Oil and natural gas operations have been shown to contaminate surface and ground water with endocrine-disrupting chemicals. In the current study, we fill several gaps in our understanding of the potential environmental impacts related to this process. We measured the endocrine-disrupting activities of 24 chemicals used and/or produced by oil and gas operations for five nuclear receptors using a reporter gene assay in human endometrial cancer cells. We also quantified the concentration of 16 of these chemicals in oil and gas wastewater samples. Finally, we assessed reproductive and developmental outcomes in male C57BL/6J mice after the prenatal exposure to a mixture of these chemicals. We found that 23 commonly used oil and natural gas operation chemicals can activate or inhibit the estrogen, androgen, glucocorticoid, progesterone, and/or thyroid receptors, and mixtures of these chemicals can behave synergistically, additively, or antagonistically in vitro. Prenatal exposure to a mixture of 23 oil and gas operation chemicals at 3, 30, and 300 μg/kg · d caused decreased sperm counts and increased testes, body, heart, and thymus weights and increased serum testosterone in male mice, suggesting multiple organ system impacts. Our results suggest possible adverse developmental and reproductive health outcomes in humans and animals exposed to potential environmentally relevant levels of oil and gas operation chemicals.

We conducted a critical review of human papillomavirus (HPV) integration into the host genome in oral/oropharyngeal cancer, reviewed the literature for HPV-induced cancers, and obtained current data for HPV-related oral and oropharyngeal cancers. In addition, we performed studies to identify HPV integration sites and the relationship of integration to viral-host fusion transcripts and whether integration is required for HPV-associated oncogenesis. Viral integration of HPV into the host genome is not required for the viral life cycle and might not be necessary for cellular transformation, yet HPV integration is frequently reported in cervical and head and neck cancer specimens. Studies of large numbers of early cervical lesions revealed frequent viral integration into gene-poor regions of the host genome with comparatively rare integration into cellular genes, suggesting that integration is a stochastic event and that site of integration may be largely a function of chance. However, more recent studies of head and neck squamous cell carcinomas (HNSCCs) suggest that integration may represent an additional oncogenic mechanism through direct effects on cancer-related gene expression and generation of hybrid viral-host fusion transcripts. In HNSCC cell lines as well as primary tumors, integration into cancer-related genes leading to gene disruption has been reported. The studies have shown that integration-induced altered gene expression may be associated with tumor recurrence. Evidence from several studies indicates that viral integration into genic regions is accompanied by local amplification, increased expression in some cases, interruption of gene expression, and likely additional oncogenic effects. Similarly, reported examples of viral integration near microRNAs suggest that altered expression of these regulatory molecules may also contribute to oncogenesis. Future work is indicated to identify the mechanisms of these events on cancer cell behavior.

Background: The molecular drivers of human papillomavirus-related head and neck squamous cell carcinoma (HPV + HNSCC) are not entirely understood. This study evaluated the relationship between HPV integration, expression of E6/E7, and patient outcomes in p16+ HNSCCs. Methods: HPV type was determined by HPV PCR-MassArray, and integration was called using detection of integrated papillomavirus sequences polymerase chain reaction (PCR). We investigated whether fusion transcripts were produced by reverse transcriptase polymerase chain reaction (RT-PCR). E6/E7 expression was assessed by quantitative RT-PCR. We assessed if there was a relationship between integration and E6/E7 expression, clinical variables, or patient outcomes. Results: Most samples demonstrated HPV integration, which sometimes resulted in a fusion transcript. HPV integration was positively correlated with age at diagnosis and E6/E7 expression. There was a significant difference in survival between patients with vs without integration. Conclusions: Contrary to previous reports, HPV integration was associated with improved patient survival. Therefore, HPV integration may act as a molecular marker of good prognosis. K E Y W O R D S head and neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV), integration, oropharynx, survival

Background Sinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown. Methods We evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788–6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion. Results Overall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/− CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling. Conclusion Collectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

Background and objectives Human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) is increasing globally. In Taiwan, HPV-positive OPSCC is obscured by tobacco, alcohol, and betel quid use. We investigated the role of high-risk HPV (hrHPV) in a large retrospective Taiwan OPSCC cohort. Methods and results The cohort of 541 OPSCCs treated at Chang Gung Memorial Hospital from 1998–2016 consisted of 507 men (94%) and 34 women (6%). Most used tobacco (81%), alcohol (51%), and betel quid (65%). Formalin-fixed, paraffin-embedded tissue was used for p16 staining (a surrogate marker for HPV) and testing for HPV DNA presence and type by Multiplex HPV PCR-MassArray. HPV DNA and/or p16 staining (HPV-positive) was found in 28.4% (150/528) tumors. p16 and HPV DNA were strongly correlated (F < 0.0001). HPV16 was present in 82.8%, and HPV58 in 7.5% of HPV-positive tumors. HPV was associated with higher age (55.5 vs. 52.7 years, p = 0.004), lower T-stage (p = 0.008) better overall survival (OS) (hazard ratio [HR] 0.58 [95% CI 0.42–0.81], p = 0.001), and disease-free survival (DFS) (HR 0.54 [95% CI 0.40–0.73], p < 0.0001). Alcohol was strongly associated with recurrence and death (OS: HR 2.06 [95% CI 1.54–2.74], p < 0.0001; DFS: HR 1.72 [95% CI 1.33–2.24], p < 0.0001). OS and DFS in HPV-positive cases decreased for alcohol users (p < 0.0001). Obscured by the strong alcohol effect, predictive associations were not found for tobacco or betel quid. Conclusions As with HPV-positive OPSCC globally, HPV is an increasingly important etiological factor in Taiwanese OPSCC. HPV-positive OPSCC has considerable survival benefit, but this is reduced by alcohol, tobacco, and betel quid use. hrHPV is a cancer risk factor in males and females. Vaccinating both sexes with a multivalent vaccine including HPV58, combined with alcohol and tobacco cessation policies will be effective cancer-prevention public health strategies in Taiwan.

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and CD36 as targets of viral integration are involved in cell migration and ECM-receptor interactions, which provides a possible mechanism for clonal migration from one tonsil to another.

Background Human papillomavirus (HPV) is a well‐established driver of malignant transformation at a number of sites, including head and neck, cervical, vulvar, anorectal, and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discovering viral‐host breakpoints from low‐read‐coverage sequencing data. Methods To overcome this limitation, the authors developed SearcHPV, a new HPV detection pipeline based on targeted capture technology, and applied the algorithm to targeted capture data. They performed an integrated analysis of SearcHPV‐defined breakpoints with genome‐wide linked‐read sequencing to identify potential HPV‐related structural variations. Results Through an analysis of HPV+ models, the authors showed that SearcHPV detected HPV‐host integration sites with a higher sensitivity and specificity than 2 other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer‐related genes, including TP63, MYC, and TRAF2, and near regions of large structural variation. The authors further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral‐host junction breakpoint sequences. They found that viral integration occurred through a variety of DNA repair mechanisms, including nonhomologous end joining, alternative end joining, and microhomology‐mediated repair. Conclusions In summary, SearcHPV is a new optimized tool for the accurate detection of HPV‐human integration sites from targeted capture DNA sequencing data.

Background: Sinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.Methods: We evaluate survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We perform exome sequencing to characterize a series of SMARCB1 wild type tumors and cell line including identification of high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA was utilized for validation of a novel PGAP3-SRPK1 gene fusion. Results: We discover recurrent aberrations to the SWI/SNF and FAT gene families. We also validate a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling. Conclusion: Collectively, these data demonstrate recurrent alterations in the SWI/SNF and FAT gene families and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.