scholarly journals Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA

2021 ◽  
Author(s):  
James C. Willey ◽  
Tom Morrison ◽  
Brad Austermiller ◽  
Erin L. Crawford ◽  
Daniel J. Craig ◽  
...  
Keyword(s):  
Quality Control ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Circulating Tumor Dna ◽  
Primary Objective ◽  
Nucleotide Position ◽  
Targeted Next Generation Sequencing ◽  
Next Generation Sequencing Ngs ◽  
Rate Limit ◽  
Tumor Dna

SUMMARYThe primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients

2019 ◽  
Vol 57 (10) ◽  
pp. 1501-1510 ◽  
Author(s):  
Beili Wang ◽  
Shengchao Wu ◽  
Fei Huang ◽  
Minna Shen ◽  
Huiqin Jiang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Circulating Tumor Dna ◽  
Concordance Rate ◽  
Tissue Samples ◽  
Percent Agreement ◽  
Pre Treatment ◽  
Tumor Dna

Abstract Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

Quantifying the evolution of tumor architecture using serial circulating tumor DNA.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 600-600
Author(s):  
Jason Henry ◽  
Jonathan M. Loree ◽  
John H. Strickler ◽  
Kanwal Pratap Singh Raghav ◽  
Van K. Morris ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Limit Of Detection ◽  
Circulating Tumor Dna ◽  
Tumor Evolution ◽  
Disease Course ◽  
Serial Sampling ◽  
Blood Specimens ◽  
Serial Samples ◽  
Tumor Dna ◽  
Over Time

600 Background: There is limited data regarding changes in the genomic landscape in individual patients over time as serial tissue biopsy has risk and is of uncertain clinical benefit. The advent of circulating tumor DNA (ctDNA) allows for safe and repeated molecular sampling with the potential to investigate evolution of tumor architecture over the disease course. Methods: From 5/15 to 12/17, 116 patients with metastatic CRC had between three to 12 blood specimens taken over the treatment course. Plasma was tested using targeted NGS assay (Guardant360, Guardant Health, 68 gene). To account for variations in the amount of ctDNA in serial samples, a window of evaluable allele frequency was established for each patient as the fold change between the max allele frequency (mAF) and limit of detection for serial samples with the lowest mAF. Mutations not falling within this window were excluded from analysis. Substantial treatment induced selective pressure (SP) was defined as a decrease in the mutant mAF of > 50% in patients with at least an initial mAF of 1%. Results: 116 patients with a total of 317 serial blood samples were evaluable after accounting for ctDNA variations over time. Specimens were collected a median of 12 months apart, with a median of three specimens per patient. Thirteen patients (11%) did not have any changes in mutations on serial sampling, however the remainder of patients gained an average of 1.1 mutations per time point (mut/tp), and lost 1.0 mut/tp. 31% of patients demonstrated evidence of substantial treatment-induced SP. These patients were more likely to demonstrate a change in clonal architecture of the tumor (46% greater rate than those without SP, P = 0.04), predominantly through gain of new clones. In contrast, clonal hematopoiesis alterations that may be induced by chemotherapy, such as JAK2V617F, were neither gained or lost. Conclusions: After correction for variations over time in the total amount of ctDNA in circulation, we identify numerous changes in tumor architecture with serial sampling. For the first time in colorectal cancer we demonstrate that when treatment-induced SP is applied the rate of tumor evolution is increased, demonstrating potential value of monitoring changes in tumor architecture over the disease course.

scholarly journals Alectinib-Induced Pleural and Pericardial Effusions in ALK-Positive NSCLC

2021 ◽  
pp. 1323-1327
Author(s):  
Maiken Parm Ulhoi ◽  
Boe Sandahl Sorensen ◽  
Peter Meldgaard
Keyword(s):  
Progressive Disease ◽  
Heart Function ◽  
Circulating Tumor Dna ◽  
Driver Mutations ◽  
First Line ◽  
Alk Positive ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing ◽  
Mild Toxicity

Alectinib is the first-line targeted treatment for advanced ALK-positive non-small-cell lung cancer. Although it has a relatively mild toxicity profile, adverse events (AEs) do occur. We present a case of alectinib-induced bilateral pleural effusions and pericardial effusion that has not previously been reported. The patient developed severe dyspnea 3 months after starting alectinib. He underwent thorough clinical examination including evaluations of heart function. The heart function was normal. There was no sign of pneumonitis or progressive disease on the CT scans. Cytology samples of the pleural fluid from multiple thoracocenteses were examined and showed no malignant cells. Next-generation sequencing (NGS) analysis of circulating tumor DNA from sequential blood samples was also carried out. NGS identified no known driver mutations associated with the effusions. Hence, the effusions were suspected to be an alectinib-induced AE. Alectinib was withdrawn, and the patient commenced brigatinib. The effusions subsequently regressed.

scholarly journals Differential Mutation Detection Capability Through Capture-Based Targeted Sequencing in Plasma Samples in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Jian Gao ◽  
Lei Xi ◽  
Rentao Yu ◽  
Huailong Xu ◽  
Min Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Circulating Tumor Dna ◽  
Tumor Burden ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Tumor Protein P53 ◽  
Next Generation Sequencing Ngs ◽  
Coding Variants ◽  
G Protein Coupled ◽  
Generation Sequencing

Circulating tumor DNA (ctDNA) is a promising biomarker for accurate monitoring and less invasive assessment of tumor burden and treatment response. Here, targeted next-generation sequencing (NGS) with a designed gene panel of 176 cancer-relevant genes was used to assess mutations in 90 ctDNA samples from 90 patients with multiple types of liver disease and 10 healthy donor samples for control. Using our ctDNA detection panel, we identified mutations in 98.89% (89/90) of patient plasma biopsy samples, and 19 coding variants located in 10 cancer-related genes [ACVR2A, PCLO, TBCK, adhesion G protein-coupled receptor (ADGRV1), COL1A1, GABBR1, MUC16, MAGEC1, FASLG, and JAK1] were identified in 96.7% of patients (87/90). The 10 top mutated genes were tumor protein p53 (TP53), ACVR2A, ADGRV1, MUC16, TBCK, PCLO, COL11A1, titin (TTN), DNAH9, and GABBR1. TTN and TP53 and TTN and DNAH9 mutations tended to occur together in hepatocellular carcinoma samples. Most importantly, we found that most of those variants were insertions (frameshift insertions) and deletions (frameshift deletions and in-frame deletions), such as insertion variants in ACVR2A, PCLO, and TBCK; such mutations were detected in almost 95% of patients. Our study demonstrated that the targeted NGS-based ctDNA mutation profiling was a useful tool for hepatocellular carcinoma (HCC) monitoring and could potentially be used to guide treatment decisions in HCC.

scholarly journals Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabriella Taques Marczynski ◽  
Ana Carolina Laus ◽  
Mariana Bisarro dos Reis ◽  
Rui Manuel Reis ◽  
Vinicius de Lima Vazquez
Keyword(s):  
Somatic Mutations ◽  
Limit Of Detection ◽  
Progression Free Survival ◽  
Circulating Tumor Dna ◽  
Advanced Melanoma ◽  
Plasma Samples ◽  
Free Survival ◽  
Melanoma Patients ◽  
Digital Polymerase Chain Reaction ◽  
Tumor Dna

Abstract BRAF, NRAS and TERT mutations occur in more than 2/3 of melanomas. Its detection in patient’s blood, as circulating tumor DNA (ctDNA), represents a possibility for identification and monitoring of metastatic disease. We proposed to standardize a liquid biopsy platform to identify hotspot mutations in BRAF, NRAS and TERT in plasma samples from advanced melanoma patients and investigate whether it was associated to clinical outcome. Firstly, we performed digital polymerase chain reaction using tumor cell lines for validation and determination of limit of detection (LOD) of each assay and screened plasma samples from healthy individuals to determine the limit of blank (LOB). Then, we selected 19 stage III and IV patients and determined the somatic mutations status in tumor tissue and track them in patients’ plasma. We established a specific and sensitive methodology with a LOD ranging from 0.13 to 0.37%, and LOB ranging from of 0 to 5.201 copies/reaction. Somatic mutations occurred in 17/19 (89%) patients, of whom seven (41%) had ctDNA detectable their paired plasma. ctDNA detection was associated with shorter progression free survival (p = 0.01). In conclusion, our data support the use of ctDNA as prognosis biomarker, suggesting that patients with detectable levels have an unfavorable outcome.

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