Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA.
Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0267389 |
| _version_ | 1828790221569785856 |
|---|---|
| author | Jonathan Poh Kao Chin Ngeow Michelle Pek Kian-Hin Tan Jing Shan Lim Hao Chen Choon Kiat Ong Jing Quan Lim Soon Thye Lim Chwee Ming Lim Boon Cher Goh Yukti Choudhury |
| author_facet | Jonathan Poh Kao Chin Ngeow Michelle Pek Kian-Hin Tan Jing Shan Lim Hao Chen Choon Kiat Ong Jing Quan Lim Soon Thye Lim Chwee Ming Lim Boon Cher Goh Yukti Choudhury |
| author_sort | Jonathan Poh |
| collection | DOAJ |
| description | Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing. |
| first_indexed | 2024-12-12T01:25:29Z |
| format | Article |
| id | doaj.art-ece18644792f47e0852a1f56ab83766e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T01:25:29Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ece18644792f47e0852a1f56ab83766e2022-12-22T00:43:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-01174e026738910.1371/journal.pone.0267389Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA.Jonathan PohKao Chin NgeowMichelle PekKian-Hin TanJing Shan LimHao ChenChoon Kiat OngJing Quan LimSoon Thye LimChwee Ming LimBoon Cher GohYukti ChoudhuryNext-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.https://doi.org/10.1371/journal.pone.0267389 |
| spellingShingle | Jonathan Poh Kao Chin Ngeow Michelle Pek Kian-Hin Tan Jing Shan Lim Hao Chen Choon Kiat Ong Jing Quan Lim Soon Thye Lim Chwee Ming Lim Boon Cher Goh Yukti Choudhury Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. PLoS ONE |
| title | Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. |
| title_full | Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. |
| title_fullStr | Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. |
| title_full_unstemmed | Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. |
| title_short | Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA. |
| title_sort | analytical and clinical validation of an amplicon based next generation sequencing assay for ultrasensitive detection of circulating tumor dna |
| url | https://doi.org/10.1371/journal.pone.0267389 |
| work_keys_str_mv | AT jonathanpoh analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT kaochinngeow analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT michellepek analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT kianhintan analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT jingshanlim analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT haochen analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT choonkiatong analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT jingquanlim analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT soonthyelim analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT chweeminglim analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT boonchergoh analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna AT yuktichoudhury analyticalandclinicalvalidationofanampliconbasednextgenerationsequencingassayforultrasensitivedetectionofcirculatingtumordna |