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.

How to Prepare Cytological Samples for Molecular Testing

“…As a matter of the fact, most NGS assays have a lower limit of mutation detection of 10%, which requires at least 20% of neoplastic cells. 39 Besides NGS technological improvements, automated allele-specific real-time PCR technology is also advancing at a rapid pace. In particular, the fully automated molecular diagnostic system Idylla (Biocartis, Mechelen, Belgium) is a fascinating technology.…”

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

“…Therefore, different NGS platforms use FNC to harvest cells from different tumors [92,93,94,95,96,97,98,99,100]. These studies demonstrated that NGS-based mutational profiling can be performed with a few nanograms of DNA (∼40 ng/μl), which can be easily obtained from FNC in different tumors [93,94,95,96,97,98]. Therefore, NGS may enhance the molecular FNC potential, providing mutational information on genes with high diagnostic and predictive relevance, such as EGFR, BRAF, K/N/HRAS, KIT, PTEN, CDKN2A, just to mention some, thus contributing to personalized therapies.…”

The use of endobronchial ultrasound guided transbronchial needle aspiration specimens for next generation sequencing in non-small cell lung cancer: a clinical perspective

“…The Viewpoint The use of endobronchial ultrasound guided transbronchial needle aspiration specimens for next generation sequencing in non-small cell lung cancer: a clinical perspective adequacy of a cell block cannot be reliably assessed intraprocedurally. The importance of adequate sample cellularity is highlighted by prior studies which describe the successful technique of extracting DNA from cytology slides prepared with fine needle aspiration (FNA) tumor samples (17)(18)(19)(20)(21). These studies demonstrate a direct relationship between successful NGS testing and increased slide cellularity.…”