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Supplemental Material Biomarker methods IGF1R gene copy number was determined by both a silver in situ hybridization assay (SISH) and by fluorescent in situ hybridization assay (FISH). The details of the SISH assay have been described previously.1 Briefly, the tissue slides were probed with the IGF1R specific DNA probe obtained from Ventana Medical Systems Inc (Tucson, AZ) according to manufacturer’s instructions and using the Benchmark XT automated slide stainer with appropriate secondary and ultraView SISH Detection reagents. Following precipitation of the silver particles within the nuclei, a single black dot represents a single copy of the IGF1R gene, whereas a cluster of black dots concentrated so closely together that single dots cannot be resolved are considered amplified genes. A single black dot was given a score of one and clusters, if present, were scored as 6 or 12 for small and big clusters, respectively. A certified pathologist visually scanned the entire tissue specimen and counted signals within ~15 nuclei in 4 fields with high copy number for a total of 50 nuclei. For the FISH assay, slides from 12 specimens were subjected to a dual-color FISH assay using the IGF-1R/CEP15 probe set, following standard protocol in the laboratory. A FISH probe for the IGF-1R gene (located on 15q26.3) was prepared with the insert from the BAC clone RP11-262P8 obtained from CHORI (Oakland, CA). DNA was extracted, amplified, tested for the presence of the gene sequences by PCR-based reactions. DNA was labeled with SpectrumRed (SR) conjugated dUTPs in aliquots of 1ug using the Vysis Nick Translation Kit (Des Plaines, IL) according to the manufacturer’s instructions. The labeled DNA was precipitated with herring sperm and human Cot-1 DNA and the pellet was resuspended in 10 ul of c-DenHyb (Insitus Biotechnologies, Albuquerque, NM). CEP 15 labeled in SpectrumGreen (SG) was purchased from Abbott Molecular (Des Plaines, IL). The samples were incubated at 56oC for 4 hours, soaked in CitriSolv wash series 3 times for 10 minutes each and allowed to air dry. The tissue area to be hybridized was marked with a diamond scriber guided by the marks on the H-E stained slides. The slides were incubated in 2xSSC at 75oC for 5-15 min and in 0.6 mg/ml Proteinase K at 45 oC for 5-15 min. After washing and dehydrating the specimens in 70% EtOH, 85% EtOH and 100% EtOH for 2 min each, the slides were allowed to air dry. The appropriate volume of the probe set probe set was applied to the selected hybridization areas, which were covered with a coverslip and sealed with rubber cement. DNA codenaturation was performed at 85oC for 15 min and hybridization allowed to occur at 37 oC for 16-24 hours. Post-hybridization washes were performed though incubations in 2xSSC/0.3% NP-40 at 74oC for 2 min and 2xSSC for 2 min each, followed by dehydration. Finally, 14 ul of DAPI/anti-fade (0.3 ug/ml in Vectashield mounting medium) was applied to the slide and the area covered with a 22x50 mm coverslip. Analysis was performed on epifluorescence microscope using a single interference filter sets for green (FITC), red (Texas red), and blue (DAPI) as well as dual (red/green) and triple (blue, red, green) band pass filters. Monochromatic images were captured by a CCD camera (Cool Snap, Photometrics) and merged using dedicated software CytoVision (Genetix). Slides were scored by one trained reader and independently reviewed IGF1-R protein expression was evaluated on the tissue specimens by immunohistochemistry using the G11 clone and ultraView detection kit from Ventana Medical Systems Inc (Tucson, AZ) according the manufacture’s protocol for the Benchmark XT autostainer. The final HScore was determined by a certified pathologist but summing the products of the five staining intensity categories (0-4) by the percentage of positive cells (0-100%) for a final score of 0-400. Biomarker Results Figure 1. Hazard ratio and confidence intervals for biomarker association with PFS Legend: N = number of patients with biomarker measured; HR: Hazard ratio; CI = confidence interval. A Cox Proportional Hazard model was used regarding the effect of biomarker status on PFS. Diagnostics for the functional forms of variables were not performed due to low sample size; free serum IGF, IGFR1 FISH, and IGFR1 SISH values and IGF IHC H scores were included individually as continuous covariates. No multivariate analysis was performed. All tests and confidence intervals were two-sided with conclusions based on a significance level of 0.05. Statistical analyses were performed using SAS/BASE and SAS/STAT software, Version 9.2 of the SAS System for Windows. Summary: Small number of patients limit possible conclusions. Figure 2. Representation of IHC and SISH for IGF1R Gene Copy number Table 1. Raw Data for Biomarkers PT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Dose cohort 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 IGF IHC H score (up to 400) IGF SISH IGF FISH 105 2.16 1.98 100 100 75 100 210 160 280 20 90 100 2.24 1.84 2 2.88 2.36 2.14 2.62 3.33 2.16 1.9 5.32 3.58 1.16 1.78 2.44 3.16 2.92 1.6 3.5 PFS (days) OS (days) 22 37 27 52 112 21 217 57 432 34 21 23 20 245 40 40 21 39 31 65 92 135 408 105 541 275 432 56 392 236 44 303 138 70 93 69 Table 2. Free IGF-1 levels and Tumor Characteristics Number of patients Male Median Age Ever Smokers EGFR mutation EGFR wild type EGFR status not assessed Quartile 1-3 Quartile 4 10 patients 2 (30%) 66 years 8 (80%) 2 (20%) 7 (70%) 1 (10%) 4 patients 3 (75%) 57 years 3 (75%) 0 (0%) 3 (75%) 1 (25%) Table 1. Summary Statistics of Erlotinib and OSI-420 Stratified by Course and Day Drug/ Metabolite Conc. (ng/ml) Erlotinib OSI-420 Course 1 2 1 2 Day 8 1 8 1 N 10 7 9 7 Mean 458.71 489.34 228.01 237.10 Min 17.1 42.4 67.7 5.29 StDev 182.19 230.78 198.86 230.54 Max 680 683 693 553 1. Badzio A, Wynes MW, Dziadziuszko R, et al. Increased insulin-like growth factor 1 receptor protein expression and gene copy number in small cell lung cancer. J Thorac Oncol 2010;5:1905-1911. Med 501.5 601 132 108