Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: Promises and challenges for routine clinical diagnostics

Rashmi Kanagal-Shamanna, Bryce P. Portier, Rajesh R. Singh, Mark J. Routbort, Kenneth D. Aldape, Brian A. Handal, Hamed Rahimi, Neelima G. Reddy, Bedia A. Barkoh, Bal M. Mishra, Abhaya V. Paladugu, Jawad H. Manekia, Neda Kalhor, Sinchita Roy Chowdhuri, Gregg A. Staerkel, L. Jeffrey Medeiros, Rajyalakshmi Luthra, Keyur P. Patel

Research output: Contribution to journalArticlepeer-review

189 Scopus citations


Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

Original languageEnglish (US)
Pages (from-to)314-327
Number of pages14
JournalModern Pathology
Issue number2
StatePublished - Feb 2014

ASJC Scopus subject areas

  • Pathology and Forensic Medicine


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