Detection of human papillomavirus (HPV) in laryngeal carcinoma cell lines provides evidence for a heterogeneic cell population

The role of human papillomavirus (HPV) has been studied in laryngeal carcinomas with contradictory results. To evaluate the causal relationship between HPV infection and epithelial malignancies of the larynx, 27 laryngeal carcinoma cell lines from 22 patients were studied. Also, paraffin-embedded biopsy samples of the original tumours were available from 12 patients. First, Southern blot hybridisation (SBH) was used for the analysis of 18 cell lines and 12 original tumour sections were studied by in situ hybridisation (ISH) to detect HPV. Further, cell lines and tumour biopsy samples were investigated with polymerase chain reaction (PCR) using three sets of consensus primers directed to L1 and E1 ORFs (open reading frames) and type-specific primers to HPV 16 E6 region. The adjacent apparently normal epithelium of one original biopsy sample showed positive signals for HPV by ISH. All other samples were HPV negative with these methods. The study was then extended to 27 laryngeal carcinoma cell lines, including the 18 cell lines studied earlier. A new nested PCR method was used with MY as external and general primers (GP) as internal primers for the cell lines and original tumour samples to achieve a maximal sensitivity. Subsequent SBH was performed to confirm the specificity of PCR products with both low- and high-risk HPV oligonucleotide probe mixtures and also with the HPV 16 oligoprobe. With this method, seven of 27 (26%) cell lines and seven of 12 (58%) tumour samples were found to harbour high-risk HPV. In two cases both the original tumour sample and the derived cell line showed HPV positivity. These results indicate that HPV copy numbers are low and only a minority of tumour cells harbour HPV DNA, explaining partly the controversial results reported earlier.