Abstract 3147: Non-invasive detection of aristolochic acid exposure using ultra-sensitive duplex sequencing
Authors: Lo Fang Yin , Valentine Charles , Schmidt Elizabeth , Williams Lindsey , Boot Arnoud , Rozen Steve , Salk Jesse 
Published: September 2020

Aristolochic acids (AA) are a potent class of mutagen that occur in plants of the genera Aristolochia and Asarum. Exposures to AA are caused both by intentional ingestion of traditional herbal medicines containing Aristolochia and through inadvertent consumption from contaminated crops. The carcinogenic potential and epidemiologic connection of AA exposure with upper-tract urothelial cancers (UTUC), liver cancer and kidney failure have been known for years; however, the extent of AA exposure and its influence on cancer risk and mortality have yet to be fully explored in many world populations.

Using whole exome sequencing of tumor samples, a recent study determined that 78% of HCCs from Taiwan exhibit distinct mutation signatures of AA exposure (Ng, Poon, Huang, et al., 2017). Exposure to AA is geographically widespread and there is a major opportunity for primary and secondary prevention of AA-associated diseases if it were possible to detect the exposure through non-invasively sampled body fluids such as blood or urine sediment.

Although AA adducts can be measured in body fluids, this measurement only reflects recent ingestion, not the life-integrated exposure that better approximates disease risk. A mutation burden-based assay could circumvent this problem; however, a major challenge is that AA-mediated mutations are extremely rare in bulk tissue or fluid samples from non-tumor sources. Duplex Sequencing (DS) is a tag-based NGS error-correction method that enables the detection of ultra-rare mutations that exist at the mutation frequency of normal tissues (~10e-8). DS can be applied to DNA from any tissue of any organism without the need for tumor formation or ex vivo cloning.

We developed a DS-based assay that measures mutations across a 50 kb genome-representative panel and applied it to the tissues and liquid biopsies of cancer patients of known AA-exposure status. We sampled the blood, urine, kidney, normal-adjacent urothelium and ureteral tumors from 12 UTUC patients where 6 of the patients were exposed to AA, and 6 were not.

Under blinded conditions, we positively identified AA mutation signatures in the normal adjacent urothelium tissues of 5 AA-exposed patients. Remarkably, we also detected AA signatures in all available blood and urine samples from the same patients. The proportion of mutations attributed to AA-exposure ranged from 2.5% in blood to 67.8% in kidney. These mutations, on the order of one-in-a-million, were exposure-related, not tumor derived. We did not detect AA mutation signatures in any samples from the non-AA-exposed patients.

To our knowledge, this is the first time an AA mutation signature has been detected in non-invasively sampled body fluids. DS is a promising approach for the detection of mutagenic signatures caused by environmental carcinogens, and we foresee it being a powerful metric that can be used to measure life-integrated carcinogenic processes and cancer risk.

Citation Format: Fang Yin Lo, Charles Valentine, Elizabeth Schmidt, Lindsey Williams, Arnoud Boot, Steve Rozen, Jesse Salk. Non-invasive detection of aristolochic acid exposure using ultra-sensitive duplex sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3147.