Spectrochipii

Following the assessment of the percentage of tumor cells in relation with the sample area (including non tumoral liver and stroma of the tumor), the block was subsequently cut at 30 μm and macrodissected if containing less than 10% of tumor cells (Supplementary Figure 1). DNA extraction was performed using the QIAmp DNA Mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer instructions. For the purpose of the study, DNA extraction was performed within all the tumor areas sampled from a given CLM. All the samples were retrospectively and completely tested for the relevant genes in primary colorectal tumor and CLM (i.e., KRAS, NRAS, BRAF and PIK3CA). Somatic gene mutations were detected using the MassARRAY iPLEX platform (Sequenom, San Diego, US), which involves a three-step process consisting of the initial PCR reaction, inactivation of unincorporated nucleotides by shrimp alkaline phosphatase and a single-base primer extension. Then, the products are nano-dispensed onto a matrix-loaded silicon chip (SpectroChipII, Sequenom, San Diego, US) and finally, the mutations are detected by MALDI–TOF (matrix-assisted laser desorption-ionization–time of flight) mass spectrometry. The experimental sensitivity of the assay was estimated to be below 1% for each gene mutation.

High-throughput multiplex mutation profiling was performed using the MassARRAY® LungCarta Panel Version 1.0 (Sequenom, San Diego, CA, USA). This panel permits screening of 214 mutations across 26 oncogenes and tumor suppressors with a limit of sensitivity of approximately 10% with the use of 480 ng DNA [17 (link)]. DNA was amplified using the OncoCarta PCR primer mix, unincorporated nucleotides were inactivated by shrimp alkaline phosphatase, and a single base extension reaction was performed using extension primers that hybridize adjacent to the mutation. Multiplexed reactions were spotted onto the SpectroChipII (Sequenom) using the MassARRAY Nanodispenser. Peaks with different mass were resolved by matrix-assisted laser desorption/ionization time-of-flight on the MassARRAY Compact Analyzer. A predefined ratio of expected normal allele to mutant allele at a specific nucleotide position allows mutations to be detected using primer extensions at that specific position. Because of the multiplexing capabilities of this assay, multiple mutations are detected simultaneously using one panel. Further details of the multiplex methodology can be found in the protocol provided by the manufacturer.