A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

2020 ◽  
Vol 15 ◽  
Author(s):  
Zheng Jiang ◽  
Hui Liu ◽  
Siwen Zhang ◽  
Jia Liu ◽  
Weitao Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
High Sensitivity ◽  
Next Generation ◽  
Tissue Samples ◽  
Next Generation Sequencing Technology ◽  
Medication Selection ◽  
Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

scholarly journals Blood-Based Liquid Biopsy for Comprehensive Cancer Genomic Profiling Using Next-Generation Sequencing: An Emerging Paradigm for Non-invasive Cancer Detection and Management in Dogs

2021 ◽  
Vol 8 ◽  
Author(s):  
Kristina M. Kruglyak ◽  
Jason Chibuk ◽  
Lisa McLennan ◽  
Prachi Nakashe ◽  
Gilberto E. Hernandez ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
Genomic Profiling ◽  
Next Generation ◽  
Blood Draw ◽  
Genomic Alterations ◽  
Tissue Samples ◽  
Non Invasive ◽  
Generation Sequencing

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

Detection of actionable gene mutations in breast cancer by amplicon-based next-generation sequencing liquid biopsy.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 1035-1035
Author(s):  
Jing Shan Lim ◽  
Yukti Choudhury ◽  
Wai Min Phyo ◽  
Chaitanya Gupta ◽  
Yiliang Ho ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
Pik3ca Mutation ◽  
Genetic Alterations ◽  
Driver Mutations ◽  
Next Generation ◽  
Pik3ca Mutations ◽  
Generation Sequencing

1035 Background: The PI3K-inhibitor, alpelisib, was approved for the treatment of hormone receptor (HR)-positive, HER2-negative breast cancers with PIK3CA mutations based on findings from the SOLAR-1 trial. PIK3CA-positivity in tumor tissue was 29% (341/1172), but was only 15% (177/1172) in plasma cell-free DNA (cfDNA). The lower mutation detection rate observed in cfDNA may limit the clinical application of liquid biopsy in breast cancer. Amplicon-based next-generation sequencing (NGS) approach may confer improved sensitivity, allowing more effective profiling. We conducted a study to evaluate the use of this technology. Methods: Plasma cfDNA from 113 breast cancer patients (82.3% metastatic) underwent real-world testing in a CAP and ISO15189 accredited central laboratory. We analysed genetic alterations in cfDNA using an amplicon-based NGS technology. The presence of PIK3CA and other mutations relevant to breast cancer were correlated to molecular subtypes and treatment histories. Results: At least one mutation was detected in 70.8% of cases. Mutations were more frequent in metastatic cases (77.4%) compared to non-metastatic cases (27.3%). Across all patients, mutations in PIK3CA (33.6%), TP53 (32.7%), ESR1 (22.1%), GATA3 (7.1%) and ERBB2 (7.1%) were most frequently detected, in accordance with tumor tissue genotyping studies. PIK3CA mutations were more common in HR+ HER2- patients (44.4% vs 28.6% of other patients). Among PIK3CA-mutant cases, multiple PIK3CA mutations were present in 18.4% of cases, and hotspot mutations H1047R (34.2%), E542K (26.3%) and E545K (15.8%) were most frequent. An association was seen between PIK3CA mutation and prior treatment with CDK4/6 inhibitors (palbociclib, ribociclib) or mTOR inhibitor (everolimus), with 58% of PIK3CA-mutant cases having received these treatment previously compared to only 20% of PIK3CA-wild type (wt) cases. In addition, 75% of previously treated ESR1-mutant cases had specifically received hormonal treatment, compared to 60 % of ESR1-wt cases that received any treatment. Conclusions: We report similar PIK3CA mutation frequencies (~30%) with amplicon-based NGS on cfDNA compared to tumor tissue testing in breast cancer. Importantly, other driver mutations were also observed at similar frequencies as external tissue studies, implying high sensitivity as the primary reason for performance. This supports the clinical utility of an amplicon-based NGS-based approach to liquid biopsy for the sensitive detection of actionable mutations in breast cancer.

scholarly journals Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling

PLoS ONE ◽  
2018 ◽  
Vol 13 (3) ◽  
pp. e0193802 ◽  
Author(s):  
Vincent Plagnol ◽  
Samuel Woodhouse ◽  
Karen Howarth ◽  
Stefanie Lensing ◽  
Matt Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Liquid Biopsy ◽  
High Sensitivity ◽  
Molecular Profiling ◽  
Next Generation ◽  
Analytical Validation ◽  
Generation Sequencing

scholarly journals Detection of activating mutations in liquid biopsy of Egyptian breast cancer patients using targeted next-generation sequencing: a pilot study

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Neemat Kassem ◽  
Hebatallah Kassem ◽  
Loay Kassem ◽  
Mohamed Hassan
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Cancer Patients ◽  
Liquid Biopsy ◽  
Missense Mutations ◽  
Next Generation ◽  
Breast Cancer Patients ◽  
Activating Mutations ◽  
Generation Sequencing

Abstract Background Breast cancer (BC) is the 2nd most prevalent malignancy worldwide and is the most prevalent cancer among Egyptian women. The number of newly described cancer-associated genes has grown exponentially since the emergence of next-generation sequencing (NGS) technology. We aim to identify activating mutations in liquid biopsy of Egyptian breast cancer patients using targeted NGS technology. We also demonstrate the microsatellite instability (MSI) status using BAT25, BAT26, and NR27 markers which are tested on the Bioanalyzer 2100 system. Results Twenty-one variants were detected in 15 genes: 7 Substitution-Missense, 12 Substitution-coding silent, and 2 Substitution-intronic. Regarding ClinVar database, out of 21 variants there were 14 benign variants, 3 variants with conflicting interpretations of pathogenicity, 3 variants not reported, and 1 drug response variant. TP53 p.(Pro72Arg) missense mutations were found in 75% of patients. PIK3CA p.(Ile391Met), KDR p.(Gln472His) missense mutations were detected in 25% of patients each. Two patients revealed APC gene missense mutation with p.(Ile1307Lys) and p.(Glu1317Gln) variants. Only one patient showed ATM p.(Phe858Leu) gene mutation and one showed FGFR3 p.(Ala719Thr) variant. Regarding microsatellite instability (MSI) status, 2/8 (25%) patients were MSS, 3/8 (37.5%) patients were MSI-L, and 3/8 (37.5%) patients were MSI-HI. Conclusion It is essential to use and validate minimally invasive liquid biopsy for activating mutations detection by next-generation sequencing especially in patients with inoperable disease or bone metastasis. This work should be extended with larger patient series with comparison of genetic mutations in liquid-based versus tissue-based biopsy and longer follow up period.

scholarly journals Targeted Next-Generation Sequencing of Liquid Biopsy Samples from Patients with NSCLC

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Hestia Mellert ◽  
Jordan Reese ◽  
Leisa Jackson ◽  
Victoria Maxwell ◽  
Chérie Tschida ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Liquid Biopsy ◽  
High Sensitivity ◽  
Next Generation ◽  
Small Cell Lung ◽  
Chain Reaction ◽  
Targeted Next Generation Sequencing ◽  
Polymerase Chain ◽  
Test Process ◽  
Generation Sequencing

Liquid biopsy tests have become an integral part of the molecular diagnosis of patients with non-small cell lung cancer (NSCLC). We describe a new test panel that uses very low input (20 ng) of cell-free nucleic acids extracted from human plasma, which is designed to yield results in less than 72 h. In this study, we performed novel amplicon-based targeted next-generation sequencing with a semiconductor-based system, the Ion GeneStudio S5 Prime. The analytic performance of the assay was evaluated using contrived and retrospectively collected clinical specimens. The cumulative percent coefficient of variation for the new test process was very precise at 8.4% for inter-day, 4.0% for inter-operator and 3.4% for inter-instrument. We also observed significant agreement (95.7–100%) with an orthogonal, high-sensitivity droplet digital™ Polymerase Chain Reaction (ddPCR) test. This method offers a valuable supplement to assessing targeted mutations from blood while conserving specimens and maintaining sensitivity, with rapid turn-around times to actionable results.

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