Quantitative assessment of mutant allele burden in solid tumors by semiconductor-based next-generation sequencing

Bryce P. Portier, Rashmi Kanagal-Shamanna, Rajyalakshmi Luthra, Rajesh Singh, Mark J. Routbort, Brian Handal, Neelima Reddy, Bedia A. Barkoh, Zhuang Zuo, L. Jeffrey Medeiros, Kenneth Aldape, Keyur P. Patel

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


Objectives: Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms. Methods: We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides. Results: Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more. Conclusions: NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.

Original languageEnglish (US)
Pages (from-to)559-572
Number of pages14
JournalAmerican Journal of Clinical Pathology
Issue number4
StatePublished - Apr 2014


  • AmpliSeq cancer panel
  • Ion torrent
  • Massarray
  • Mutant allele burden
  • Mutant allele frequency
  • Next-generation sequencing
  • Sequenom

ASJC Scopus subject areas

  • Pathology and Forensic Medicine


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