Screening for EGFR and KRAS Mutations in Endobronchial Ultrasound Derived Transbronchial Needle Aspirates in Non-Small Cell Lung Cancer Using COLD-PCR

Abstract

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)–polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18–21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.

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

“…The genes tested have included EGFR KRAS, BRAF and PIK3CA in the lung specimens and BRAF, RET, RAS, TRK and PPRg in the thyroid nodules. 6,7,10,23,[30][31][32][33][34][35][36][37][38] Due to the low analytical sensitivity of direct sequencing, ultrasensitive techniques such as high resolution melting, realtime PCR and pyrosequencing have been utilized to improve detection sensitivity. 7 However, assessment of multiple genes in cytological specimens is sometimes not possible, because traditional platforms require large amounts of DNA.…”

“…Fortunately, most FNA procedures routinely have either a concurrent biopsy or a follow-up surgical excision, which provide adequate tissue for molecular assessment. 10,11 In some instances, however, cytological specimens may be the only material available for molecular testing. If the tumor is locally advanced or metastatic, surgical resection is not performed.…”

“…1 Additionally, use of COLD-PCR/direct sequencing for EGFR and KRAS detection in lung aspirates and locked nucleic acid-based PCR/sequencing assay in thyroid aspirates has improved the sensitivity of mutation detection. 10,12 Massively parallel or next-generation sequencing (NGS) technology is increasingly being used for mutational analysis of tumors for both clinical and research applications. The advent of affordable bench top NGS platforms such as Ion Personal Genome Machine (PGM) has facilitated multi-gene mutational profiling using only nanograms (ng) of DNA.…”

Cell block samples from endobronchial ultrasound transbronchial needle aspiration provide sufficient material for ancillary testing in lung cancer—a quaternary referral centre experience

“…One concern, however, is that there may be less tissue in an EBUS sample and that this could be insufficient to perform the required ancillary IHC and molecular testing. Current evidence from other studies suggests that EBUS-transbronchial needles aspiration (TBNA) samples are usually sufficient for molecular genetic testing with diagnostic rates between 72-95.5% (5)(6)(7)(8). Santis et al [2011] had a diagnostic rate of 95.5% with the aid of rapid on site examination (ROSE) (8).…”

Development and Clinical Application of Liquidchip Luminex Assay in the Detection of Epidermal Growth Factor Receptor and K-Ras Mutation

“…Therefore, it is critical to accurately determine the mutation status of EGFR and K-ras for the selection of patients who may benefit from TKI therapy [7]. Recently, direct sequencing, allele-specific PCR, amplificationrefractory mutation sequencing (ARMS), H&E-staining, and quantitative real time PCR are available for detection of gene mutation [2,5,8,9]. Nevertheless, these techniques are relatively expensive, technically difficult, long procedure for routine application in clinic, and also depend on the quality of the samples [10].…”