Home Medicine Effective quality management practices in routine clinical next-generation sequencing
Article
Licensed
Unlicensed Requires Authentication

Effective quality management practices in routine clinical next-generation sequencing

  • Francine B. de Abreu , Jason D. Peterson , Christopher I. Amos , Wendy A. Wells and Gregory J. Tsongalis EMAIL logo
Published/Copyright: February 12, 2016
Become an author with De Gruyter Brill
Submit Manuscript Author Information
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 5

Abstract

Background:

Molecular technologies have allowed laboratories to detect and establish the profiles of human cancers by identifying a variety of somatic variants. In order to improve personalized patient care, we have established a next-generation sequencing (NGS) test to screen for somatic variants in primary or advanced cancers. In this study, we describe the laboratory quality management program for NGS testing, and also provide an overview of the somatic variants identified in over 1000 patient samples as well as their implications in clinical practice.

Methods:

Over the past one-and-a-half years, our laboratory received a total of 1028 formalin-fixed, paraffin-embedded (FFPE) tumor tissues, which consisted of non-small-cell lung carcinomas (NSCLCs), colon adenocarcinomas, glioma/glioblastomas, melanomas, breast carcinomas, and other tumor types. During this time period, we implemented a series of quality control (QC) checks that included (1) pre-DNA extraction, (2) DNA quantification, (3) DNA quality, (4) library quantification, (5) post-emulsification PCR, and (6) post-sequencing metrics. At least 10 ng of genomic DNA (gDNA) were used to prepare barcoded libraries using the AmpliSeq CHPv2. Samples were multiplexed and sequenced on Ion Torrent 318 chips using the Ion PGM System. Variants were identified using the Variant Caller Plugin, and annotation and functional predictions were performed using the Golden Helix SVS.

Results:

A total of 1005 samples passed QC1–3, and following additional library preparation QC checkpoints, 877 samples were sequenced. Samples were classified into two categories: wild-type (127) and positive for somatic variants (750). Somatic variants were classified into clinically actionable (60%) and non-actionable (40%).

Conclusions:

The use of NGS in routine clinical laboratory practice allowed for the detection of tumor profiles that are essential for the selection of targeted therapies and identification of applicable clinical trials, contributing to the improvement of personalized patient care in oncology.

Keywords: massively parallel sequencing; next-generation sequencing; solid tumor; somatic variant
Corresponding author: Gregory J. Tsongalis, PhD, Department of Pathology, Dartmouth Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756, USA, Phone: +1-603-650-5498, E-mail:

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Ross JS, Cronin M. Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol 2011;136:527–39.10.1309/AJCPR1SVT1VHUGXWSearch in Google Scholar PubMed

2. Yu B, O’Toole SA, Trent RJ. Somatic DNA mutation analysis in targeted therapy of solid tumours. Transl Pediatr 2015;4:125–38.Search in Google Scholar

3. Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med 2013;15:733–47.10.1038/gim.2013.92Search in Google Scholar PubMed PubMed Central

4. Aziz N, Zhao Q, Bry L, Driscoll DK, Funke B, Gibson JS, et al. College of American pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med 2015;139:481–93.10.5858/arpa.2014-0250-CPSearch in Google Scholar PubMed

5. Tsongalis GJ, Peterson JD, de Abreu FB, Tunkey CD, Gallagher TL, Strausbaugh LD, et al. Routine use of the ion torrent AmpliSeqTM Cancer Hotspot Panel for identification of clinically actionable somatic mutations. Clin Chem Lab Med 2013;13:1–8.Search in Google Scholar

6. Kanagal-Shamanna R, Portier BP, Singh RR, Routbort MJ, Aldape KD, Handal BA, et al. Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics. Mod Pathol 2014;27:314–27.10.1038/modpathol.2013.122Search in Google Scholar PubMed

7. Van Allen EM, Wagle N, Stojanov P, Perrin DL, Cibulskis K, Marlow S, et al. Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine. Nat Med 2014;20:682–8.10.1038/nm.3559Search in Google Scholar PubMed PubMed Central

8. Gailey MP, Stence AA, Jensen CS, Ma D. Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin-fixed, paraffin-embedded tissue. Cancer Cytopathol 2015;123:30–9.10.1002/cncy.21476Search in Google Scholar PubMed

9. Dumur CI. Available resources and challenges for the clinical annotation of somatic variations. Cancer Cytopathol 2014;122:730–6.10.1002/cncy.21471Search in Google Scholar PubMed PubMed Central

10. Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, et al. ClinGen – the clinical genome resource. N Engl J Med 2015;372:2235–42.10.1056/NEJMsr1406261Search in Google Scholar PubMed PubMed Central

11. Johnson DB, Dahlman KH, Knol J, Gilbert J, Puzanov I, Means-Powell J, et al. Enabling a genetically informed approach to cancer medicine: a retrospective evaluation of the impact of comprehensive tumor profiling using a targeted next-generation sequencing panel. Oncologist 2014;19:616–22.10.1634/theoncologist.2014-0011Search in Google Scholar PubMed PubMed Central

12. Janne PA, Smith I, McWalter G, Mann J, Doughrty B, Walker J, et al. Impact of KRAS codon subtypes from a randomised phase II trial of selumetinib plus docetaxel in KRAS mutant advanced non-small-cell lung cancer. Br J Cancer 2015;113:199–203.10.1038/bjc.2015.215Search in Google Scholar PubMed PubMed Central

13. Schwaederle M, Daniels GA, Piccioni DE, Fanta PT, Schwab RB, Shimabukuro KA, et al. On the road to precision cancer medicine: analysis of genomic biomarker actionability in 439 patients. Mol Cancer Ther 2015;14:1488–94.10.1158/1535-7163.MCT-14-1061Search in Google Scholar PubMed

14. Schwaederle M, Parker BA, Schwab RB, Fanta PT, Boles SG, Daniels GA, et al. Molecular tumor board: the University of California San Diego moores cancer center experience. Oncologist 2014;19:631–6.10.1634/theoncologist.2013-0405Search in Google Scholar PubMed PubMed Central

15. Tafe LJ, Gorlov I, de Abreu FB, Lefferts JA, Liu X, Pettus JR, et al. Implementation of a Molecular tumor board: the impact on treatment decisions for the first 35 patients evaluated at Dartmouth-Hitchcock Medical Center. Oncologist 2015;20:1011–8.10.1634/theoncologist.2015-0097Search in Google Scholar PubMed PubMed Central

16. Burrel RA, McGranahan N, Bartek J, Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature 2013;501:338–45.10.1038/nature12625Search in Google Scholar PubMed

17. Burrel RA, Swanton C. Tumor heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 2014;8:1095–111.10.1016/j.molonc.2014.06.005Search in Google Scholar PubMed PubMed Central

18. Pribluda A, de la Cruz CC, Jackson EL. Intratumoral heterogeneity: from diversity comes resistance. Clin Cancer Res 2015;21:2916–22.10.1158/1078-0432.CCR-14-1213Search in Google Scholar PubMed

19. Jamal-Hanjani M, Quezada SA, Larkin J, Swanton C. Translational implication of tumor heterogeneity. Clin Cancer Res 2015;21:1258–66.10.1158/1078-0432.CCR-14-1429Search in Google Scholar PubMed PubMed Central

20. Swanton C. Cancer evolution: the final frontier of precision medicine? Ann Oncol 2014;25:549–51.10.1093/annonc/mdu005Search in Google Scholar PubMed PubMed Central

21. Janku F. Tumor heterogeneity in the clinic: is it a real problem? Ther Adv Med Oncol 2014;6:43–51.10.1177/1758834013517414Search in Google Scholar PubMed PubMed Central

22. Jorgensen JT. Clinical application of companion diagnostics. Trends Mol Med 2015;21:405–7.10.1016/j.molmed.2015.05.003Search in Google Scholar PubMed

23. Stewart EL, Mascaux C, Pham NA, Sakashita S, Sykes J, Kim L, et al. Clinical utility of patient derived xenografts to determine biomarkers of prognosis and map resistance pathways in EGFR-mutant lung adenocarcinoma. J Clin Oncol 2015;33:2472–80.10.1200/JCO.2014.60.1492Search in Google Scholar PubMed

Received: 2015-11-30
Accepted: 2016-1-14
Published Online: 2016-2-12
Published in Print: 2016-5-1

©2016 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Protein S100B: from cancer diagnostics to the evaluation of mild traumatic brain injury
  4. Reviews
  5. Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease
  6. Oxidative damage and the pathogenesis of menopause related disturbances and diseases
  7. Opinion Paper
  8. EFLM WG-Preanalytical phase opinion paper: local validation of blood collection tubes in clinical laboratories
  9. Genetics and Molecular Diagnostics
  10. Effective quality management practices in routine clinical next-generation sequencing
  11. Analysis of PMP22 duplication and deletion using a panel of six dinucleotide tandem repeats
  12. A novel exonuclease (TaqMan) assay for rapid haptoglobin genotyping
  13. General Clinical Chemistry and Laboratory Medicine
  14. Heparinate but not serum tubes are susceptible to hemolysis by pneumatic tube transportation
  15. Criteria of adequacy for vitamin D testing and prevalence of deficiency in clinical practice
  16. A simple clot based assay for detection of procoagulant cell-derived microparticles
  17. Measurement of factor XIII (FXIII) activity by an automatic ammonia release assay using iodoacetamide blank-procedure: no more overestimation in the low activity range and better detection of severe FXIII deficiencies
  18. Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children?
  19. Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay
  20. Reference Values and Biological Variations
  21. Preanalytical, analytical, gestational and pediatric aspects of the S100B immuno-assays
  22. Establishment of reference intervals of clinical chemistry analytes for the adult population in Saudi Arabia: a study conducted as a part of the IFCC global study on reference values
  23. First data on the biological variation and quality specifications for plasma ammonia concentrations in healthy subjects
  24. Cancer Diagnostics
  25. Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome
  26. Cardiovascular Diseases
  27. Galectin-3, osteopontin and successful aging
  28. Platelet volume is associated with the Framingham risk score for cardiovascular disease in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)
  29. Infectious Diseases
  30. Diagnostic values of CD64, C-reactive protein and procalcitonin in ventilator-associated pneumonia in adult trauma patients: a pilot study
  31. Corrigendum
  32. Corrigendum to: Accuracy of GFR estimating equations combining standardized cystatin C and creatinine assays: a cross-sectional study in Sweden
  33. Letters to the Editor
  34. Theranos phenomenon – part 3
  35. Biological variation of high sensitivity cardiac troponin-T in stable dialysis patients: implications for clinical practice
  36. Biological variation of high sensitivity cardiac troponin-T in stable dialysis patients: implications for clinical practice
  37. Impact of specimen mixing methods on presepsin point-of-care test results using whole blood
  38. Clinical laboratories have a critical role in test strip lot management in glucose point-of-care testing
  39. Hemoglobin A2-Leuven (α2δ2 143(H21) His>Asp): a novel delta-chain variant potentially interfering in hemoglobin A1c measurement using cation exchange HPLC
  40. Eryptosis is induced by hyperthermia in hereditary spherocytosis red blood cells
  41. Investigation of sensitivity for coagulation factor deficiency in APTT and PT: how to perform it?
  42. Underestimation of hepcidin concentration by time of flight mass spectrometry and competitive ELISA in hepcidin p.Gly71Asp heterozygotes
  43. Congress Abstracts
  44. ISMD2016 Eleventh International Symposium on Molecular Diagnostics
https://doi.org/10.1515/cclm-2015-1190
Keywords for this article
massively parallel sequencing; next-generation sequencing; solid tumor; somatic variant

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Protein S100B: from cancer diagnostics to the evaluation of mild traumatic brain injury
  4. Reviews
  5. Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease
  6. Oxidative damage and the pathogenesis of menopause related disturbances and diseases
  7. Opinion Paper
  8. EFLM WG-Preanalytical phase opinion paper: local validation of blood collection tubes in clinical laboratories
  9. Genetics and Molecular Diagnostics
  10. Effective quality management practices in routine clinical next-generation sequencing
  11. Analysis of PMP22 duplication and deletion using a panel of six dinucleotide tandem repeats
  12. A novel exonuclease (TaqMan) assay for rapid haptoglobin genotyping
  13. General Clinical Chemistry and Laboratory Medicine
  14. Heparinate but not serum tubes are susceptible to hemolysis by pneumatic tube transportation
  15. Criteria of adequacy for vitamin D testing and prevalence of deficiency in clinical practice
  16. A simple clot based assay for detection of procoagulant cell-derived microparticles
  17. Measurement of factor XIII (FXIII) activity by an automatic ammonia release assay using iodoacetamide blank-procedure: no more overestimation in the low activity range and better detection of severe FXIII deficiencies
  18. Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children?
  19. Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay
  20. Reference Values and Biological Variations
  21. Preanalytical, analytical, gestational and pediatric aspects of the S100B immuno-assays
  22. Establishment of reference intervals of clinical chemistry analytes for the adult population in Saudi Arabia: a study conducted as a part of the IFCC global study on reference values
  23. First data on the biological variation and quality specifications for plasma ammonia concentrations in healthy subjects
  24. Cancer Diagnostics
  25. Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome
  26. Cardiovascular Diseases
  27. Galectin-3, osteopontin and successful aging
  28. Platelet volume is associated with the Framingham risk score for cardiovascular disease in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)
  29. Infectious Diseases
  30. Diagnostic values of CD64, C-reactive protein and procalcitonin in ventilator-associated pneumonia in adult trauma patients: a pilot study
  31. Corrigendum
  32. Corrigendum to: Accuracy of GFR estimating equations combining standardized cystatin C and creatinine assays: a cross-sectional study in Sweden
  33. Letters to the Editor
  34. Theranos phenomenon – part 3
  35. Biological variation of high sensitivity cardiac troponin-T in stable dialysis patients: implications for clinical practice
  36. Biological variation of high sensitivity cardiac troponin-T in stable dialysis patients: implications for clinical practice
  37. Impact of specimen mixing methods on presepsin point-of-care test results using whole blood
  38. Clinical laboratories have a critical role in test strip lot management in glucose point-of-care testing
  39. Hemoglobin A2-Leuven (α2δ2 143(H21) His>Asp): a novel delta-chain variant potentially interfering in hemoglobin A1c measurement using cation exchange HPLC
  40. Eryptosis is induced by hyperthermia in hereditary spherocytosis red blood cells
  41. Investigation of sensitivity for coagulation factor deficiency in APTT and PT: how to perform it?
  42. Underestimation of hepcidin concentration by time of flight mass spectrometry and competitive ELISA in hepcidin p.Gly71Asp heterozygotes
  43. Congress Abstracts
  44. ISMD2016 Eleventh International Symposium on Molecular Diagnostics
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 7.12.2025 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2015-1190/pdf
Scroll to top button