title: Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics.
abstract: 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.
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kanagal-Shamanna
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Bryce P
authors.identifier: 000000024479221X
authors.initials: BP
authors.lastname: Portier
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Rajesh R
authors.initials: RR
authors.lastname: Singh
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Routbort
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Aldape
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Handal
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Hamed
authors.initials: H
authors.lastname: Rahimi
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Neelima G
authors.initials: NG
authors.lastname: Reddy
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Bedia A
authors.initials: BA
authors.lastname: Barkoh
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Bal M
authors.initials: BM
authors.lastname: Mishra
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Abhaya V
authors.initials: AV
authors.lastname: Paladugu
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Jawad H
authors.initials: JH
authors.lastname: Manekia
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Neda
authors.initials: N
authors.lastname: Kalhor
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Sinchita Roy
authors.initials: SR
authors.lastname: Chowdhuri
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Gregg A
authors.initials: GA
authors.lastname: Staerkel
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: L Jeffrey
authors.initials: LJ
authors.lastname: Medeiros
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
authors.affiliation: Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Keyur P
authors.initials: KP
authors.lastname: Patel
mesh_terms: D044963:Biopsy, Fine-Needle; D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
chemical_list: D004247:DNA