology/Principal Findings We chose from previous epidemiological studies three series of subjects (n = 276, age range 40–65 years) unselected for hepatic disease. These subjects were tested for the presence of HCV antibodies and HCV-RNA in plasma and in the peripheral blood mononuclear cells (PBMCs) by using commercial systems. All subjects tested negative for HCV antibodies and plasma HCV-RNA and showed normal levels of liver enzymes; 9/276 patients (3.3%) were positive for HCV-RNA in PBMCs, identifying a subset of subjects with potential occult HCV infection. We could determine the HCV type for 8 of the 9 patients finding type 1a (3 patients), type 1b (2 patients), and type 2a (3 patients). Conclusions The results of this study show evidence that occult HCV infection may occur in a population unselected for hepatic disease. A potential risk of HCV infection spread by subjects harbouring occult HCV infection should be considered. Design of prospective studies focusing on the frequency of infection in the general population and on the clinical evolution of occult HCV infection will be needed to verify this unexpected finding.