Post-mortem Plasma Cell-Free DNA Sequencing: Proof-of-Concept Study for the "Liquid Autopsy"

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.

Figure 1

Size distributions of cell-free DNA (cfDNA) extracted from post-mortem plasma. cfDNA extracted from 12 post-mortem plasma samples was analyzed using the Agilent 2100 Bioanalyzer DNA 1000 kit (Agilent Technologies, Santa Clara, CA). The electropherograms showed a fragmented pattern with integral multiples the size of a nucleosome plus linker DNA, which is the typical size distribution of cfDNA.

Figure 2

Pathologic features of the prostatic cancer case. (a) Prostate core needle biopsy. Histologically, adenocarcinoma with solid and glandular structures was observed. The tumor cells were negative for ERG based on immunohistochemistry analyses (inset). (b) Prostate at autopsy. Macroscopically, the prostate was of normal size (inset). Histologically, scattered foci of cancer with degenerative changes were present. (c) Liver at autopsy. The liver was extremely enlarged with numerous cancer metastases. (d) Histology of the liver metastasis. The metastatic lesion was predominantly composed of poorly differentiated carcinoma growing in solid nests. (e) Vertebrae at autopsy. Whitish and sclerotic changes were observed in almost all vertebrae, suggestive of osteogenic metastases. (f) Histology of the vertebral metastasis. Thick trabeculae were formed, and the intratrabecular spaces were filled with cancer cells. (g,h) Histology of the lymph node metastases. Poorly differentiated carcinoma composed of atypical cells with enlarged nuclei growing in sheets was observed.

Figure 3

Evaluation of somatic mutations detected in cell-free DNA (cfDNA) by whole-exome sequencing. (a) Composition of mutations detected in plasma cfDNA. The classification of mutations was based on whole-exome sequences from eight frozen metastatic tumor tissues (Table 1). This figure is drawn using Excel for Mac (16.16.18) and PowerPoint for Mac (16.16.18), Microsoft Cooperation, Redmond, WA, USA. (b,c) Variant allele frequencies (VAFs) of mutations detected in cfDNA. Box plots indicate the VAF values of mutations classified by category. Each box shows the median (central line), inter-quartile range (IQR; box), and ±1.5 × IQR (whiskers). Mutations classified as “unique to one” (b) were subdivided by tumor sample and are shown in panel (c).

Figure 4

Sanger sequencing of the DNA extracted from formalin-fixed paraffin-embedded metastases. (a) Systemic distribution of clones harboring representative cell-free DNA (cfDNA)-specific mutations (N = 8) and mutations commonly detected in eight frozen metastases (N = 3). WT, wild type; NA, data not available due to unsuccessful sequencing. This figure is drawn using Excel 2016 and PowerPoint 2016, Microsoft Cooperation, Redmond, WA, USA. (b) One of the cfDNA-specific mutations, PRKAR1A (c. 996T > G), was identified in one of the liver metastases (Liver 10). Other metastatic nodules, even those that were in close proximity (Liver 11, 12, and 13), harbored wild-type PRKAR1A. Another cfDNA-specific mutation, BCL11B (c. 391G > A), was identified in one of the paraaortic lymph node metastases.

Figure 5

Scheme of liquid autopsy. The concept of liquid autopsy is illustrated and compared to conventional autopsy and rapid autopsy. FFPE, formalin-fixed paraffin-embedded; WES, whole exome sequencing; WGS, whole genome sequencing. This figure is drawn using PowerPoint 2016, Microsoft Cooperation, Redmond, WA, USA.