Post-mortem Plasma Cell-Free DNA Sequencing: Proof-of-Concept Study for the "Liquid Autopsy".
Ontology highlight
ABSTRACT:
SUBMITTER: Takai E
PROVIDER: S-EPMC7005783 | biostudies-literature | 2020 Feb
REPOSITORIES: biostudies-literature
Publications
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]
Similar Datasets
Project description:Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1-10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.
| S-EPMC6624284 | biostudies-literature
Project description:Human cytomegalovirus (HCMV) primary infections of pregnant women can lead to congenital infections of the fetus that could have severe impacts on the health of the newborn. Recent studies have shown that 10-100?billion DNA fragments per milliliter of plasma are circulating cell-free. The study of this DNA has rapidly expanding applications to non-invasive prenatal testing (NIPT). In this study, we have shown that we can detect viral specific reads in the massively parallel shotgun sequencing (MPSS) NIPT data. We have also observed a strong correlation between the viral load of calibration samples and the number of reads aligned on the reference genome. Based on these observations we have constructed a statistical model able to quantify the viral load of patient samples. We propose to use this new method to detect and quantify circulating DNA virus like HCMV during pregnancy using the same sequencing results as NIPT data. This method could be used to improve the NIPT diagnosis.
| S-EPMC5847603 | biostudies-literature
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.
| S-EPMC5506259 | biostudies-literature
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.
| S-EPMC6829814 | biostudies-literature
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.
| S-EPMC6147047 | biostudies-literature
Project description:This research was designed as a pilot proof-of-concept study to evaluate the use of low-dose methadone in post-herpetic neuralgia patients who remained refractory after first and second line post-herpetic neuralgia treatments and had indications for adding an opioid agent to their current drug regimens.This cross-over study was double blind and placebo controlled. Ten opioid naïve post-herpetic neuralgia patients received either methadone (5 mg bid) or placebo for three weeks, followed by a 15-day washout period and a second three-week treatment with either methadone or placebo, accordingly. Clinical evaluations were performed four times (before and after each three-week treatment period). The evaluations included the visual analogue scale, verbal category scale, daily activities scale, McGill pain questionnaire, adverse events profile, and evoked pain assessment. All patients provided written informed consent before being included in the study. ClinicalTrials.gov: NCT01752699 RESULTS: Methadone, when compared to placebo, did not significantly affect the intensity of spontaneous pain, as measured by the visual analogue scale. The intensity of spontaneous pain was significantly decreased after the methadone treatment compared to placebo on the category verbal scale (50% improved after the methadone treatment, none after the placebo, p=0.031). Evoked pain was reduced under methadone compared to placebo (50% improved after the methadone treatment, none after the placebo, p=0.031). Allodynia reduction correlated with sleep improvement (r=0.67, p=0.030) during the methadone treatment. The side effects profile was similar between both treatments.Methadone seems to be safe and efficacious in post-herpetic neuralgia. It should be tried as an adjunctive treatment for post-herpetic neuralgia in larger prospective studies.
| S-EPMC3714859 | biostudies-literature
Project description:Public 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.This 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.We 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).We 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.Policy Research Programme, Department of Health, UK.
| S-EPMC3262166 | biostudies-literature
Project description:Cell-free mitochondrial DNA (cfmtDNA) is detectable in almost all human body fluids and has been associated with the onset and progression of several complex traits. In-life assessments indicate that reduced cfmtDNA is a feature of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. However, whether this feature is conserved across all neurodegenerative diseases and how it relates to the neurodegenerative processes remains unclear. In this study, we assessed the levels of ventricular cerebrospinal fluid-cfmtDNA (vCSF-cfmtDNA) in a diverse group of neurodegenerative diseases (NDDs) to determine if the in-life observations of reduced cfmtDNA seen in lumbar CSF translated to the post-mortem ventricular CSF. To investigate further, we compared vCSF-cfmtDNA levels to known protein markers of neurodegeneration, synaptic vesicles and mitochondrial integrity. Our data indicate that reduced vCSF-cfmtDNA is a feature specific to Parkinson's and appears consistent throughout the disease course. Interestingly, we observed increased vCSF-cfmtDNA in the more neuropathologically severe NDD cases, but no association to protein markers of neurodegeneration, suggesting that vCSF-cfmtDNA release is more complex than mere cellular debris produced following neuronal death. We conclude that vCSF-cfmtDNA is reduced in PD, but not other NDDs, and appears to correlate to pathology. Although its utility as a prognostic biomarker is limited, our data indicate that higher levels of vCSF-cfmtDNA is associated with more severe clinical presentations; suggesting that it is associated with the neurodegenerative process. However, as vCSF-cfmtDNA does not appear to correlate to established indicators of neurodegeneration or indeed indicators of mitochondrial mass, further work to elucidate its exact role is needed.
| S-EPMC7499424 | biostudies-literature
Project description:BackgroundNew strategies for collecting post-mortem tissue are necessary, particularly in areas with emerging infections. Minimally invasive autopsy (MIA) has been proposed as an alternative to conventional autopsy (CA), with promising results. Previous studies using MIA addressed the cause of death in adults and children in developing countries. However, none of these studies was conducted in areas with an undergoing infectious disease epidemic. We have recently experienced an epidemic of yellow fever (YF) in Brazil. Aiming to provide new information on low-cost post-mortem techniques that could be applied in regions at risk for infectious outbreaks, we tested the efficacy of ultrasound-guided MIA (MIA-US) in the diagnosis of patients who died during the epidemic.Methodology/principal findingsIn this observational study, we performed MIA-US in 20 patients with suspected or confirmed YF and compared the results with those obtained in subsequent CAs. Ultrasound-guided biopsies were used for tissue sampling of liver, kidneys, lungs, spleen, and heart. Liver samples from MIA-US and CA were submitted for RT-PCR and immunohistochemistry for detection of YF virus antigen. Of the 20 patients, 17 had YF diagnosis confirmed after autopsy by histopathological and molecular analysis. There was 100% agreement between MIA-US and CA in determining the cause of death (panlobular hepatitis with hepatic failure) and main disease (yellow fever). Further, MIA-US obtained samples with good quality for molecular studies and for the assessment of the systemic involvement of the disease. Main extrahepatic findings were pulmonary hemorrhage, pneumonia, acute tubular necrosis, and glomerulonephritis. One patient was a 24-year-old, 27-week pregnant woman; MIA-US assessed the placenta and provided adequate placental tissue for analysis.ConclusionsMIA-US is a reliable tool for rapid post-mortem diagnosis of yellow fever and can be used as an alternative to conventional autopsy in regions at risk for hemorrhagic fever outbreaks with limited resources to perform complete diagnostic autopsy.
| S-EPMC6675127 | biostudies-literature
Project description:Major questions of neurological and psychiatric mechanisms involve the brain functions on a molecular level and cannot be easily addressed due to limitations in access to tissue samples. Post mortem studies are able to partly bridge the gap between brain tissue research retrieved from animal trials and the information derived from peripheral analysis (e.g., measurements in blood cells) in patients. Here, we wanted to know how fast DNA degradation is progressing under controlled conditions in order to define thresholds for tissue quality to be used in respective trials. Our focus was on the applicability of partly degraded samples for bisulfite sequencing and the determination of simple means to define cut-off values. After opening the brain cavity, we kept two consecutive pig skulls at ambient temperature (19-21°C) and removed cortex tissue up to a post mortem interval (PMI) of 120 h. We calculated the percentage of degradation on DNA gel electrophoresis of brain DNA to estimate quality and relate this estimation spectrum to the quality of human post mortem control samples. Functional DNA quality was investigated by bisulfite sequencing of two functionally relevant genes for either the serotonin receptor 5 (SLC6A4) or aldehyde dehydrogenase 2 (ALDH2). Testing our approach in a heterogeneous collective of human blood and brain samples, we demonstrate integrity of measurement quality below the threshold of 72 h PMI. While sequencing technically worked for all timepoints irrespective of conceivable DNA degradation, there is a good correlation between variance of methylation to degradation levels documented in the gel (R (2) = 0.4311, p = 0.0392) for advancing post mortem intervals (PMI). This otherwise elusive phenomenon is an important prerequisite for the interpretation and evaluation of samples prior to in-depth processing via an affordable and easy assay to estimate identical sample quality and thereby comparable methylation measurements.
| S-EPMC4435253 | biostudies-literature