Research Projects HUMAN HERPESVIRUS-6 (HHV-6) ETIOPATHOGENETIC ROLE IN THE DEVELOPMENT OF CERVICAL CANCER BY COMBINING IMMUNOHISTOCHEMISTRY, LASER MICRODISSECTION AND SINGLE CELL-REAL-TIME PCR ANALYSIS
Human papillomaviruses (HPVs) are necessary, but not sufficient, for the development of cervical cancer (CC). Human herpesviruses (HHVs) have been suggested as possible cofactors in the oncogenesis of CC. Besides herpes simplex virus, which has long been suspected to act as an “initiator” in the development of CC, others HHVs such human herpesvirus-6 (HHV-6) have been suggested as possible candidates in the development of cervical displasia. Although all these viruses are ubiquitous and have been detected in the cervix, epidemiological data regarding their association with genital HPVs and their role in CC have not reached a consensus. In particular, it has been shown in vitro that HHV-6 can enhance the expression of HPV oncoproteins E6 and E7. As these mechanisms are relevant in the development of CC, the ability of these HHVs to persist in cervical epithelial cells might be crucial. However, the available epidemiological data are conflicting. HHV-6 has been proposed as a cofactor because it can transactivate the HPV genome and it has been detectedin CC.
In our recent study (Broccolo et al; J. Med Virology. 2008, 80:147-153) we demonstrated that the positive rate for HHV-6 cervical specimens was 25% (range 14–40%) and that the prevalence of HHV-6 DNA resulted significantly higher in patients with HSIL compared to normal women (P=0.017). Furthermore, although the HHV-6 level did not differ significantly between patients with pathological findings and women with normal cytology as well as between HPV positive and negative samples, a very high HHV-6 viral load was found only in women with HSIL HPV positive.
In this study we evaluate if HHV-6 genital infections, besides those caused by high-risk human papillomavirus (HR-HPV) genotypes, could be implicated in the development of cervical cancer (CC). HHV-6 viral load will be determined also in HHV-6- antigens positive cells isolated by laser microdissection in cone biopsy (conization) after immunohistochemistry (IHC) for HHV-6 early (p41 and IE-2) and late (gp116/64/54) antigens.
IDENTIFICATION AND QUANTITATION OF ONCOGENIC HPV NUCLEIC ACID BY MEANS OF REAL-TIME PCR ASSAYS
In recent years it has been established that infection by oncogenic human papillomavirus (HPV) is a necessary condition for cervical carcinogenesis. Persistent infection is considered to be the true precursor of neoplastic progression. Currently, however, HPV infections are monitored primarily by qualitative HPV DNA detection assays which are often not type specific and therefore in the clinical management of the patients do not distinguish between persistent and transient infections, the latter being extremely frequent in sexually active women. Qualitative unspecific viral detection therefore represents an inefficient means of identifying women at risk of developing cervical cancer (CC). There is therefore a need to establish a suitable clinical marker able to distinguish persistent from transient infection in order to better identify those women at risk of neoplastic progression. Thus, HPV detection and typing techniques have been proposed as an adjunct to, or a replacement for, the current cytological screening regime. Clearly the success of such strategies will depend on the development of rapid, reliable, sensitive, and specific HPV detection methods applicable in the clinical setting. HPV viral load has been proposed as a surrogate marker of persistent infection but its use in identifying women at risk of developing CC remains controversial. Furthermore, several studies have shown that in cervical carcinogenesis, the expression of HPV of specific E6 and E7 oncogene transcripts is required for cell transformation and immortalisation. Moreover, the presence of E6 and E7 has been found to increase with increasing severity of cervical disease. Consequently, persistent expression of these oncogenes may serve as an indicator of progression to neoplastic precursor lesions and CC. Finally a better understanding of the circulating HPV genotypes in different geographical areas has become very important, particularly in view of the recent introduction of HPV vaccines. Screening programmes will in fact need to be continued particularly in areas where other oncogenic genotypes are found to prevalent, as immunization will only protect against HPV types targeted by the vaccine.
The present project refers to the development of a flow chart based on Real-Time PCR assays for identification and quantification of oncogenic HPV. Method for the identification and quantification of oncogenic HPV nucleic acids comprising:
a) first line screening by means of a SYBR Green I Real-time PCR assay to determine the total viral DNA load and to identify the presence of one or more of 13 high risk HPV genotypes in clinical samples;
b) second line assays to be applied to samples positives for a):
4 independent TaqMan Real-time PCR assays to determine the presence and the viral load of the most common oncogenic HPV types: 16, 18 and/or 45, 31, 33 and/or 58;
6 independent SYBR Green I RT Real-time PCR assays to determine the presence in the sample of the oncogenic transcripts E6/E7 of HPV types 16, 18, 31, 33, 45, 58.
|
