Detection of HPV16 African variants and quantitative analysis of viral DNA methylation in oropharyngeal squamous cell carcinomas.
BACKGROUND
HPV16 is the most prevalent genotype in oropharyngeal squamous cell carcinomas (OPSCCs), but there are few information about intratype variants prevalence and their clinical relevance in these tumors. Moreover, since HPV DNA detection alone is not sufficient to determine a favorable prognosis for OPSCCs, additional diagnostic/prognostic biomarkers are necessary.


OBJECTIVE
To assess the prevalence of HPV intratype variants and evaluate the role of HPV16 DNA methylation as prognostic marker in OPSCCs.


METHODS
We analyzed 81 biopsies of OPSCCs for HPV detection and genotyping with a pyrosequencing-based HPV DNA-test. We also quantified viral DNA methylation frequency in the LCR by pyrosequencing in patients with transcriptionally-active HPV16-infection, analyzing it in function of available clinical/virological data.


RESULTS
The overall HPV prevalence was 74.1% and HPV16 was confirmed the most prevalent genotype (51/60, 85.0%). Interestingly, we detected, for the first time, HPV16 African variants in 10/51 cases (19.6%), pointing out their clinical relevance also out of the anogenital district. Regarding viral DNA methylation, the E2BS1 showed a significantly higher mean methylation frequency compared to E2BS3/4, as previously observed in high-grade precancerous lesions of the uterine cervix. On the other hand, patient with a mean methylation frequency in the early promoter >10% showed only a tendency to have a worse prognosis.


CONCLUSIONS
Our results highlight both the involvement of HPV16 African variants in OPSCCs development and the necessity of further studies to deepen the role of viral methylation in these tumors, in order to better define similarities/differences compared to cervical cancer.
Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Section of Microbiology, S.Orsola-Malpighi Hospital, University of Bologna, via Massarenti 9, 40138 Bologna, Italy; Unit of Microbiology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Daniela
D
Barbieri
Unit of Otolaryngology, Department of Specialist Surgery and Anesthesiology, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Aurel
A
Nebiaj
Unit of Microbiology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Rosaria
R
Strammiello
Unit of Pathology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Roberto
R
Agosti
Unit of Otolaryngology, Department of Specialist Surgery and Anesthesiology, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Silvia
S
Sciascia
Unit of Microbiology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy; Department of Pharmacy and Biotechnology (FaBiT), S.Orsola-Malpighi Hospital, University of Bologna, via Massarenti 9, 40138 Bologna, Italy.
Giorgio
G
Gallinella
Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Section of Microbiology, S.Orsola-Malpighi Hospital, University of Bologna, via Massarenti 9, 40138 Bologna, Italy; Unit of Microbiology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Maria Paola
MP
Landini
Unit of Otolaryngology, Department of Specialist Surgery and Anesthesiology, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy.
Umberto
U
Caliceti
Unit of Microbiology, Department of Hematology, Oncology and Laboratory Medicine, S.Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy. Electronic address: simona.venturoli@unibo.it.
Simona
S
Venturoli
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000453:epidemiology / Q000473:pathology / Q000821:virology*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004279:DNA, Viral; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D009959:Oropharyngeal Neoplasms / Q000453:epidemiology / Q000473:pathology / Q000821:virology*; D030361:Papillomavirus Infections / Q000150:complications / Q000821:virology*; D015995:Prevalence; D011401:Promoter Regions, Genetic; D012307:Risk Factors; D016019:Survival Analysis
D004279:DNA, Viral
DNA methylation; HPV genotyping; Molecular markers; Oropharyngeal cancer; Pyrosequencing