Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling.

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Nitrated Polycyclic Aromatic Hydrocarbon (Nitro-PAH) Signatures and Somatic Mutations in Diesel Exhaust-Exposed Bladder Tumors.

BACKGROUND: Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors. METHODS: Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures. RESULTS: We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2-10.5; Ptrend < 0.001; Pheterogeneity = 0.002]. In muscle-invasive tumors, we observed a positive association between diesel exposure and the nitro-PAH signatures of 1,6-dintropyrene (RR, 1.93; 95% CI, 1.28-2.92) and 3-nitrobenzoic acid (RR, 1.97; 95% CI, 1.33-2.92). CONCLUSIONS: The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer. IMPACT: This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Analysis of Several Common APOBEC-type Mutations in Bladder Tumors Suggests Links to Viral Infection.

FGFR3 and PIK3CA are among the most frequently mutated genes in bladder tumors. We hypothesized that recurrent mutations in these genes might be caused by common carcinogenic exposures such as smoking and other factors. We analyzed 2,816 bladder tumors with available data on FGFR3 and/or PIK3CA mutations, focusing on the most recurrent mutations detected in ≥10% of tumors. Compared to tumors with other FGFR3/PIK3CA mutations, FGFR3-Y375C was more common in tumors from smokers than never-smokers (P = 0.009), while several APOBEC-type driver mutations were enriched in never-smokers: FGFR3-S249C (P = 0.013) and PIK3CA-E542K/PIK3CA-E545K (P = 0.009). To explore possible causes of these APOBEC-type mutations, we analyzed RNA sequencing (RNA-seq) data from 798 bladder tumors and detected several viruses, with BK polyomavirus (BKPyV) being the most common. We then performed IHC staining for polyomavirus (PyV) Large T-antigen (LTAg) in an independent set of 211 bladder tumors. Overall, by RNA-seq or IHC-LTAg, we detected PyV in 26 out of 1,010 bladder tumors with significantly higher detection (P = 4.4 × 10-5), 25 of 554 (4.5%) in non-muscle-invasive bladder cancers (NMIBC) versus 1 of 456 (0.2%) of muscle-invasive bladder cancers (MIBC). In the NMIBC subset, the FGFR3/PIK3CA APOBEC-type driver mutations were detected in 94.7% (18/19) of PyV-positive versus 68.3% (259/379) of PyV-negative tumors (P = 0.011). BKPyV tumor positivity in the NMIBC subset with FGFR3- or PIK3CA-mutated tumors was also associated with a higher risk of progression to MIBC (P = 0.019). In conclusion, our results support smoking and BKPyV infection as risk factors contributing to bladder tumorigenesis in the general patient population through distinct molecular mechanisms. PREVENTION RELEVANCE: Tobacco smoking likely causes one of the most common mutations in bladder tumors (FGFR3-Y375C), while viral infections might contribute to three others (FGFR3-S249C, PIK3CA-E542K, and PIK3CA-E545K). Understanding the causes of these mutations may lead to new prevention and treatment strategies, such as viral screening and vaccination.

Insights into the Mechanisms and Structure of Breakage-Fusion-Bridge Cycles in Cervical Cancer using Long-Read Sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and this is one of the first analyses of these events using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes and only one BFB breakpoint showed chromothripsis. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Indeed, YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that cervical cancer patients with YAP1/BIRC2/BIRC3-amplification, especially those of African American ancestry, might benefit from targeted therapy. In summary, we uncovered new insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Targeted Deep Sequencing of Bladder Tumors Reveals Novel Associations between Cancer Gene Mutations and Mutational Signatures with Major Risk Factors.

PURPOSE: Exome- and whole-genome sequencing of muscle-invasive bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non-muscle-invasive bladder cancer with detailed risk factor information. EXPERIMENTAL DESIGN: We examined the relationship between smoking and other bladder cancer risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in bladder cancer was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case-control study. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RR) and 95% CIs, evaluating signatures and risk factors. RESULTS: Non-silent KDM6A mutations were more common in females than males (OR = 1.83; 95% CI, 1.05-3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2-Signature mutations compared with former (P = 0.024) and never smoking (RR = 1.40; 95% CI, 1.09-1.80; P = 0.008), former smoking was associated with greater APOBEC-Signature13 mutations (P = 0.05), and never smoking was associated with greater APOBEC-Signature2 mutations (RR = 1.54; 95% CI, 1.17-2.01; P = 0.002). There was evidence that smoking duration (the component most strongly associated with bladder cancer risk) was associated with ERCC2-Signature mutations and APOBEC-Signature13 mutations among current (P trend = 0.005) and former smokers (P = 0.0004), respectively. CONCLUSIONS: These data quantify the contribution of bladder cancer risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers.

Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells.

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells-a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes--activation of ISGs, decreased proliferation, and increased cell death--could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance.