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

Abstract

Background Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urological 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. Methods Here we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multi-sample 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 aristolochic acid nephropathy. Results 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 ~10x. Analysis at 3–9x 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 non-synonymous 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. Conclusion 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. Impact 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.

Mutational Processes in Hepatocellular Carcinoma: The Story of Aristolochic Acid

“…22,40,51 Moreover, mutational signature analyses using whole exome and whole genome sequencing from a large panel of cancers identified aristolochic acid signature in bladder transitional cell carcinoma, clear renal cell carcinoma, and HCC. 22,51,52 A typical mutational fingerprint of exposure to aristolochic acid has been described with a high number of mutations (median of 233 nonsynonymous mutations per tumor in urothelial carcinoma due to aristolochic acid vs. 70 per tumor in urothelial carcinoma not related to aristolochic acid) with frequent T ! A transversions (also described as A:T to T:A taking into account the two strands of DNA) in a CTG trinucleotide context (mutational signature 22).…”

Genome-scale mutational signatures of aflatoxin in cells, mice and human tumors

“…Genomic DNA was extracted from macro-dissected paraffin sections, and 250 nanograms of each DNA sample were sheared by Covaris (Covaris, Inc.) to ~300 bp fragments, as previously described (Castells et al 2015). We prepared libraries with the Kapa LTP Library Preparation Kit (Kapa Biosystems) according to the manufacturer's recommendations.…”

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

“…( a ) Upper panels: spectra in upper urinary tract urothelial carcinomas (UTUC) of patients from Taiwan, China and from Balkan Endemic nephropathy (BEN) regions of Europe, two populations known to be exposed to AA. 75, 76, 97 The lower panel shows that exposure of Hupki MEF to AA 92 induces a similar mutational profile. Pooled data from multiple samples are shown for each data set.…”

“…95, 96 Another source of experimentally induced genome-wide mutation patterns is potentially available from past in vivo toxicology projects. There is an untapped reservoir of archived tumour samples from animal carcinogen tests that can be mined using robust protocols for extraction and NGS of DNA derived from formalin-fixed, paraffin-embedded tumours already developed for human studies, 77, 97, 98 allowing immediate access to information from this valuable source.…”

“…Two representative cases of upper urinary tract urothelial tumours from regions of either low or high risk of exposure to the carcinogen aristolochic acid 97 were analysed using whole-exome sequencing. The single-base substitution distribution spectra are shown on top.…”