Detection of tumor-derived DNA mutations in cerebrospinal fluid of patients with primary or metastatic brain tumors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 2070-2070
Author(s):  
Jianfei Wang ◽  
Wenbo Han ◽  
Chen Tian ◽  
Ying Hu ◽  
Yanhui Chen ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Brain Tumors ◽  
Dna Analysis ◽  
Frequency Variation ◽  
Sequencing Data ◽  
Metastatic Brain Tumors ◽  
Single Nucleotide Variants ◽  
Alternative Analysis ◽  
Cell Free Dna ◽  
Free Dna

2070 Background: Because detecting tumor-derived cell free DNA (cfDNA) in the blood of patients with primary or metastatic brain tumors is challenging, here we studied whether cerebrospinal fluid (CSF) could be serve as an alternative “liquid biopsy” by enabling measurement of circulating DNA within CSF to characterize tumor specific mutations. Methods: The paired cfDNA in CSF and plasma were collected from 20 patients with brain tumors and was subjected to enrichment for a 1.15M size panel cover exon regions from 1,086 genes. Followed by next generation sequencing on an Illumina X10 platform, the captured sequencing data was further processed using bioinformatics analysis to identify somatic mutations, including single nucleotide variants (SNV) and short insertions/deletions (indels). Results: The mutation profiles of 48 tumor associated genes in cfDNA were compared between the CSF and plasma. Our results showed that both average somatic mutation number and frequency identified in the cerebrospinal fluid was much higher than that in the corresponding plasma samples (25 vs. 18 & 1.39% vs. 0.55%). Among the twenty cases, one more potential actionable mutation, EGFR exon 19 deletion mutation with a 25.38% allele frequency variation, was only detected in the CSF cfDNA of a patient with brain metastasis lung cancer. Conclusions: Tumor mutations were detectable in CSF cfDNA of patients with different primary and metastatic brain tumors. Thus cerebrospinal fluid cell free DNA analysis could be a potential alternative analysis for patients with primary or metastatic brain tumors.

Urinary cell-free DNA analysis for tumor mutation detection in patients with oligometastatic colorectal cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15545-e15545
Author(s):  
Bruna Pellini ◽  
Nadja Pejovic ◽  
Peter Harris ◽  
Wenjia Feng ◽  
Abul Usmani ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Analysis ◽  
Mononuclear Cells ◽  
Circulating Tumor Dna ◽  
Detection Sensitivity ◽  
Single Nucleotide Variants ◽  
Curative Intent ◽  
Peripheral Blood Mononuclear ◽  
Cell Free Dna ◽  
Free Dna

e15545 Background: Cell-free DNA (cfDNA) analysis from blood is a useful liquid biopsy strategy. However, it requires phlebotomy and moderate volumes of plasma. Here, we explored the feasibility of urinary cfDNA (ucfDNA) analysis in patients with oligometastatic colorectal cancer (CRC). Methods: We applied error-corrected targeted next-generation sequencing (NGS) (AVENIO ctDNA surveillance assay) to 10 patients with oligometastatic CRC to the liver who underwent curative-intent surgery. 40 paired samples (tumor, plasma, urine supernatant, peripheral blood mononuclear cells (PBMCs)) were collected, from which DNA was extracted. Plasma and urine cfDNA samples (acquired preoperatively) were sequenced to a median de-duplicated depth of 8,065X with on-target rate of 71%. Similarly high depth targeted NGS was applied to tumor and PBMC samples. Variants were called from plasma cfDNA and tumoral DNA and queried in ucfDNA using methods derived from CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) with integrated digital error suppression (iDES). Clonal hematopoiesis was accounted for by removing mutations also present in PBMCs. Results: Patients had a median of 2 tumors resected (range 1-7) with average sum of longest diameters of 8.7 cm (range 0.6-11). We used an average of 10 ml of blood and 37 ml of urine for cfDNA extraction. Circulating tumor DNA (ctDNA) was detected in all preoperative plasma samples (n = 10) at a mean mutant allele fraction (MAF) of 2.35% (range 0.09-11.78). Plasma ctDNA MAFs correlated significantly with tumor SLD (Pearson r = 0.81, P = 0.03). The average number of single nucleotide variants detected in plasma cfDNA, tumor and ucfDNA was 8 (range 1-42), 4 (range 1-7) and 2 (range 0-11), respectively. Indicative of concordance, 79% of the mutations called in tumor were detected in plasma. Tumor-derived DNA was detected in the preoperative urine of 6 patients (60%) with a MAF of 0.05% (range 0.03-0.12), ~35-fold less than in plasma (MAF of 1.78%, range 0.09-7.94) (P = 0.004). 38% of mutations identified in tumor and 26% from plasma were detected in ucfDNA for these 6 patients. In two patients, targetable mutations (BRAF p.Asp594Gly and PIK3CA p.Glu545Lys) present in tumor were also detected in ucfDNA, with the latter not detected in plasma. Conclusions: Our results indicate that ucfDNA analysis in patients with oligometastatic CRC is feasible; however, the mutational levels and detection sensitivity were much lower than from plasma. Yet, there may be situations where actionable mutations are missed in plasma but detected in urine.

scholarly journals GENE-17. TUMOR-DERIVED CELL-FREE DNA MAY BE FREQUENTLY DETECTED BY CLINICAL TARGETED SEQUENCING OF CEREBROSPINAL FLUID IN CHILDREN WITH BRAIN TUMORS

2019 ◽  
Vol 21 (Supplement_2) ◽  
pp. ii84-ii85
Author(s):  
Kristine Karvonen ◽  
Christina Lockwood ◽  
Bonnie Cole ◽  
Shannon Stasi ◽  
Jeff Stevens ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Brain Tumors ◽  
Targeted Sequencing ◽  
Cell Free Dna ◽  
Free Dna

Tumor methylation patterns to measure tumor fraction in cell-free DNA.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3052-3052
Author(s):  
Colin Melton ◽  
Pranav Singh ◽  
Oliver Venn ◽  
Earl Hubbell ◽  
Samuel Gross ◽  
...  
Keyword(s):  
Cancer Detection ◽  
Classification Performance ◽  
Whole Genome ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Spearman's Rho ◽  
Spearman’S Rho ◽  
Methylation Patterns

3052 Background: Cell-free DNA (cfDNA) tumor fraction (TF), the proportion of tumor molecules in a cfDNA sample, is a direct measurement of signal for cfDNA cancer applications. The Circulating Cell-free Genome Atlas study (CCGA; NCT02889978) is a prospective, multi-center, observational, case-control study designed to support development of a methylation-based, multi-cancer detection test in which a classifier is trained to distinguish cancer from non-cancer. Here we leveraged CCGA data to examine the relationship between cfDNA containing tumor DNA methylation patterns, TF, and cancer classification performance. Methods: The CCGA classifier was trained on whole-genome bisulfite sequencing (WGBS) and targeted methylation (TM) sequencing data to detect cancer versus non-cancer. 822 samples had biopsy WGBS performed; of those, 231 also had cfDNA targeted methylation (TM) and cfDNA whole-genome sequencing (WGS). Biopsy WGBS identified somatic single nucleotide variants (SNV) and methylation variants (MV; defined as methylation patterns in sequenced DNA fragments observed commonly in biopsy but rarely [ < 1/10,000] in the cfDNA of non-cancer controls [n = 898]). Observed tumor fragment counts (SNV in WGS; MV in TM), were modeled as a Poisson process with rate dependent on TF. TF and classifier limits of detection (LOD) were each assessed using Bayesian logistic regression. Results: Across biopsy samples, a median of 2,635 MV was distributed across the genome, with a median of 86.8% shared with ≥1 participant, and a median of 69.3% targeted by the TM assay. TF LOD from MV was 0.00050 (95% credible interval [CI]: 0.00041 - 0.00061); MV and SNV estimates were concordant (Spearman’s Rho: 0.820). MV TF estimates explained classifier performance (Spearman’s Rho: 0.856) and allowed determination of the classifier LOD (0.00082 [95% CI: 0.00057 - 0.00115]). Conclusions: These data demonstrate the existence of methylation patterns in tumor-derived cfDNA fragments that are rarely found in individuals without cancer; their abundance directly measured TF, and was a major factor influencing classification performance. Finally, the low classifier LOD (~0.1%) motivates further clinical development of a methylation-based assay for cancer detection. Clinical trial information: NCT02889978 .

BIOM-04. SENSITIVE DETECTION OF LEPTOMENINGEAL DISEASE USING CELL-FREE DNA FROM CEREBROSPINAL FLUID

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi10-vi10
Author(s):  
Michael White ◽  
Robert Klein ◽  
Brian Shaw ◽  
Albert Kim ◽  
Megha Subramanian ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Brain Metastases ◽  
Dna Analysis ◽  
Leptomeningeal Disease ◽  
Cell Free Dna ◽  
Free Dna ◽  
Cerebrospinal Fluid Cytology ◽  
Low Sensitivity ◽  
Fluid Cytology

Abstract Leptomeningeal disease is a devastating complication of cancer that is frequently underdiagnosed due to the low sensitivity of cerebrospinal fluid cytology, the current gold-standard diagnostic method. We performed genomic sequencing on cerebrospinal fluid specimens obtained from patients with suspected or confirmed leptomeningeal disease to identify tumor-derived cell-free DNA. From the same fluid draw, cerebrospinal fluid cytology was assayed for comparison. 30 patients underwent cytology and cell-free DNA analysis. This study consisted of two patient populations: 22 patients with cytology-confirmed leptomeningeal disease without parenchymal tumors abutting their cerebrospinal fluid and 8 patients with parenchymal brain metastases with no evidence of leptomeningeal disease. The primary outcome was the diagnostic accuracy of cell-free DNA, defined as the number of correct diagnoses out of the total number of tests assayed. A total of 30 patients, 23 female and 7 male, with a median age of 51 participated in this study. Participants mostly presented with metastatic solid malignancies. In patients previously diagnosed with leptomeningeal disease via cytology with no parenchymal tumor abutting cerebrospinal fluid, cell-free DNA was accurate in diagnosis of leptomeningeal disease in 45 of 48 follow-up samples (94%; 95% CI, 83%-99%). Cytology was accurate in 36 of 48 follow-up samples (75%; 95% CI, 60%-86%). Cell-free DNA was significantly more accurate (P=.02) and sensitive (P=.02) than cytology in patients without parenchymal tumors abutting the cerebrospinal fluid. In three patients with parenchymal brain metastases abutting the cerebrospinal fluid and no suspicion for leptomeningeal disease, cytology was negative in all three patients; whereas, cell-free DNA was positive in all three. This study demonstrates the improved sensitivity and accuracy of cell-free DNA in diagnosing leptomeningeal disease with the exception of parenchymal tumors abutting cerebrospinal fluid. Overall, these results will lead to improved diagnosis of leptomeningeal disease and potentially earlier intervention and clinical trial enrollment.

scholarly journals Cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Zimeng Ye ◽  
Zac Chatterton ◽  
Jahnvi Pflueger ◽  
John A Damiano ◽  
Lara McQuillan ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Liquid Biopsy ◽  
Somatic Mutations ◽  
Somatic Mosaicism ◽  
Autism Spectrum ◽  
Brain Diseases ◽  
Cell Free Dna ◽  
Free Dna ◽  
Subcortical Band Heterotopia ◽  
Fluid Cell

Abstract Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.

scholarly journals Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

2020 ◽  
Vol 21 (23) ◽  
pp. 9083
Author(s):  
Catherine Taylor ◽  
Simi Chacko ◽  
Michelle Davey ◽  
Jacynthe Lacroix ◽  
Alexander MacPherson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Nsclc Patient ◽  
Egfr Mutations ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Nsclc Patients ◽  
Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3043-3043
Author(s):  
Grace Q. Zhao ◽  
Yun Bao ◽  
Heng Wang ◽  
Wanping Hu ◽  
John Coller ◽  
...  
Keyword(s):  
Cancer Detection ◽  
Dna Analysis ◽  
Residual Disease ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Assay Sensitivity ◽  
Cell Free Dna ◽  
Target Sequencing ◽  
Free Dna ◽  
Dual Analysis

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with > 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with > 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

scholarly journals TBIO-18. LIQUID BIOPSY DETECTION OF GENOMIC ALTERATIONS IN PEDIATRIC BRAIN TUMORS FROM CELL FREE DNA IN PERIPHERAL BLOOD, CSF, AND URINE

2018 ◽  
Vol 20 (suppl_2) ◽  
pp. i184-i184
Author(s):  
Melanie Pages ◽  
Denisse Rotem ◽  
Gregory Gydush ◽  
Sarah Reed ◽  
Justin Rhoades ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Peripheral Blood ◽  
Liquid Biopsy ◽  
Pediatric Brain Tumors ◽  
Genomic Alterations ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pediatric Brain
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