Post-mortem Plasma Cell-Free DNA Sequencing: Proof-of-Concept Study for the \u201cLiquid Autopsy\u201d

Erina Takai,Misato Kirikawa,Mamoru Kato,Yasushi Totoki,Daichi Maeda,Hiromi Nakamura,Yukitsugu Kudo‐Asabe ,Makoto Yoshida,Shinichi Yachida,Shohei Ikoma,Hiroto Katoh,Shumpei Ishikawa,Tatsuhiro Shibata,Tomonori Habuchi,Yukinobu Ito,Zhuo Li,Takamitsu Inoue,R Nakamura ,Akiko Nakamura ,Akiteru Goto

doi:10.1038/s41598-020-59193-y

Abstract

Recent genomic studies on cancer tissues obtained during rapid autopsy have provided insights into the clonal evolution and heterogeneity of cancer. However, post-mortem blood has not been subjected to genetic analyses in relation to cancer. We first confirmed that substantial quantities of cell-free DNA were present in the post-mortem plasma of 12 autopsy cases. Then, we focused on a pilot case of prostate cancer with multiple metastases for genetic analyses. Whole-exome sequencing of post-mortem plasma-derived cell-free DNA and eight frozen metastatic cancer tissues collected during rapid autopsy was performed, and compared their mutational statuses. The post-mortem plasma cell-free DNA was successfully sequenced and 344 mutations were identified. Of these, 160 were detected in at least one of the metastases. Further, 99% of the mutations shared by all metastases were present in the plasma. Sanger sequencing of 30 additional formalin-fixed metastases enabled us to map the clones harboring mutations initially detected only in the plasma. In conclusion, post-mortem blood, which is usually disposed of during conventional autopsies, can provide valuable data if sequenced in detail, especially regarding cancer heterogeneity. Furthermore, post-mortem plasma cell-free DNA sequencing (liquid autopsy) can be a novel platform for cancer research and a tool for genomic pathology.

Highlights

Evidence of the clonal evolution of cancer is rapidly accumulating, and tumor heterogeneity is recognised as a critical issue in the era of personalised cancer medicine[1,2]
All metastases were composed of poorly differentiated carcinoma that grew in solid nests and sheets (Fig. 2)
We investigated the contribution of tumor DNA from different lesions to post-mortem plasma cell-free DNA (cfDNA) by comparing the variant allele frequencies (VAFs) of mutations that were unique to a single metastasis

Summary

Introduction

Evidence of the clonal evolution of cancer is rapidly accumulating, and tumor heterogeneity is recognised as a critical issue in the era of personalised cancer medicine[1,2]. To precisely evaluate the clonal evolution and heterogeneity of cancer, sequence analysis of multiple cancer lesions is essential. The genotypes of cell-free DNA (cfDNA) in post-mortem blood samples have never been analyzed in relation to cancer. Analyses of cfDNA are expected to provide an overview of the somatic changes that occur in multiple cancer clones involving various organs, including primary and metastatic sites. We analyzed cfDNA in post-mortem plasma by next-generation sequencing. We further assessed the feasibility of post-mortem cfDNA sequencing and examined the relationship between the mutational status of cfDNA and systemic tumor heterogeneity in a pilot case of prostate cancer. We discuss the significance of post-mortem plasma sequencing, the concept of “liquid autopsy,” and the potential of liquid autopsy data to expand the applications of liquid biopsies

Methods

Results

Conclusion