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Comprehensive molecular characterization of acquired resistance to antiEGFR monoclonal antibodies in patients with metastatic colorectal cancer 1Filippo Pietrantonio, 1,2 Claudio Vernieri, 3,4Giulia Siravegna, 1Alessia Mennitto, 1Rosa Berenato, 5Federica Perrone, 5Annunziata Gloghini, 5Elena Tamborini, 6Sara Lonardi, 1Federica Morano, 5Benedetta Picciani, 5Adele Busico, 5Chiara Costanza Volpi, 1Antonia Martinetti, 5Francesca Battaglin, 1Ilaria Bossi, 5Alessio Pellegrinelli, 5Massimo Milione, 6Chiara Cremolini, 1Maria Di Bartolomeo, 3,4Alberto Bardelli and 1,8Filippo de Braud Supplementary Methods 1 Next generation sequencing Formalin-fixed, paraffin-embedded samples were sliced in 5um paraffine sections and manually microdissected to isolate the highest percentage of neoplastic cells as possible. Sample were treated with xylene and 100% ethanol to remove paraffin and then DNA was isolated using the GeneRead DNA FFPE kit (Qiagen, Hilden, Germany, http://www.qiagen.com Cat. n. 180134). DNA amount and quality were identified using Nano Drop (Invitrogen, Life Technologies) platform following the manufacturer’s instructions. We performed deep sequencing of 50 gene’s hotspot regions contained in the Hotspot Cancer Panel v2 (Life Technologies) using the Ion Torrent Personal Genome Machine platform (Life Technologies). Briefly, we used 10 ng of genomic DNA to amplify 50 gene’s hotspot regions using the Ion AmpliSeq Library Kit2.0 (Life Technologies) according to the manufacturer’s manual (MAN0006735 rev 5.0). Amplicons were ligated to P1 and Barcode adapters using DNA Ligase. Barcoded libraries were purified using AMPure Beads XP (Beckman coulters) and PCR-amplified for a total of five cycles. After a second round of purification with AMPure Beads, the amplified libraries were size and quality assessed using the Agilent Bio Analyzer DNA High Sensitivity kit (Agilent Technologies) and quantified using the Qbit dsDNA HS kit (Invitrogen, Life Technologies). Emulsion PCR and sample enrichment were performed using the Ion One touch 2 instrument according to the manufacturer’s instruction. Briefly, an input concentration of DNA library obtained with the first amplification step was added to the emulsion PCR master mix and the Ion sphere particles (ISPs) and a double phase (oil/water) PCR weas performed. Then ISPs were recovered ant template positive ISPs were enriched using Dynabeads MyOne Streptavidin C1 beads (Life Technologies). 316 Chips were used to sequence samples on Ion torrent PGM using Ion PGM 200 sequencing kit following the manufacturer’s instructions. 2 Data from the PGM sequencing were initially processed using the Ion Torrent platformspecific software Torrent Suite to generate sequence reads, alignment of the reads on the reference genome Hg19, trim adapter sequences, filter and remove poor signal-profile reads. The variant calling from the sequencing data was generated using the Variant Caller plugin. We applied some filters to that plugin to eliminate erroneous base calling: we set an average coverage depth >100, each variant coverage >20, a variant frequency on each sample >8, and a quality value >30. Filtered variants were visually examinated using the Integrative Genomic Viewer (IGV) tool to taste their level of quality and to confirm the variant presence on both “+” and “-“ strand. Finally, we annotate resulting variations using the Ensembl Variant Effect Predictor pipeline (Mc. Laren et al.), COSMIC database, dbSNP database. and MyCancerGenome database (http://www.mycancergenome.org/). Immunohistochemistry for HER-2 and MET Immunoistochemistry (IHC) was performed on 3 μm formalin fixed paraffin-embedded (FFPE) tissue sections. MET protein expression was detected by using a rabbit monoclonal anti-MET antibody (dilution 1:200; clone SP44, Spring Bioscience, Pleasanton, CA), directed against the synthetic peptide derived from C-terminus of human MET displaying membranous and/or cytoplasmic epitope. IHC was carried out on a BenchMark Ultra Platform (Ventana Medical Systems, Tucson, AZ) by using the OptiviewDAB Detection Kit (Ventana Medical Systems). MET IHC was evaluated according to a semi-quantitative assessment (H-score) which combines staining intensity (scored from 0 to 4) with the percentage of positive cells (scored 0–100%). Each individual intensity level is multiplied by the percentage of cells and all values are added to obtain the final IHC score, ranging from 0 to 400. Scores from 0 to 200 are considered negative/low expression and scores from 201 to 400 are considered positive/high expression. 3 HER2 protein expression was evaluated by using the anti-human c-erbB-2 A0485 polyclonal antibody (dilution 1:1500; Dako Denmark A/S, Glostrup, Denmark) on a Dako Autostainer Plus by using the EnVision® FLEX+ (Dako) detection system. HER2 immunoreactivity was evaluated according to previously described scoring systems (1, 2). Dual color silver in situ hybridization (SISH) for HER-2 and MET The MET and HER2 gene status was assessed by bright field dual-color SISH analysis on 3 μm FFPE tissue sections by using the INFORM HER2 Dual ISH DNA Probe Cocktail (Ventana Medical Systems), and the MET DNP Probe along with the Chromosome 7 DIG Probe (Ventana Medical Systems). Dual color ISH was performed on a BenchMark Ultra Platform (Ventana Medical Systems) according to the manufacture’s protocol. The signals were counted in at least 40 non overlapping tumor cells nuclei from each case. Small or large clusters were considered to be 6 signals and 12 signals, respectively. MET gene amplification was defined as positive when: a) MET/CEP7 ratio was > 2 or b) average number of MET signals per tumor cell nucleus was > 6, or c) tumor cells containing > 5 signals were > 50% of tumor cells, or d) tumor cells containing > 5 signals with a ratio > 2 were > 10% of tumor cells. HER2 gene amplification was defined as positive when: a) HER2/CEP17 ratio was > 2 or b) average number of HER2 signals per tumor cell nucleus was > 6 (3). References 1. Valtorta E, Martino C, Sartore-Bianchi A, Penaullt-Llorca F, Viale G, Risio M, et al. Assessment of a HER2 scoring system for colorectal cancer: results from a validation study. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2015;28:1481-91. 2. Hofmann M, Stoss O, Shi D, Buttner R, van de Vijver M, Kim W, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 2008;52:797-805. 3. Ruschoff J, Hanna W, Bilous M, Hofmann M, Osamura RY, Penault-Llorca F, et al. HER2 testing in gastric cancer: a practical approach. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2012;25:637-50. 4 5