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Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

ABSTRACT: Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors.Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy.LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding.LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas.By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures.

SUBMITTER: Castells X

PROVIDER: S-EPMC4806408 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

Castells Xavier X Karanović Sandra S Ardin Maude M Tomić Karla K Xylinas Evanguelos E Durand Geoffroy G Villar Stephanie S Forey Nathalie N Le Calvez-Kelm Florence F Voegele Catherine C Karlović Krešimir K Mišić Maja M Dittrich Damir D Dolgalev Igor I McKay James J Shariat Shahrokh F SF Sidorenko Viktoria S VS Fernandes Andrea A Heguy Adriana A Dickman Kathleen G KG Olivier Magali M Grollman Arthur P AP Jelaković Bojan B Zavadil Jiri J

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 20150917 12

<h4>Background</h4>Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors.<h4>Methods</h4>Here, we report a low-coverage whole-exome sequenc ...[more]

PMID: 26383547

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Project description:Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk.

Dataset Information

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

Publications

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

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