Supplementary table S2: Technical Description of Biomarker Testing Performed

IHC analysis
IHC analysis was performed on formalin-fixed paraffin-embedded (FFPE) tumor samples using commercially available detection kits, automated staining techniques including BenchMark XT (Ventana Medical Systems, Inc., Tucson, AZ) and AutostainerLink 48 (Dako North America, Inc., Carpinteria, CA), and commercially available antibodies.
Below is a list of all IHC antibodies used with supplier information.
Product Name / Vendor / Catalog # / Clone
ALK / Ventana / 790-4383 / D5F3
AR / LEICA / NCL-AR-318 / AR27
cMet / VENTANA / 790-4430 / SP44
EGFR / Zymed/ Invitrogen / 28-0005 / 31G7
ER / VENTANA / 790-4325 / SP1
ERCC1 / ABCAM / AB2356 / 8F1
H3K36me3 / ABCAM / AB9050 / polyclonal
HER2/neu / VENTANA / 790-2991 / 4B5
MGMT / INVITROGEN / 18-7337 / MT23.2
MLH-1 / VENTANA / 790-4535 / M1
MSH-2 / VENTANA (CELL MARQUE) / 760-4265 / G219-1129
MSH-6 / VENTANA / 790-4455 / 44
PBRM1 (PB1/BAF180) / Bethyl Laboratories / A301-591A / polyclonal
PD-1 / VENTANA (CELL MARQUE) / 760-4895 / NAT105
PD-L1/CD274 / Spring Bioscience / M4424 / SP142
PGP (MDR1) / INVITROGEN / 18-7243 / C494
PMS-2 / VENTANA (CELL MARQUE) / 760-4531 / EPR3947
PR / VENTANA / 790-4296 / IE2
PTEN / DAKO / M 3627 / 6H2.1
SPARC-MONO / R&D SYSTEMS / MAB941 / 122511
SPARC-POLY / EXALPHA / X1867P / polyclonal
TLE3 / Sigma / HPA054116 / polyclonal
TLE3 / Lifespan / LS-C7310 / polyclonal
TOPO1 / LEICA / NCL-TOPO1 / 1D6
TOPO2A / LEICA / NCL-TOPO11A / 3F6
TS / INVITROGEN / 18-0405 / TS106/4H4B1
TUBB3 / COVANCE / PRB-435P / polyclonal
FISH and CISH
FISH and CISH was used to evaluate HER2/neu [HER2/CEP17 probe], EGFR [EGFR/CEP7 probe], and cMET [cMET/CEP7 probe] (Vysis PathVysion FISH assay, Abbott Laboratories, Abbott Park, IL). HER2/neu and cMET status were evaluated by CISH using the INFORM HER2 Dual ISH DNA Probe Cocktail, and the Chromosome 7 DIG Probe (Ventana Medical Systems, Inc., Tucson, AZ). The same scoring system was applied as for FISH. Either the absolute gene copy number in tumor cells or a gene:CEP17 signal ratio was used to score results in both methods.
HER2 CISH test was carried out using the INFORM DUAL HER2 ISH Assay (Ventana). Control was CEP17. Cutoff was HER2/CEP17 ratio >=2.0. cMET CISH was carried out using a probe specific for cMET – pericentromeric region of chromosome 7 (Ventana). Positivity for increased gene copy number for cMET CISH has been defined as mean of ≥5 copies of MET gene per cell in NSCLC, because the gene copy number threshold for other tumor types has not been determined. TOP2A CISH was carried out using a probe specific for TOP2A – pericentromeric region of chromosome 17 (Ventana). Control was CEP17. Cutoff was TOP2A/CEP17 ratio >=2.0 or the presence of the mean of ≥ 6 copies of the TOP2A in cancer cells. EGFR CISH was carried out using a probe specific for EGFR – pericentromeric region of chromosome 7 (Ventana).
Sequencing
Direct NGS analysis was performed on genomic DNA isolated from FFPE tumor samples using the MiSeq platform (Illumina, Inc., San Diego, CA). Specific regions of 45 genes of the genome were amplified using the TruSeq Amplicon Cancer Panel (Illumina, Inc., San Diego, CA). Mutation analysis by Sanger sequencing included selected regions of BRAF, KRAS, cKIT, EGFR, and PIK3CA genes and was performed by using M13-linked PCR primers designed to amplify target sequences. The depth of coverage was >1000X. Depth of coverage in DNA sequencing refers to the number of times a nucleotide is read/analyzed during the sequencing process. Coverage is the average number of reads representing a given nucleotide in the reconstructed sequence. 100% of NGS samples were microdissected after pathologist identification of tumour cells.