Anna Gillio-Tos

Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial

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.

Results at Recruitment From a Randomized Controlled Trial Comparing Human Papillomavirus Testing Alone With Conventional Cytology as the Primary Cervical Cancer Screening Test

BACKGROUND In the first recruitment phase of a randomized trial of cervical cancer screening methods (New Technologies for Cervical Cancer Screening [NTCC] study), we compared screening with conventional cytology with screening by human papillomavirus (HPV) testing in combination with liquid-based cytology. HPV-positive women were directly referred to colposcopy if aged 35 or older; if younger, they were retested after 1 year. METHODS In the second recruitment phase of NTCC, we randomly assigned women to conventional cytology (n = 24,661) with referral to colposcopy if cytology indicated atypical squamous cells of undetermined significance or more severe abnormality or to testing for high-risk HPV DNA alone by Hybrid Capture 2 (n = 24,535) with referral to colposcopy if the test was positive at a concentration of HPV DNA 1 pg/mL or greater. For the main endpoint of the study, histologic detection of cervical intraepithelial neoplasia of grade 2 or more (CIN2+), we calculated and compared sensitivity and positive predictive value (PPV) of the two screening methods using HPV DNA cutoffs of 1 pg/mL and 2 pg/mL. All statistical tests were two-sided. RESULTS For women aged 35-60 years, the relative sensitivity of HPV testing for detection of CIN2+ at a cutoff of 1 pg/mL vs conventional cytology was 1.92 (95% CI = 1.28 to 2.87) and the relative PPV was 0.80 (95% CI = 0.55 to 1.18). At a cutoff of 2 pg/mL HPV DNA, the relative sensitivity was 1.81 (95% CI = 1.20 to 2.72) and the relative PPV was 0.99 (95% CI = 0.67 to 1.46). In this age group, there was no evidence of heterogeneity between study phases. Among women aged 25-34 years, the relative sensitivity for detection of CIN2+ of HPV testing at a cutoff of 1 pg/mL vs cytology was 3.50 (95% CI = 2.11 to 5.82), statistically significantly larger (P = .019) than that observed in phase 1 at this age (1.58; 95% CI = 1.03 to 2.44). CONCLUSIONS For women aged 35-60 years, HPV testing with a cutoff of 2 pg/mL achieves a substantial gain in sensitivity over cytology with only a small reduction in PPV. Among women aged 25-34 years, the large relative sensitivity of HPV testing compared with conventional cytology and the difference between relative sensitivity during phases 1 and 2 suggests that there is frequent regression of CIN2+ that are detected by direct referral of younger HPV-positive women to colposcopy. Thus, triage test or repeat testing is needed if HPV is to be used for primary testing in this context.

Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial

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.

Risk of high-grade cervical intraepithelial neoplasia during follow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of the NTCC randomised controlled trial

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.

Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples

Aims: Archival fixed paraffin‐embedded tissue (PET) is a valuable source for population‐based molecular genetic studies but the extraction of high quality DNA is still a problematic issue. The present study tested the grade of DNA fragmentation and the DNA adequacy for genetic investigations in a large series of tissue specimens that were formalin‐ or Bouin‐fixed and paraffin‐embedded between 1979 and 1983. Specific aims were to: (1) estimate the amount of archival tissue samples from which DNA is recoverable by conventional methods and the influence of variables (origin, fixative, section size) on DNA recovery; and (2) evaluate the feasibility of genetic investigations in large scale population studies. Methods: DNA was extracted in 2005 from 365 PET samples from Italy and Spain and subjected to PCR analysis targeting fragments of 152, 268 and 676 bp of the &bgr;‐globin gene. Results: Amplification of a 152 bp fragment was obtained in 252/365 (69%) PET samples, a 268 bp fragment in 62/365 (17%), a 676 bp fragment in 19/365 (5%) and no amplification for any fragment was obtained in 113/365 (31%). A second processing of newly cut sections performed in a 25% simple random sample gave comparable results, with substantial concordance between the first and second tests (kappa value 0.62 [95% CI 0.59–0.64]). Conclusions: The results of this study show that DNA can be efficiently extracted from PETs archived for more than 20 years, and that large scale population studies based on PCR amplification of short target sequences are feasible.

Association Between Hypermethylated Tumor and Paired Surgical Margins in Head and Neck Squamous Cell Carcinomas

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.

Hypermethylation, risk factors, clinical characteristics, and survival in 235 patients with laryngeal and hypopharyngeal cancers

It has been established that promoter hypermethylation occurs in several genes during the pathogenesis of head and neck cancer. The authors investigated the role played by the hypermethylation of 4 cancer‐related genes in the survival of patients who had laryngeal and hypopharyngeal cancer and in the occurrence of second primary tumors.

Occult HCV Infection: An Unexpected Finding in a Population Unselected for Hepatic Disease

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.

Concurrent infections with multiple human papillomavirus (HPV) types in the New Technologies for Cervical Cancer (NTCC) screening study

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 <0.01. 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.

High prevalence of oral human papillomavirus infection in Fanconi's anemia patients.

BACKGROUND Fanconis 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.