Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Abstract

Malignant melanoma, the deadliest form of skin cancer, is characterised by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Therefore patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real time polymerase chain reaction (PCR) and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild type cells using as little as 20 ng/µl of RNA material, without prior PCR amplification and use of labels.The identification of alterations in specific signalling pathways and recurrent oncogenic mutations in particular types of cancers has led in the past decade to the explosion of targeted therapy approaches. In cutaneous melanoma, a significant improvement in overall survival has been achieved by vemurafenib and similar drugs that selectively inhibit tumours carrying a mutated BRAF 2 gene 1,2 . Additional drugs for combination therapies with higher efficacies and fewer side effects are in clinical trials 3 . BRAF is one of three RAF genes (rapidly accelerated fibrosarcoma A, B and C) encoding cytoplasmic protein serine/threonine kinases belonging to the mitogen-activated protein kinase (MAPK) signal transduction cascade, a pathway controlling various cellular processes such as proliferation, migration and survival 4,5 . BRAF somatic mutations are present in half of cutaneous melanomas. Over 90% of the mutations are a single T to A transversion at position 1799 in the BRAF coding sequence (cT1799A), which converts a valine amino acid residue at position 600 in the protein to a glutamic acid (V600E). This mutation renders the protein constitutively active, resulting in a deregulated MAPK pathway 6 and thus uncontrolled cell growth and cancer. BRAF mutations are also present in other neoplasms, including hairy cell leukemias, thyroid and colon carcinomas 7,8,9 . As the presence of the cT1799A/V600E BRAF (hereafter BRAF V600E ) mutation determines eligibility to BRAF inhibitor treatment, molecular screening of tumour biopsies is now carried out routinely. with sensitivity comparable to COBAS TM , silicon nanowire field-effect transistors 12 and a threedimensional gold nanowire platform 13 . The latter technologies have only been shown to work using synthetic oligonucleotide targets, larger gene fragments or still rely on an initial PCR amplification. In particular, they have not been applied to the direct identification of a mutated messenger RNA (mRNA) sequence in total RNA (constituted primarily by ribosomal RNA and containing all mRNAs transcribed from gen...

Microcantilever arrays functionalised with spiropyran photoactive moieties as systems to measure photo-induced surface stress changes

“…Further advances in nanomechanics allowed the use of microcantilevers for the detection of biomarkers and the transduction of DNA hybridisation into a nanomechanical response [9][10][11], along with the use of silicon cantilevers for the detection of volatile organic compounds [5]. Silicon cantilever technology also uses microcantilevers for the detection of malignant melanoma [12,13]. Recent developments in cantilever sensor technology have demonstrated the chemo response of cantilever sensors in the detection of methane using thermally sensitive microcantilevers [14].…”

Effect of Conformational Entropy on the Nanomechanics of Microcantilever-Based Single-Stranded DNA Sensors

“…The centroidal axis of the cantilever is taken as the coordinate axis x; its vertical direction along the cantilever thickness is consistent with that of the coordinate axis y. Here, l, b and h are the length, width and thickness, respectively; s h , 1 h and 2 h represent the respective thicknesses, and…”

“…Such devices can either be static-mode (bending) sensors or dynamic-mode (resonance frequency variation) sensors. The low-cost sensors exhibit fast response, high sensitivity and suitability for parallelization into arrays and allow for a wide range of analyses, such as gene mutation [1], DNA hybridization [4] and protein-ligand interactions [5].…”

Fast Diagnostics of BRAF Mutations in Biopsies from Malignant Melanoma

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