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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.
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