HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analysed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines, and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression, and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

The 8p12 locus (containing the FGFR1 tyrosine kinase gene) is frequently amplifi ed in squamous cell lung cancer. However, it is currently unknown which of the 8p12-amplifi ed tumors are also sensitive to fi broblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifi cations, the 8p12 region included multiple centers of amplifi cation, suggesting marked genomic heterogeneity. FGFR1 -amplifi ed tumor cells were dependent on FGFR ligands in vitro and in vivo . Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of FGFR1 -amplifi ed tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with FGFR1-amplifi ed and MYC-overexpressing tumors may benefi t from FGFR inhibitor therapy. Thus, both cell-autonomous and non-cell-autonomous mechanisms of transformation modulate FGFR dependency in FGFR1 -amplifi ed lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors. SIGNIFICANCE:Amplifi cation of FGFR1 is one of the most frequent candidate targets in lung cancer. Here, we show that multiple factors affect the tumorigenic potential of FGFR1 , thus providing clinical hypotheses for refi nement of patient selection. Cancer Discov; 4(2);

Cutaneous human papillomaviruses (HPV) have been reported in cutaneous squamous cell carcinoma (SCC). We conducted a clinic-based case–control study to investigate the association between genus-beta HPV DNA in eyebrow hairs (EBH) and SCC. EBH from 168 SCC cases and 290 controls were genotyped for genus-beta HPV DNA. SCC tumors from a subset of cases (n = 142) were also genotyped. Viral load was determined in a subset of specimens positive for a single HPV type. Associations with SCC were estimated by odds ratios (OR) and 95% confidence intervals (CI) adjusted for age and sex using logistic regression. Statistical tests were two-sided. EBH DNA prevalence was greater in cases (87%) than controls (73%) (p < 0.05), and the association with SCC increased with the number of HPV types present, (≥4 types vs. HPV-negative: OR = 2.02, 95% CI = 1.07–3.80; ptrend = 0.02). Type-specific associations were observed between SCC and DNA in EBH for HPV23 (OR = 1.90, 95% CI = 1.10–3.30) and HPV38 (OR = 1.84, 95% CI = 1.04–3.24). Additionally, when compared with the controls, the DNA prevalence in EBH was significantly higher among cases for 11 of the 25 genus-beta types tested, when accounting for DNA for the same HPV type in the tumor (ORs = 3.44–76.50). Compared to controls, the mean viral DNA load in EBH among the selected cases was greater for HPV5, HPV8 and HPV24, but lower for HPV38. SCC cases were more likely than controls to have HPV DNA+ EBH for single and multiple HPV types, providing additional support for the potential role of genus-beta HPV infections in SCC development.