HPV prevalence, viral load and physical state of HPV‐16 in cervical smears of patients with different grades of CIN

Abstract

Human papillomavirus (HPV) infection is the most important event in malignant transformation of human cervical epithelium. We analysed in cervical smears, HPV genotypes with a focus on single/multiple infections, then characteristics of HPV-16 infections (presence of other genotypes, viral load and physical state) according to the grade of histological lesions. The purpose of this study was to know if these parameters could allow to differentiate histological diagnoses. DNA was extracted from 363 cervical samples corresponding to 24 cases without lesion, 96 CIN1, 92 CIN2, 144 CIN3 and 7 cancers. Our results show that HPV-16 was predominant and its prevalence increased with the severity of lesions (CIN1: 27.1%; CIN3: 65.3%). In addition, we showed that the frequency of single infections, as compared with multiple infections, increased with the severity of the lesion (CIN1: 25.0%; CIN3: 54.8%). Among HPV-16 positive samples (n 5 170), we found that viral load, determined on cervical samples by real-time PCR, did not vary significantly according to the different CIN grades. Concerning HPV-16 integration, the mixed and integrated HPV-16 forms, already present in women with normal histology, increased to the benefit of pure episomal forms with the severity of lesions (normal cervix: 28.6%; CIN3: 73.8%). Thus, our data raise the question of the viral load as a valuable clinical parameter to discriminate between lesion grades. Moreover, we emphasize integration as an early event in cervical carcinogenesis, increasing with the severity of lesions. Finally, this study underlines the importance of single versus multiple infections linked to the severity of CIN. ' 2007 Wiley-Liss, Inc.Key words: HPV; cervical cancer; genotyping; viral load; viral DNA integration Cervical cancer is the second most frequent malignant tumour of women worldwide. The recognized association of human papillomaviruses (HPV) as the most significant etiological factor in cervical cancer is now well established.1,2 More than 40 genotypes infect the anogenital area and are associated with a large spectrum of diseases from benign proliferation to invasive cancers. The low-risk (LR-) HPV are linked to benign lesions whereas high-risk (HR)-HPV, principally types 16 and 18, are associated with malignant lesions. The most common HR-HPV type in squamous cell cervical cancer is HPV-16, found in over 50% of the cases. 3,4 Recent studies have suggested that HR-HPV viral load, notably for HPV-16, may be a molecular biomarker of risk for developing cervical (pre-) cancerous lesions. Indeed several studies found a high correlation between high viral load and HSIL (High grade Squamous cell Intraepithelial Lesion) 5-8 whereas other reports concluded that there was no correlation.9-13 Also, despite multiple investigations, using various study designs and methodologies, the clinical utility of HR-HPV load remains unclear.The physical status of HR-HPV in pre-neoplastic cervical lesions may be a further promising marker for the progression to cervical cancer. Molecular...

Integración, carga viral y niveles de ARN mensajero de E2 de VPH 16 en la progresión de lesiones intraepiteliales cervicales

“…Al comparar las medias de carga viral en cada grupo histológico se observó una diferencia estadísticamente significativa, con un incremento a mayor severidad de la enfermedad. Los estudios en los que se ha empleado la PCR cuantitativa para evaluar la carga viral han mostrado de manera consistente que a mayor grado de la lesión es significativamente mayor la carga viral (Carcopino et al, 2006;Flores et al, 2006;Cricca et al, 2007;Saunier et al, 2008;Shukla et al, 2014), aunque en algunos estudios no se ha encontrado esta diferencia (Andersson et al, 2005;Briolat et al, 2007;Boulet et al, 2009). Cabe señalar que una comparación directa entre nuestros resultados y los obtenidos previamente por otros grupos es difícil dadas las diferencias en las técnicas de tiempo real empleadas (Taqman, SYBR Green), y las múltiples formas de presentación de los resultados (copias/ célula, copias/ng de ADN, copias/muestra).…”

“…El genoma viral integrado, del cual son transcritos los genes tempranos E6 y E7, ha sido detectado en aproximadamente 90 % de los cánceres cervicales (Klaes et al, 1999). En varios estudios se ha demostrado que mediante PCR en tiempo real dirigida a los genes E2 y E6 es posible evaluar el estado físico del ADN de VPH 16 de manera confiable (Peitsaro et al, 2002;Andersson et al, 2005;Fujii et al, 2005;Briolat et al, 2007;Saunier et al, 2008). Estos estudios en general muestran que a mayor grado de lesión hay una mayor proporción de lesiones con el genoma del VPH 16 integrado.…”

Human papillomavirus viral load in cervical intraepithelial neoplasia as a prognostic factor in a Mexican population

“…Figure 1 shows a dispersion of values in the CIN2 samples in which some samples displayed an even greater number of viral copies per cell than that in CIN3. This result can be explained by the frequent misclassification of lesion grade in samples (Briolat et al, 2007). The CIN2 grade has the highest level of misclassification owing to variability in interpretation by the observer; CIN2 can represent the spectrum of late CIN1, CIN2, or early CIN3 (Briolat et al, 2007;Xi et al, 2008); true CIN2 is thought of as an early phenotype of high-grade CIN, a true cervical precancerous lesion (Einstein, 2008).…”

“…On the spectrum of cervical disease, CIN2 and 3 are considered cervical cancer precursors. Several studies have consistently reported that the persistence of HR-HPV in the genital tract is necessary for the development and progression of cervical dysplastic lesions (Ho et al, 1995;Remmink et al, 1995;Nobbenhuis et al, 1999;Briolat et al, 2007;Xi et al, 2008;Sargent et al, 2010). Conversely, high-grade squamous intraepithelial lesions were shown to be unlikely to develop in HR-HPVnegative women in a 2-year follow-up cohort, and smears showed that mild or borderline cell atypia returns to normal (Nobbenhuis et al, 2001;Zielinski et al, 2001).…”

“…Because persistent HR-HPV infection is the major risk factor for cervical carcinoma and considering the sensitive and reproducible methods available for HR-HPV DNA, HR-HPV status could be part of the screening and management of women at risk for progression to CIN2, CIN3, or worse (Briolat et al, 2007). However, the use of HPV testing combined with cervical cytology to detect cervical cancer or its precursors is still controversial and insufficient (Cain and Howett, 2000;Fiander et al, 2007;Boulet et al, 2009;Hesselink et al, 2009;Grce et al, 2010).…”