Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness ofp16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH andp16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, whilep16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

A very rare case of HPV-53-related cervical cancer, in a 79-year-old woman with a previous history of negative Pap cytology Roberta Zappacosta,1 Giuseppe Lattanzio,2 Patrizia Viola,2 Manuel Maria Ianieri,3 Daniela Maria Pia Gatta,1 Sandra Rosini11Cytopathology Unit, Experimental and Clinical Sciences Department, Gabriele dâ??Annunzio University of Chieti-Pescara, Chieti, Italy; 2Surgical Pathology Unit, 3Obstetrics and Gynecology Unit, SS Annunziata Hospital, Chieti, Italy The introduction of organized cervical cancer (CC) screening programs has drastically reduced the prevalence of CC. However the incidence is still too high, especially among elderly women. All guidelines strongly recommend a regular Papanicolaou (Pap) testing for young and middle-aged patients. On the other hand, many international professional societies no longer advise screening in women who have undergone hysterectomy, and in women aged 65 years and above, who have a previous history of regular Pap smears. Here we report the case of poorly differentiated CC, involving the pelvic lymph nodes and urinary bladder, occurring in a 79-year-old woman who regularly underwent Pap tests, with no reported cytological abnormalities. In this very rare case, the CC cells, as well as cells from metastatic lymph nodes and cells from urinary specimens, molecularly showed human papilloma virus (HPV)-53. With the limitations of a single case, this report brings important information to prevent CC in elderly patients: the utility of molecular tests to increase sensitivity of Pap smears in postmenopausal women; the importance of HPV-53 as one of the four â??emergentâ?? genotypes having a possible role in oncogenesis; and the presence of HPV-53 in lymph node metastases from cervical carcinoma, which would support the role of this virus in the maintenance of malignant status.Keywords: old women, molecular tests, cervical cancer screening, HPV-DNA test, HPV genotyping

Human Papillomavirus (HPV) E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001–2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/μL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC) cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A “one sample for all” approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

HPV-DNA testing has entered in clinical practice. Three important questions remain controversial: 1) which is the best HPV-DNA technology? 2) Which age group should be targeted? 3) Is HPV-DNA testing predictive of disease outcome? The answers to these queries represent the endpoints of this study. The population of this retrospective study consisted of 272 women, each one having: baseline cytological diagnosis of Low-grade Squamous Intraepithelial Lesion (LSIL); baseline HPV-DNA reports by Hybrid Capture 2 (HC2) and MY09/11 consensus primers PCR; follow-up duration over 3-years; cytological report of disease status at follow-up time. Firstly, we assessed the concordance and the performances of both HPV-DNA testing, then we correlated respectively HPV-DNA results and age of patients to disease outcome. DNA testing methods agreed in 83.4% of cases (K=0.66). Baseline HPV-DNA result was not significantly associated to disease outcome (p=0.06). Within HPV-DNA positive group, we found no evidence of correlation between age and LSIL prognosis (p=0.89). Confining the analysis to age-stratified HPV-DNA negative women, the differences were statistically significant (p=0.01). In conclusion, HPV-DNA testing gives no information about the real behaviour of cervical abnormalities. These findings suggest the demand for additive markers, reflecting the risk of progression, in prevention strategy and clinical approach.