High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods

Dan Su, Dadong Zhang, Kaiyan Chen, Jing Lu, Junzhou Wu, Xinkai Cao, Lisha Ying, Qihuang Jin, Yizhou Ye, Zhenghua Xie, Lei Xiong, Weimin Mao, Fugen Li

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis. Methods: Genomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity. Results: At sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99% and 98.7% respectively, and in 20% of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100% sensitivity and specificity. On ALK fusion detection, about 86% IHC-identified cases could be identified by targeted NGS and all ALK fusion detected by targeted NGS were confirmed by IHC. For HER2-amplification, 14 HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3% of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detected EGFR hotspot mutations in 215 NSCLC patients. Conclusions: DNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.

Original languageEnglish (US)
Article number121
JournalJournal of Experimental and Clinical Cancer Research
Volume36
Issue number1
DOIs
StatePublished - Sep 7 2017

Fingerprint

DNA
Immunohistochemistry
Neoplasms
Fluorescence In Situ Hybridization
Mutation
DNA Copy Number Variations
Sensitivity and Specificity
Cell Line
Gene Rearrangement
Cell Count
Genome

Keywords

  • Amplification-refractory mutation system
  • Clinical tumor samples
  • Fluorescence in situ hybridization
  • Immunohistochemistry
  • Targeted next generation sequencing

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods. / Su, Dan; Zhang, Dadong; Chen, Kaiyan; Lu, Jing; Wu, Junzhou; Cao, Xinkai; Ying, Lisha; Jin, Qihuang; Ye, Yizhou; Xie, Zhenghua; Xiong, Lei; Mao, Weimin; Li, Fugen.

In: Journal of Experimental and Clinical Cancer Research, Vol. 36, No. 1, 121, 07.09.2017.

Research output: Contribution to journalArticle

Su, Dan ; Zhang, Dadong ; Chen, Kaiyan ; Lu, Jing ; Wu, Junzhou ; Cao, Xinkai ; Ying, Lisha ; Jin, Qihuang ; Ye, Yizhou ; Xie, Zhenghua ; Xiong, Lei ; Mao, Weimin ; Li, Fugen. / High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods. In: Journal of Experimental and Clinical Cancer Research. 2017 ; Vol. 36, No. 1.
@article{30e9476833894dad8ad96e2f498d3eb4,
title = "High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods",
abstract = "Background: Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis. Methods: Genomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity. Results: At sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99{\%} and 98.7{\%} respectively, and in 20{\%} of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100{\%} sensitivity and specificity. On ALK fusion detection, about 86{\%} IHC-identified cases could be identified by targeted NGS and all ALK fusion detected by targeted NGS were confirmed by IHC. For HER2-amplification, 14 HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3{\%} of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detected EGFR hotspot mutations in 215 NSCLC patients. Conclusions: DNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.",
keywords = "Amplification-refractory mutation system, Clinical tumor samples, Fluorescence in situ hybridization, Immunohistochemistry, Targeted next generation sequencing",
author = "Dan Su and Dadong Zhang and Kaiyan Chen and Jing Lu and Junzhou Wu and Xinkai Cao and Lisha Ying and Qihuang Jin and Yizhou Ye and Zhenghua Xie and Lei Xiong and Weimin Mao and Fugen Li",
year = "2017",
month = "9",
day = "7",
doi = "10.1186/s13046-017-0591-4",
language = "English (US)",
volume = "36",
journal = "Journal of Experimental and Clinical Cancer Research",
issn = "0392-9078",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods

AU - Su, Dan

AU - Zhang, Dadong

AU - Chen, Kaiyan

AU - Lu, Jing

AU - Wu, Junzhou

AU - Cao, Xinkai

AU - Ying, Lisha

AU - Jin, Qihuang

AU - Ye, Yizhou

AU - Xie, Zhenghua

AU - Xiong, Lei

AU - Mao, Weimin

AU - Li, Fugen

PY - 2017/9/7

Y1 - 2017/9/7

N2 - Background: Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis. Methods: Genomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity. Results: At sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99% and 98.7% respectively, and in 20% of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100% sensitivity and specificity. On ALK fusion detection, about 86% IHC-identified cases could be identified by targeted NGS and all ALK fusion detected by targeted NGS were confirmed by IHC. For HER2-amplification, 14 HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3% of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detected EGFR hotspot mutations in 215 NSCLC patients. Conclusions: DNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.

AB - Background: Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This study is to set up basic parameters of targeted NGS for clinical diagnosis and to understand advantage of targeted NGS in comparison with the conventional methods of molecular diagnosis. Methods: Genomic DNA from 1000 Genomes Project and DNA from cancer cell lines have been used to establish the basic parameters for targeted NGS. The following confirmation was conducted by clinical samples. The multiple variants tested by amplification-refractory mutation system (ARMS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were evaluated by targeted NGS to determine the sensitivity. Furthermore, the multiple variants detected by targeted NGS were confirmed by current conventional methods to elucidate the specificity. Results: At sequencing depth of 500×, the maximal sensitivities on detecting single nucletic variances (SNVs) and small insertions/deletions (Indels) can reach 99% and 98.7% respectively, and in 20% of cancer cells, CNV detection can reach to the maximal level. The following confirmation of the sensitivity and specificity was conducted by a large cohort of clinical samples. For SNV and indel detection in clinical samples, targeted NGS can identify all hotspot mutations with 100% sensitivity and specificity. On ALK fusion detection, about 86% IHC-identified cases could be identified by targeted NGS and all ALK fusion detected by targeted NGS were confirmed by IHC. For HER2-amplification, 14 HER2-amplification cases identified by target NGS were all confirmed by FISH and about 93.3% of Her-2 IHC (3+) cases were identified by targeted NGS. Finally, the targeted NGS platform developed here has accurately detected EGFR hotspot mutations in 215 NSCLC patients. Conclusions: DNA from cancer cell lines is better than standard DNA as a reference to establish basic parameters for targeted NGS. Comparison of the conventional methods using a large cohort of patient samples confirmed the high preformance of targeted NGS on detecting DNA alterations.

KW - Amplification-refractory mutation system

KW - Clinical tumor samples

KW - Fluorescence in situ hybridization

KW - Immunohistochemistry

KW - Targeted next generation sequencing

UR - http://www.scopus.com/inward/record.url?scp=85029008655&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85029008655&partnerID=8YFLogxK

U2 - 10.1186/s13046-017-0591-4

DO - 10.1186/s13046-017-0591-4

M3 - Article

VL - 36

JO - Journal of Experimental and Clinical Cancer Research

JF - Journal of Experimental and Clinical Cancer Research

SN - 0392-9078

IS - 1

M1 - 121

ER -