HPV e carcinoma della cervice uterina

The identification of Human Papillomavirus (HPV) as a necessary cause for the development of cervical cancer and the detection of the different HPV oncogenic types through molecular techniques has made it possible the study of new strategies for the screening (triage, primary test) that can permit to identify with greater accuracy women at risk to develop a high-grade lesion or an invasive carcinoma. Given this information, we have performed search and typing of HPV sequences for triage of women with ASC-US diagnosis in a multicentric study that has involved five screening centres of the Veneto region to evaluate sensitivity and specificity of HPV test in comparison to the Pap test repetition or colposcopy . The use of HPV test as a primary screening test is the object of the NTCC (New Technologies for Cervical Cancer) randomized trial. It has been conducted on 95.000 women enrolled from 9 cervical screening programmes in Italy; the study has the goal to evaluate the efficacy of HPV test, in comparison to conventional Pap test, in detecting high risk lesions. We also conducted a study of type-specific prevalence in invasive carcinomas of the uterine cervix. The results show that the HC2 test is more sensitive and more specific than colposcopy and or Pap test for the triage of women with an ASC-US diagnosis. In the NTCC study the HC2 test showed greater sensitivity compared to Pap test but lower specificity; it has been observed that increasing the cut-off value from 1 to 2 pg/ml the test shows better specificity maintaining comparable sensitivity. In both studies women with a negative HC2 test have shown a very low risk to develop high grade lesions. The typing results, in all the studies, pointed out HPV 16 as the most frequent type and its elevated association with high-grade lesions. Analyzing HPV 16 variants, it has emerged that most of the high grade lesions and cervical carcinomas are caused by the European variant T350G (L83V). The technical aspects to consider for HPV search and typing are: the method of specimen collection, set of primers and typing method. The different strategies used have shown peculiar characteristics of sensitivity and specificity on identifying the different HPV types, and the use of a strategy with multiple methods has allowed, in both studies, to type more than 90% of the HC2+ specimens. All the typing methods have shown an elevated reliability in the detection of HPV 16 and HPV 18.L’identificazione del Papillomavirus Umano (HPV) come causa necessaria per lo sviluppo del carcinoma della cervice uterina e la rilevazione dei diversi tipi di HPV con diverso potenziale oncogeno mediante tecniche molecolari hanno reso possibile lo studio di nuove strategie nello screening (triage, test primario) basate su test molecolari che possono permettere di individuare con maggiore accuratezza le donne a rischio di sviluppare una lesione di alto grado o un carcinoma invasivo. Con questa informazione abbiamo effettuato la ricerca e la tipizzazione di sequenze HPV in diverse casistiche: come triage delle donne con diagnosi di ASC-US in uno studio multicentrico che ha coinvolto i servizi di screening di cinque ULSS del Veneto per valutare sensibilità e specificità del test HPV rispetto alla ripetizione del Pap test o alla colposcopia. L’utilizzo del test HPV nello screening come test primario è invece l’oggetto dello studio Nuove Tecnologie per lo screening del Carcinoma Cervicale (NTCC). Si tratta di un trial multicentrico randomizzato su 95.000 donne, in 9 centri di screening in Italia, il cui principale obiettivo è valutare se, rispetto al Pap test tradizionale, il test per la ricerca dei tipi ad alto rischio dell’HPV (HC2, Digene) permette di migliorare l’efficacia dello screening. Vengono qui presentati i dati di tipizzazione dei campioni HC2 positive delle donne arruolate a Padova. È stato condotto anche uno studio di valutazione della prevalenza tipo-specifica in carcinomi invasivi della cervice uterina. I risultati mostrano che il test HC2 nello studio ASC-US è più sensibile e più specifico rispetto alla colposcopia e alla ripetizione del Pap test. Nello studio NTCC il test HC2 ha mostrato una sensibilità più alta del Pap test ma una minore specificità; si è visto che aumentando il valore soglia da 1 a 2pg/ml migliora la specificità mantenendo una buona sensibilità. In entrambi gli studi le donne con un risultato negativo al test HC2 hanno mostrato un rischio molto basso di sviluppare lesioni di alto grado. Per quanto riguarda la tipizzazione i risultati indicano, in tutte le casistiche, l’HPV 16 come tipo più frequente e una sua elevata associazione con lesioni di alto grado e carcinomi. Analizzando le varianti virali dei campioni HPV 16 è emerso che la maggioranza delle lesioni di alto grado e i carcinomi cervicali sono associati alla variante virale europea T350G (L83V). Gli aspetti tecnici da considerare per la ricerca e la tipizzazione dell’HPV sono tipo e metodo di prelievo e conservazione del campione, set dei primers utilizzati, metodo di tipizzazione. Le diverse strategie da noi impiegate hanno mostrato caratteristiche peculiari di sensibilità e specificità nell’identificare i tipi di HPV, e va sottolineato che l’utilizzo di una strategia con più di una metodica ha permesso, in entrambi gli studi, di tipizzare più del 90% dei campioni. Tutti i metodi di tipizzazione hanno mostrato un’elevata affidabilità nella rilevazione di HPV 16 e HPV 18

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature. c) 2004 Wiley-Liss, Inc.Funded by: Fondo Investigación Sanitaria, Ministerio de Sanidad, España. Grant Numbers: FIS 98/1349, 01/09587; CITMA-CSIC. Grant Number: 2003CU0015 and International Union Agains Cancer. Grant Number: ICRETT 584/200.Peer Reviewe

Efficacy of self-sampling in promoting participation to cervical cancer screening also in subsequent round

Offering self-sampling devices improves participation of under-screened women. We evaluated participation in routine screening following the self-sampling intervention in two organized population-based screening programmes located in North-East Italy. Data on participation at 3-years-interval after a randomized clinical trial assessing the response to two strategies offering self-samplers (sent at home or offered free at local pharmacy) with a control action (sending reminders for a cervical specimen taken at the clinic) in 30–64 yr-old women non-respondent to the regular call-recall invitation were analyzed. Up to April 2016, 2300 women out of the 2995 recruited in the trial in 2011 were re-invited to perform a screening test at clinic; overall, 698 women adhered. Participation was similar in the three arms (29–32%), and highest (47–68%) among those who participated in the previous round. Over the two rounds, 44.6%, 32.3% and 30.3% women had at least one test in the self-sampling at home, self-sampling at pharmacy and test at the clinic arms, respectively. Our data indicate that the beneficial effect of offering self-sampling devices to nonparticipating women is maintained over time. Self-samplers are useful to increase overall coverage; their sporadic use does not seem to increase the proportion of women regularly repeating the test

Leptomeningeal Carcinomatosis of a Poorly Differentiated Cervical Carcinoma Caused by Human Papillomavirus Type 18

Cervical cancer is caused by a persistent infection with high-risk types of Papillomaviruses (hrHPV); HPV16 and HPV18 are associated with about 70% of the cases. In the last decades the introduction of a cervical cancer screening has allowed a decrease in cervical cancer incidence and mortality; regular adhesion to the screening procedures, by pap test or HPV test, and colposcopy, according to the international guidelines, prevents cancer development and allows for diagnosis at the early stages. Nowadays, in industrialized countries, it is not common to diagnose this pathology in advanced stages, and this occurrence is frequently associated with patient’s unattendance of cervical screening programs. We describe a case of delayed diagnosis of cervical cancer, posed only after the onset of the neurological symptoms caused by leptomeningeal metastases, despite a two-year history of abnormal cytology. The endocervical mass was analyzed by immunohistochemistry, and search and typing of HPV sequences was performed by PCR in the meningeal carcinomatous cells. A poorly differentiated squamous cell carcinoma was diagnosed, and HPV18 sequences were detected. This rapidly fatal case highlights the importance of following the evidence-based recommended protocols and the preventive role of the population-based cervical cancer screening programs

Diagnóstico molecular del retinoblastoma: epidemiología molecular y consejo genético

[Fundamento y objetivo]: El retinoblastoma, prototipo de cáncer hereditario, puede causar ceguera, por enucleación terapéutica, segundos tumores en pacientes con mutación germinal e incluso muerte si no se trata. El diagnóstico molecular de 213 pacientes a lo largo de 5 años ha conducido a la detección de 106 mutaciones que se analizan desde la perspectiva de la epidemiología molecular y consejo genético. [Pacientes y método]: Estudio mutacional (reacción en cadena de la polimerasa, secuenciación y análisis de microsatélites) en pacientes con retinoblastoma procedentes de España, Cuba, Colombia y Serbia. [Resultados]: Un 45% de las mutaciones analizadas son nuevas y corresponden a mutaciones de tipo de corrimiento de pauta de lectura, cambio de aminoácido o procesado del ácido ribonucleico. Todas las mutaciones sin sentido corresponden a sitios de alta mutabilidad. La tasa de detección de mutaciones en pacientes unilaterales esporádicos es alta (22%). En este grupo de pacientes se detecta una mayor incidencia (p = 0,018) de mutaciones de cambio de aminoácido y procesamiento. España y Francia muestran una incidencia mayor de mutaciones del procesado (p = 0,0003) que Alemania y el Reino Unido, países en los que predominan las mutaciones sin sentido (p = 0,0006). Las mutaciones del procesado se asocian al fenotipo de baja penetración y retraso en la aparición de tumores (p = 0,018). [Conclusiones]: La incidencia de mutaciones germinales en pacientes unilaterales y las relaciones fenotipo/genotipo analizadas indican la necesidad del consejo genético basado en el diagnóstico molecular temprano. La mejora de las técnicas diagnósticas, la caracterización funcional de mutaciones asociadas a baja penetrancia o expresividad y el estudio del transcriptoma de los tumores son objetivos necesarios para definir mejor la patogenia del retinoblastoma.Este trabajo ha sido realizado con ayudas de la Comunidad de Madrid (GR/SAL/0855/2004), Programa Bilateral CITMA-CSIC (Ref. 2003CU0015).Peer Reviewe

HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system

Abstract Background European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps. Methods We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion. Results Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator’s hands-on time. Conclusions The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs

Methylation analysis and HPV genotyping of self-collected cervical samples from women not responding to screening invitation and review of the literature.

AIM OF THE STUDY:To assess the feasibility of partial HPV genotyping and methylation analysis of CADM1, MAL, and miR124-2 genes as triage tests in assaying self-collected cervical samples positive for high-risk HPV on primary screening, and to review the literature regarding host cellular gene methylation analysis of self-collected cervical samples. MATERIAL AND METHODS:Women residing in North-East Italy who had failed to respond to the invitation to participate in an organized population-based program were invited to provide a self-sample. Their stored baseline (self-collected) and follow-up (clinician-collected) cervical samples were included in the study. DNA was extracted from HPV-positive (Qiagen's Hybrid Capture 2, HC2) samples. Partial genotyping with separate detection of HPV types 16 and 18 was performed with a hybrid capture-based method and a quantitative PCR assay. Methylation was assayed with a quantitative methylation-specific PCR. RESULTS:High-risk HPV infection was detected in 48% of baseline and 71% of follow-up HC2-positive samples. Methylation was demonstrated respectively in 15% and 23.5% of baseline and follow-up samples and chiefly involved a single gene (miR124-2). Invalid quantitative PCR results were recorded in 5% of self-collected samples. The specificity of miR124-1, MAL, and CADM1 methylation was 84%, 94%, and 98%, respectively, and the specificity of the three markers combined was 84%. Sensitivity was not estimated due to the lack of CIN2+ samples. The systematic review showed that different methylation assays yield different accuracy values. CONCLUSION:Self-collected samples are suitable for methylation assays included in reflex triage testing. The reproducibility and accuracy of the methylation tests described in the literature should be improved

PRISMA Flow-Chart: search strategy.

<p>PRISMA Flow-Chart: search strategy.</p