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SUPPLEMENTARY DATA
Patients and Methods
Hybrid capture-based (HC-based) NGS cancer gene tests (FoundationOne and
Guardant360)
Patients were referred through Teva Pharmaceutical Industries Ltd (Shoham,
Israel) to Foundation Medicine Inc. (Cambridge, MA) and underwent the
FoundationOne assay using Illumina HiSeq2000 (Illumina Inc., San Diego,
CA). The assay was carried out on hybridization-captured, adaptor ligation–
based libraries using DNA extracted from four formalin-fixed paraffinembedded (FFPE) sections cut at 10 μm. DNA sequencing was conducted for
3,769 exons of 236 cancer-related genes and 47 introns of 19 genes
frequently rearranged in cancer (a total of 1.14 million base pairs) on indexed,
adaptor-ligated, hybridization-captured libraries (Agilent SureSelect custom
kit; Agilent Technologies, Inc., Santa Clara, CA).7,18 Full sequencing was
performed using 49-bp paired reads on the Illumina HiSeq2000 to an average
depth of 843X, and evaluated for genomic aberrations including base
substitutions, deletions, insertions, copy number alterations (CNA;
amplifications and homozygous deletions), and several gene
fusions/rearrangements. The failure rate of this method was reported to be
4.9%18.
When patients' samples were exhausted, Oncotest, Teva Pharmaceutical
Industries Ltd coordinated testing of blood samples using the Guardant360
assay (Guardant Health Inc., Redwood City, CA). The Guardant360 NGS
panel detects single nucleotide variation (SNV) by sequencing 54 genes
spanning approximately 78,000 bp. This panel includes complete exon
coverage in all exons of 18 genes, critical exons in 36 genes, and CNA in
ERBB2, EGFR and MET. Specifically, two 10 ml tubes of whole blood cells
were collected from each patient in Streck cfDNA blood collection tubes.
cfDNA was isolated from 1.5-5 ml plasma, concentrated, size selected using
Agencourt Ampure XP beads (Beckman Coulter, Brea, CA) and measured by
Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA). The DNA was
extracted as previously described.20 The cfDNA was subsequently converted
to digital sequence libraries as previously described.20 These digital libraries
were amplified and subsequently enriched for target genes using biotinylated
custom baits of RNA probed following paired-end sequencing by HiSeq2500
(Illumina Inc.). The Guardant360 NGS panel targeted region was 78,000 base
pairs (78 kbp) per sample and each base was sequenced at average raw
coverage depth of 8,000X with minimum average base coverage of 3,000X.20
The failure rate of this method was reported to be 0.2%20.
During the study period (2011-2015), these HC-based NGS tests were updated
according to accelerating knowledge regarding drivers and their novel
treatments.
Standard molecular pathology diagnostic tests
Assessment of EGFR mutations was performed using real-time PCR or narrow
spectrum NGS.
For real-time PCR analysis, FFPE samples were used.
Genomic DNA was isolated from the tumor cells and extracted with the QIAamp
DNA kit (Qiagen Hilden, Germany). The optical density value of the DNA
samples was measured using a spectrophotometer and EGFR mutations were
detected using AmoyDx® EGFR 29 Mutations Detection Kit (Amoy Diagnostics
Co. ltd, Xiamen, China) which allows identification of 29 EGFR mutations in
exons 18-21 via real-time PCR according to the manufacturer instructions. For
narrow spectrum NGS, the TruSeq Amplicon Cancer Panel (TSACP; Illumina
Inc.) was used. This panel includes 212 amplicons from 48 genes, which are
simultaneously amplified in a single-tube reaction, allowing identification of
somatic mutations. The isolated DNA was used for molecular profiling
according to the manufacturer’s instructions with the MiSeq system (Illumina
Inc).
Assessment of ALK rearrangements
FFPE specimens were subjected to FISH analysis utilizing the Vysis ALK Break
Apart FISH kit (Abbott Molecular Inc., Des Plaines, IL). At least 50 nonoverlapping nuclei were analyzed and the localization of the LSI ALK 5’ probe
(green) and LSI ALK 3’ probe (orange) signals were recorded and interpreted
according to manufacturer’s guidelines.
Other FFPE specimens were subjected to immunohistochemistry. The slides
were stained with the D5F3 antibody (Cell Signaling Technology Inc., Danvers,
MA) on Benchmark XT autostainer (Ventana Medical Systems, Inc., Tucson,
AZ) with the UltraView DAB detection kit (Ventana Medical Systems, Inc). The
antibodies are sensitive to all EML4-ALK variants according to the
manufacturer's instructions.
Note: Please see online Supplementary Table 1 for a full list of the study
patients, the detected genomic drivers and treatments.