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

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

Prevalence of HPV infection among clinically healthy Italian males and genotype concordance between stable sexual partners.

BACKGROUND: HPV is one of the most common sexually transmitted infections. However little is known about its prevalence in the male population and concordance with female partners. OBJECTIVES: This cross-sectional study aimed to: (a) investigate HPV prevalence and genotype distribution among a series of stable male sexual partners of CIN/HPV positive women and (b) assess HPV infection and type-specific concordance between partners. STUDY DESIGN: 378 stable and monogamous male partners of CIN/HPV positive women were selected. Of these, 238 cases were enrolled at the same time as their female partner. All the subjects were tested by the Linear Array HPV genotyping assay. RESULTS: Overall, 153/378 men (40.5%) and 122/238 women (51.3%) were positive for at least one of the 37 HPV types detectable by the assay used. Among the HPV-positive participants, 69 of the 378 men (18.2%) and 54 of the 238 women (22.7%) harboured multiple genotypes. 75 couples (31.5%) were concordantly HPV positive, while 102 couples (42.9%) were concordantly negative (Kappa value: 0.491, p<0.0001). Among the couples in which both partners were HPV positive, 68% harboured at least one genotype in common. Results from a GEE model evidenced that when the male partner tested HPV positive for at least one genotype, this had a significant effect on the positivity of their relative female partner (p<0.0001). CONCLUSIONS: We evidenced a high prevalence of HPV male infections and a moderate concordance between partners. However, we observed a significant HPV type-specific correlation between partners, which is unlikely to be coincidental.

Identification of episomal human papillomavirus and other DNA viruses in cytological anal samples of HIV-uninfected men who have sex with men.

To date, there have been only few studies that investigated integration of anal Human Papillomavirus (HPV). Most of them were conducted on HIV-infected individuals and mainly analyzed samples from high-grade lesions and invasive cancer. We aimed to investigate HPV physical status in HIV-negative men who have sex with men (MSM) with a detectable anal HPV infection, irrespective of the presence of lesions. We also sought to explore the presence of other circular DNA viruses in the anal region. Study participants were attendees of an STI screening program, which were also screened for anal HPV infection and cytological abnormalities. HPV physical status was assessed using multiply-primed RCA. HPV16-positive samples were also analyzed using E2/E6 multiplex PCR, qRT-PCR and APOT assay. RCA and virus-specific PCR were employed to investigate the presence of other DNA viruses. Anal HPV infection was detected in 76.9% of the 230 MSM enrolled. The anal cytological reports were: 129 NILM, 37 ASC-US and 28 L-SIL (36 samples were inadequate for interpretation). HPV physical status was evaluated in the 109 anal specimens that harbored one or two different HPV genotypes. Integration was observed only in one HPV16-positive sample (0.9%), in which integrate-derived viral transcripts of type B were detected. Integration occurred in chromosome 14 q. In 22 of the 53 (41.5%) mucosal HPV-negative samples, RCA restriction results would seem to indicate the presence of circular DNA viruses. Indeed, cutaneous HPV (4 samples), MCPyV (5 samples) and TTV (4 samples) were detected. In conclusion, anal HPV integration was rarely evidenced in HIV-uninfected MSM with no or mild anal cytological abnormalities, although the integration rate may have been underestimated because of the limitations of the employed assays. Other DNA viruses were detected in the anal samples of these individuals, although the significance of this occurrence needs to be assessed.

Prognostic value of HPV E6/E7 mRNA assay in women with negative colposcopy or CIN1 histology result: a follow-up study.

Pap test, and especially HPV DNA test, identify a large group of women who do not have any clinically relevant lesions, i.e., CIN2+ (Cervical Intraepithelial Neoplasia grade 2 or worse), but who are at greater risk of getting lesions in the future. The follow up of these women needs new biomarkers with prognostic value. The objective of this study is to evaluate the prognostic value of E6/E7 mRNA over-expression assay (PreTect HPV-Proofer, Norchip) for 5 HR-HPV types (16, 18, 31, 33, and 45) for progression to CIN2+ after a negative colposcopy. This prospective study, conducted at four Italian centres, enrolled 673 women with either a negative colposcopy or a negative or CIN1 histology. The clinical end-point was histological confirmation of CIN2+. Women were classified at baseline according to mRNA results and managed according to local colposcopy protocols. At least one conclusive follow-up test was obtained for 347 women (25 months average lapse since recruitment, range 5-74). Only seven CIN2+ were detected during follow up, three among the 82 women positive for mRNA at baseline, two among the 250 negative (Fisher exact test, p = 0.02), and two among the 12 with an invalid test. Absolute CIN2+ risk was 6.7/1,000 person/years in the whole cohort. The absolute CIN2+ risk was 18.4/1,000 person/years and 3.6/1,000 person/years in mRNA-positive and mRNA-negative women, respectively. In conclusion, E6/E7 mRNA over-expression appears to be a good candidate as a prognostic biomarker to manage HR-HPV DNA-positive women with negative colposcopy or histology, particularly in order to decrease follow-up intensity in those who are negative.

Performance of the linear array HPV genotyping test on paired cytological and formalin-fixed, paraffin-embedded cervical samples.

Detection and genotyping of human papillomavirus (HPV) from formalin-fixed, paraffin-embedded (FFPE) samples may be difficult when using assays based on amplification of large fragments. The objective of the present study was to investigate the performance of the Linear Array HPV Genotyping Test (Linear Array) on FFPE cervical cone biopsy specimens using paired cytologic samples obtained immediately before the conization as a criterion standard. Thirty-nine samples of grade 2 or higher cervical intraepithelial neoplasia were selected; all of the corresponding cytological samples were positive by the Linear Array and had a report of atypical squamous cells of undetermined significance or worse. A valid Linear Array test result was obtained for 38 FFPE specimens (97.4%, 95% CI 88.0 to 99.9). Specifically, 34 were HPV-positive (89.5%, 95% CI 76.5 to 96.9) and 4 were HPV-negative (10.5%, 95% CI 3.4 to 23.5). The overall agreement of the results obtained for the cytologic and histologic paired samples was good (Cohen's κ = 0.85, SE = 0.082, P = 0.000). Further analysis of samples with negative or invalid Linear Array test results, both modifying the nucleic acids extraction protocol and using the INNO-LiPA assay, suggested that failure of the Linear Array test in HPV detection from tissues was probably due to DNA fragmentation. Parallel analysis of paired FFPE and cytologic samples is extremely useful for evaluation of the efficiency of PCR-based assays in HPV detection and genotyping from tissue samples. In the present study, false-negative results were obtained in a limited percentage of cases, our data depicting the successful performance of the Linear Array test on FFPE samples.

Human papillomavirus type 16 DNA detected in pulmonary metastases from a penile squamous cell carcinoma: a case study.

This report describe the case of a patient presenting with pulmonary metastases from a penile cancer, where the presence of the human papillomavirus (HPV) type 16 DNA both in the primary tumor and in the distant metastases confirmed the spreading of the disease, ruling out a possible primary lung squamous cell carcinoma. Indeed, according to the findings, the HPV genotyping test might help in the identification of metastatic disease from anogenital malignancies or other HPV-related cancers.

HPV type distribution in invasive cervical cancers in Italy: pooled analysis of three large studies.

OBJECTIVE: The aim of this study is to describe the prevalence of HPV types in invasive cervical cancers in Italy from 1996 to 2008. METHODS: A pooled analysis of the three largest case series typed to date was performed. HPV typing was performed on paraffin-embedded slices. Molecular analyses were performed in four laboratories. Multivariate analyses were performed to test the associations between calendar time, age, and geographical area and the proportion of types 16/18. RESULTS: Out of 574 cancers, 24 (4.2%) were HPV negative. HPV 16 and 18 were responsible for 74.4% (378/508) and 80.3% (49/61) of the squamous cancers and adenocarcinomas, respectively. Other frequent types were 31 (9.5%), 45 (6.4%), and 58 (3.3%) for squamous cancers and 45 (13.3%), 31, 35, and 58 (5.0%) for adenocarcinomas. The proportion of HPV 16 and/or 18 decreased with age (p-value for trend <0.03), while it increased in cancers diagnosed in more recent years (p-value for trend < 0.005). CONCLUSIONS: The impact of HPV 16/18 vaccine on cervical cancer will be greater for early onset cancers. In vaccinated women, screening could be started at an older age without reducing protection.

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

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

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

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

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

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

Prevalence, genotype diversity and determinants of anal HPV infection in HIV-uninfected men having sex with men.

BACKGROUND: Anal human papillomavirus (HPV) infection is very common in men having sex with men (MSM), but the available data on its burden and characteristics mainly concern HIV-infected individuals. OBJECTIVES: This study aimed to assess the prevalence, spectrum of genotypes, and determinants of the anal HPV infection in metropolitan HIV-1 uninfected MSM. STUDY DESIGN: A cohort of 258 MSM (median age 32 years, IQR 26-39) enrolled at an STI Clinic was screened for anal HPV infection using a highly sensitive PCR-based genotyping method. Medical history and behavioral data were collected. RESULTS: Overall, 74.8% of the MSM were HPV-positive, with 56.2% of the participants being infected by high-risk (HR) types. A multiple infection was detected in 65.3% of the HPV-positive MSM, with up to 10 different HPV types detected in the same sample. A broad spectrum of infecting HPV types was observed, with 36 different types found overall and HPV16 representing the most common type (17.8%). The lifetime and recent number of sexual partners as well as having receptive anal sex were significantly associated with the anal HPV infection, confirming the role of sexual behavior in risk of HPV infection. However, neither younger age at first intercourse nor inconsistent use of condom was significantly associated with the infection. CONCLUSIONS: The present findings highlight the need to create a more significant awareness about the risk of anal HPV infection among HIV-uninfected MSM and warrant the investigation of possible anal intraepithelial lesions, particularly in view of the increasing anal cancer incidence in high-risk populations.

Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test.

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases.

Comparative evaluation of different DNA extraction methods for HPV genotyping by linear array and INNO-LiPA.

In order to investigate the influence of DNA extraction on two PCR-based HPV genotyping tests (Linear Array, Roche and INNO-LiPA Extra, Innogenetics), three different procedures were used to purify DNA from 28 cervico-vaginal samples tested previously by the Hybrid Capture 2: the AmpliLute Liquid Media Extraction kit (Roche), the QIAamp DNA Blood mini kit (QIAGEN), and the NucliSENS EasyMAG automated platform (bioMérieux). All HC2-positive samples were found positive by both assays, independently of the extract used. Type-specific concordance (i.e., identical HPV type-specific profile in all the extracts of the same sample) was observed in 55% and 75% of the cases testing samples by the Linear Array and the INNO-LiPA, respectively. Using the DNA extracted with the two manual methods the results were concordant in 75% of the cases both for the Linear Array and the INNO-LiPA. When comparing the Linear Array results obtained on either of the two manual extracts with those obtained following automated extraction, 65% of the samples showed type-specific concordance in both cases. The INNO-LiPA results were concordant in 80% of the cases comparing the AmpliLute versus the automated extract, while concordant results were observed in 90% of the cases when comparing the QIAGEN versus the automated extract. In conclusion, the Linear Array and INNO-LiPA results are affected by the method of DNA extraction. Consequently, different HPV type-specific profiles may be observed using different extracts of the same sample. The use of consistent protocols for DNA purification is a priority to guarantee intra-assay reproducibility over time.

Diagnostic and prognostic validity of the human papillomavirus E6/E7 mRNA test in cervical cytological samples of HC2-positive patients.

The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL-). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL-, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL- patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2- lesions (p < 0.0001). Moreover, among the 75 HSIL- followed up patients, the mRNA displayed high longitudinal Specificity (89%), even if the sensitivity and the PPV were low (50 and 20%, respectively). The present data suggest that the mRNA test may have a diagnostic and a potentially prognostic role in HC2+/HSIL- patients.

Comparative evaluation of nm23 and p16 expression as biomarkers of high-risk human papillomavirus infection and cervical intraepithelial neoplasia 2(+) lesions of the uterine cervix.

AIMS: To investigate the clinical role of nm23 expression in identifying both high-risk human papillomavirus (HR-HPV) and high-grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2(+) (CIN2(+) )], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR-HPV infected cervical lesions. METHODS AND RESULTS: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low- and high-grade lesions and squamous carcinomas (SC). HR-HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico-vaginal samples of the same patients. In detecting CIN2(+) , p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P < 0.0001). Concerning HR-HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% versus 47%, P <0.0001), although the sensitivities were comparable (71% versus 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. In contrast, we observed a significant association between p16 overexpression and HPV16/18 genotypes. CONCLUSIONS: We confirm the diagnostic value of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV-E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2(+) or HR-HPV infection.

Human papillomavirus prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy.

BACKGROUND: Cervical cancer represents an important global public health problem. It is the 2nd most common cancer among women worldwide. Human papillomavirus (HPV) infection is now well-established as a necessary cause of invasive cervical cancer (ICC) development. Only a few studies on HPV prevalence and type-specific distribution in ICC have been conducted in Italy. AIM: To describe the prevalence of HPV and the HPV type-specific distribution in ICC cases identified in Rome, Italy. METHODS: 140 paraffin embedded tissue blocks of primary ICC diagnosed between 2001 and 2006 were identified at the Regina Elena Cancer Institute in Rome (Italy). HPV was detected through amplification of HPV DNA using SPF-10 HPV broad-spectrum primers followed by DEIA and then genotyping by LiPA25 (version 1). RESULTS: 134 cases were considered suitable for HPV DNA detection after histological evaluation; and overall, 90.3% (121/134) HPV prevalence was detected. 111 cases had a single HPV type, 4 cases had an uncharacterized type (HPVX) and 6 cases had multiple HPV infections. The five most common single HPV types among positive cases were: HPV16 (71/121; 58.7%), HPV18 (12/121; 9.9%), HPV31, HPV45 and HPV58 (5/121; 4.1% each). 2 (1.5%) of the single infections and 2 (1.5%) of the multiple infections contained low risk types. Statistically significant differences in the relative contribution of HPV18 were found when comparing squamous cell carcinomas with adenocarcinomas. CONCLUSIONS: HPV16 and HPV18 accounted for almost 70% of all the HPV positive ICC cases. The study provides baseline information for further evaluation on the impact of recently introduced HPV vaccines in Italy.

Research trends for early cancer biomarker detection in Italy: an integrated program in oncology (PIO) survey.

AIMS AND BACKGROUND: In 2007, an Italian Research Network proposed to the Ministry of Health a concerted action aimed at developing a specific pathway for the analytical and clinical validation of new biomarkers for early cancer diagnosis. The action, funded by the Italian Ministry of Health within the Integrated Program in Oncology (PIO) and coordinated by the National Cancer Institute of Bari, started in 2008 involving 37 national research teams. METHODS: To monitor the methodological and analytical needs of the studies proposed by the research teams of PIO as well as to explore the plausibility of planning external quality assessment programs for early cancer biomarker detection, the coordinating team developed an ad hoc questionnaire that was submitted to each research team. RESULTS: From the collected data it emerged that about 70% of the biomarkers under investigation were analyzed according to nonroutine laboratory practices. The biological material utilized for biomarker assessment consisted of solid tissue (normal or pathological) in 31% of studies, serum in 21%, urine in 15%, plasma in 15%, and whole blood in 11%. Specific training of personnel directly involved in the program was reported by 18% of the teams. In 2008, only 6% of laboratories involved in PIO participated in both external quality assessment and internal quality control schemes specifically designed for the biomarkers under consideration. Standard operating procedures for the determination of about half (52%) of the biomarkers proved to be lacking in at least one phase of the biomarker assessment process. CONCLUSIONS: On the basis of these results, we decided to give priority to the application of a four-phase process for the analytical validation of new potential biomarkers by setting up and applying standard operating procedures and developing external quality assessment and internal quality control schemes as specific steps of the workflow.

The Integrated Oncology Program of the Italian Ministry of Health. Analytical and clinical validation of new biomarkers for early diagnosis: network, resources, methodology, quality control, and data analysis.

In 2007, an Italian cancer research group proposed a specific concerted action aimed at the "analytical and clinica validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis." The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators' meeting, "Analytical and clinical validation of new biomarkers for early diagnosis," was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.

HPV prevalence among healthy Italian male sexual partners of women with cervical HPV infection.

Genital human papillomavirus (HPV) is the causative agent of cervical cancer and is the most common sexually transmitted infection. Only limited and controversial data are available regarding HPV transmission in male sexual partners of women with cervical intraepithelial neoplasia (CIN). The aim of this study was to investigate the prevalence and the genotype distribution of HPV in penile scrapings of a series of Italian men, who had no visible penile lesions and were partners of women who were affected, or had been affected previously by cervical intraepithelial neoplasia or who were infected with HPV. The concordance of the viral group in the infected partners was determined. A total of 77 penile scrapings were screened for HPV infection by the polymerase chain reaction, while 59 cervicovaginal brushings of their female partners were tested. 35% of evaluable male samples and 64% of female sexual partners were found to be HPV positive. In the 55 simultaneously evaluable couples, a concordance of 45% was found, 11 couples (20%) with both partners being HPV negative and 14 couples (25%) with both partners HPV positive (P=0.001). Six out of the 14 couples (43%), where both partners were HPV positive, harbored the same HPV genotype group. These data, although preliminary, could support further the hypothesis that male HPV infection is more frequent in sexual partners of HPV positive or women with cervical intraepithelial neoplasia indicating that men could represent an important source of HPV transmission between sex partners.

Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis.

p16INK4a is overexpressed in high-risk human papillomavirus (HR-HPV)-infected preneoplastic and neoplastic lesions of the uterine cervix. Our aim was to verify whether p16 is a diagnostic marker also in cervical liquid-based cytology. We performed p16 immunocytochemical analysis and the Hybrid Capture 2 (HC2) test (Digene, Gaithersburg, MD) for HR-HPV infection in 471 ThinPrep-processed (Cytyc, Boxborough, MA) cervicovaginal samples and correlated the results with histologic findings. A total of 32.3% of the specimens showed p16 immunoreactivity, whereas the HC2 test was positive in 41.2% of the cases (65.2% concordance rate). Correlating the cytologic, p16, and HPV results with histologic findings revealed HC2 as the most sensitive test for a diagnosis of cervical intraepithelial neoplasia 2 or worse, whereas cytologic examination was the most specific. The positive predictive value was significantly higher for cytologic examination than for p16 and HR-HPV testing. These data suggest that p16 evaluation in ThinPrep samples does not have better clinical effectiveness for identifying high-grade lesions than conventional morphologic examination and HPV testing.