INTRODUCTION

The discovery of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) has allowed effective targeted therapy with EGFR tyrosine kinase inhibitors in patients that harbour these mutations1.

However, the majority of NSCLC cases have wild-type EGFR and it is now known that many other mutations can drive oncogenic pathways, including KRAS and less commonly, BRAF2. BRAF is a proto-oncogene encoding a serine/threonine protein kinase which is a downstream effector protein of RAS and transduces signalling through the mitogen-activated protein kinase pathway to promote cell proliferation and survival. This pathway functions downstream of various receptor tyrosine kinases such as EGFR and is a key mediator of oncogenesis3.


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BRAF mutations are commonly seen in a range of malignancies, including hairy-cell leukemia (100%)4, melanoma (~40%)5, papillary thyroid carcinoma (30-50%) and colorectal carcinoma (~10%)6. The V600E mutation has been shown to constitutively activate BRAF which phosphorylates the downstream effectors MEK and subsequently ERK7.

ERK, in turn, activates transcription factors such as c-fos and Elk-1, driving cell cycle progression and survival8. The importance of the BRAF pathway is well established in melanoma, asBRAF inhibitors have been shown to significantly increase progression free survival of patients with advanced stage melanoma harbouring the BRAF V600E mutation9.

This raises the possibility that BRAF mutations may also be a feasible target in NSCLC. BRAF mutations in NSCLC are not well characterised in the literature due to their low prevalence.

In this study we aimed to investigate the prevalence and clinicopathological features ofBRAF mutations in NSCLC.

METHODS

Patients

We retrospectively reviewed 273 NSCLC cases that underwent mutation testing upon request of the treating oncologist at Royal Prince Alfred Hospital between March 2012 and March 2014. The patients underwent either a resection or a diagnostic procedure (biopsy or cytological specimen) and the tissue was formalin fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E).

The H&E sections were reviewed by a pathologist (SOT or WC) to ensure adequate tumour cells were present and to mark representative areas for deoxyribonucleic acid (DNA) extraction. Histological subtypes were classified according to the IASLC/ATS/ERS classification10. This study was approved by the Human Research Ethics Committee of Royal Prince Alfred Hospital.

Mutation detection

DNA was extracted from the formalin fixed paraffin embedded tissue using NucleoSpin FFPE DNA Kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s instruction with 2 hr proteinase digestion.

The quantity of the extracted DNA was assessed using Qubit® Fluorometer (Life Technologies, Mulgrave, Australia). A minimum of 300 ng of DNA was required for optimal mutational analysis.

Samples were amplified for 238 variant targets in a 24-multiplex PCR using the OncoCarta Panel v1.0 Kit (ABL1, AKT1, AKT2, BRAF,CDK, EGFR, ERBB2, FGFR1, FGFR3, FLT3, JAK2, KIT, MET, HRAS, KRAS, NRAS, PDGFR, PIK3CA, and RET) and analyzed based on the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) technology on the Sequenom MassArray platform11,12.

The targeted mutations in the 19 oncogenes comprising the OncoCarta v1.0 Panel are reported to be biologically significant in carcinogenesis or progression in a range of malignancies.

These mutational analyses and immunohistochemistry described below were performed at an Australian National Association of Testing Authorities (NATA) accredited medical laboratory.

Immunohistochemistry

BRAF V600E immunohistochemistry was performed on sections cut at four microns. Tissue was pre-treated on a Ventana Benchmark Ultra (Roche) with CC1 (Roche) for 64 minutes.

The anti-BRAF (VE1) mouse monoclonal antibody (Spring Bioscience) was used at 1:100 dilution with 16 minutes incubation.

Staining was performed using the OptiView DAB Immunohistochemistry Detection kit (Roche) for 8 minutes. Cases with 1+, 2+ and 3+ staining were regarded as positive and cases with no staining were regarded as negative.

Statistical analysis

Fisher’s exact test was used to evaluate the difference in gender distribution between BRAF wild-type and mutant patients while Welch’s t-test was used to evaluate the age difference. Data were analysed using the R environment for statistical computing13.