Dataset Information

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.

SUBMITTER: Takai E

PROVIDER: S-EPMC7005783 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Takai Erina E Maeda Daichi D Li Zhuo Z Kudo-Asabe Yukitsugu Y Totoki Yasushi Y Nakamura Hiromi H Nakamura Akiko A Nakamura Rumi R Kirikawa Misato M Ito Yukinobu Y Yoshida Makoto M Inoue Takamitsu T Habuchi Tomonori T Ikoma Shohei S Katoh Hiroto H Kato Mamoru M Shibata Tatsuhiro T Ishikawa Shumpei S Yachida Shinichi S Goto Akiteru A

Scientific reports 20200207 1

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-mort ...[more]

PMID: 32034265

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Project description:BackgroundEngland and Wales have one of the highest frequencies of autopsy in the world. Implementation of post-mortem CT (PMCT), enhanced with targeted coronary angiography (PMCTA), in adults to avoid invasive autopsy would have cultural, religious, and potential economic benefits. We aimed to assess the diagnostic accuracy of PMCTA as a first-line technique in post-mortem investigations.MethodsIn this single-centre (Leicester, UK), prospective, controlled study, we selected cases of natural and non-suspicious unnatural death referred to Her Majesty's (HM) Coroners. We excluded cases younger than 18 years, known to have had a transmittable disease, or who weighed more than 125 kg. Each case was assessed by PMCTA, followed by autopsy. Pathologists were masked to the PMCTA findings, unless a potential risk was shown. The primary endpoint was the accuracy of the cause of death diagnosis from PMCTA against a gold standard of autopsy findings, modified by PMCTA findings only if additional substantially incontrovertible findings were identified.FindingsBetween Jan 20, 2010, and Sept 13, 2012, we selected 241 cases, for which PMCTA was successful in 204 (85%). Seven cases were excluded from the analysis because of procedural unmasking or no autopsy data, as were 24 cases with a clear diagnosis of traumatic death before investigation; 210 cases were included. In 40 (19%) cases, predictable toxicology or histology testing accessible by PMCT informed the result. PMCTA provided a cause of death in 193 (92%) cases. A major discrepancy with the gold standard was noted in 12 (6%) cases identified by PMCTA, and in nine (5%) cases identified by autopsy (because of specific findings on PMCTA). The frequency of autopsy and PMCTA discrepancies were not significantly different (p=0·65 for major discrepancies and p=0·21 for minor discrepancies). Cause of death given by PMCTA did not overlook clinically significant trauma, occupational lung disease, or reportable disease, and did not significantly affect the overall population data for cause of death (p≥0·31). PMCTA was better at identifying trauma and haemorrhage (p=0·008), whereas autopsy was better at identifying pulmonary thromboembolism (p=0·004).InterpretationFor most sudden natural adult deaths investigated by HM Coroners, PMCTA could be used to avoid invasive autopsy. The gold standard of post-mortem investigations should include both PMCT and invasive autopsy.FundingNational Institute for Health Research.

Project description:BACKGROUND:During human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases. METHODS:Maternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data. RESULTS:EVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations. CONCLUSIONS:We proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.

Project description:BackgroundAlthough total plasma lipoproteome consists of proteins that have shown promises as biomarkers that can identify Alzheimer's disease (AD), effect sizes are modest. The objective of this study is to provide initial proof-of-concept that the plasma lipoproteome more likely differ between AD cases and controls when measured in individual plasma lipoprotein fractions than when measured as total in immunodepleted plasma.MethodsWe first developed a targeted proteomics method based on selected reaction monitoring (SRM) and liquid chromatography and tandem mass spectrometry for measurement of 120 tryptic peptides from 79 proteins that are commonly present in plasma lipoproteins. Then in a proof-of concept case-control study of 5 AD cases and 5 sex- and age-matched controls, we applied the targeted proteomic method and performed relatively quantification of 120 tryptic peptides in plasma lipoprotein fractions (fractionated by sequential gradient ultracentrifugation) and in immunodepleted plasma (of albumin and IgG). Unadjusted p values from two-sample t-tests and overall fold change was used to evaluate a peptide relative difference between AD cases and controls, with lower p values (<?0.05) or greater fold differences (>?1.05 or?<?0.95) suggestive of greater peptide/protein differences.ResultsWithin-day and between-days technical precisions (mean %CV [SD] of all SRM transitions) of the targeted proteomic method were 3.95% (2.65) and 9.31% (5.59), respectively. Between-days technical precisions (mean % CV [SD]) of the entire plasma lipoproteomic workflow including plasma lipoprotein fractionation was 27.90% (14.61). Ten tryptic peptides that belonged to 5 proteins in plasma lipoproteins had unadjusted p values?<?0.05, compared to no peptides in immunodepleted plasma. Furthermore, 27, 32, 17, and 20 tryptic peptides in VLDL, IDL, LDL and HDL, demonstrated overall peptide fold differences?>?1.05 or?<?0.95, compared to only 6 tryptic peptides in immunodepleted plasma. The overall comparisons, therefore, suggested greater peptide/protein differences in plasma lipoproteome when measured in individual plasma lipoproteins than as total in immunodepleted plasma. Specifically, protein complement C3's peptide IHWESASLLR, had unadjusted p values of 0.00007, 0.00012, and 0.0006 and overall 1.25, 1.17, 1.14-fold changes in VLDL, IDL, and LDL, respectively. After positive False Discovery Rate (pFDR) adjustment, the complement C3 peptide IHWESASLLR in VLDL remained statistically different (adjusted p value?<?0.05).DiscussionThe findings may warrant future studies to investigate plasma lipoproteome when measured in individual plasma lipoprotein fractions for AD diagnosis.

Project description:BackgroundPublic objection to autopsy has led to a search for minimally invasive alternatives. Imaging has potential, but its accuracy is unknown. We aimed to identify the accuracy of post-mortem CT and MRI compared with full autopsy in a large series of adult deaths.MethodsThis study was undertaken at two UK centres in Manchester and Oxford between April, 2006, and November, 2008. We used whole-body CT and MRI followed by full autopsy to investigate a series of adult deaths that were reported to the coroner. CT and MRI scans were reported independently, each by two radiologists who were masked to the autopsy findings. All four radiologists then produced a consensus report based on both techniques, recorded their confidence in cause of death, and identified whether autopsy was needed.FindingsWe assessed 182 unselected cases. The major discrepancy rate between cause of death identified by radiology and autopsy was 32% (95% CI 26-40) for CT, 43% (36-50) for MRI, and 30% (24-37) for the consensus radiology report; 10% (3-17) lower for CT than for MRI. Radiologists indicated that autopsy was not needed in 62 (34%; 95% CI 28-41) of 182 cases for CT reports, 76 (42%; 35-49) of 182 cases for MRI reports, and 88 (48%; 41-56) of 182 cases for consensus reports. Of these cases, the major discrepancy rate compared with autopsy was 16% (95% CI 9-27), 21% (13-32), and 16% (10-25), respectively, which is significantly lower (p<0·0001) than for cases with no definite cause of death. The most common imaging errors in identification of cause of death were ischaemic heart disease (n=27), pulmonary embolism (11), pneumonia (13), and intra-abdominal lesions (16).InterpretationWe found that, compared with traditional autopsy, CT was a more accurate imaging technique than MRI for providing a cause of death. The error rate when radiologists provided a confident cause of death was similar to that for clinical death certificates, and could therefore be acceptable for medicolegal purposes. However, common causes of sudden death are frequently missed on CT and MRI, and, unless these weaknesses are addressed, systematic errors in mortality statistics would result if imaging were to replace conventional autopsy.FundingPolicy Research Programme, Department of Health, UK.

Dataset Information

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

Publications

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

Similar Datasets

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