Supporting Information

Patient population. 130 fresh frozen, surgically resected lung SCC samples from 129 individual patients (LS-71 and LS-136 were duplicate samples from different areas of the same tumor) from all stages of squamous cell lung carcinoma were evaluated in this study. These samples were collected from patients from the University of Michigan Hospital between October 1991 and July 2002 with patient consent and Institutional Review Board approval. Portions of the resected lung carcinomas were sectioned and evaluated by the study pathologist by routine H&E staining. Samples chosen for analysis contained greater than 70% tumor cells. Seventy-three patients had stage I SCC. Followup data was available for all patients. The mean patient age was 68 10 (range 42-91) with approximately 45% of patients 70 years or older. Supplementary Table 1 lists the clinical data associated with all lung SCC samples used in this study. The 86 lung adenocarcinoma samples have previously been described (1). Table 1 shows the clinical information associated with both adenocarcinoma and SCC samples used for identifying and testing the prognostic signatures. Patients were censored from statistical analysis if they were alive but had less than three years of clinical follow up. The independent NSCLC dataset comprised 36 lung adenocarcinoma (27 Stage I) and 36 lung SCC samples (25 Stage I) with at least 3 years follow up (Gene Omnibus dataset: GSE3141) (2).

Microarray Analysis. Total RNA was extracted from fresh frozen tissue using the Trizol method (Gibco BRL). RNA quality was checked using the Agilent Bioanalyzer. The

mean ribosomal ratio (28s/18s) for all samples was 1.5 (range: 1.0 - 2.1).

Four

micrograms of total RNA was amplified, labeled and aRNA was fragmented and hybridized to the Affymetrix U133A GeneChip as previously described (3). Microarray data was extracted using the Affymetrix MAS 5 software. Global gene expression was scaled to an average intensity of 600 units. The data was then normalized using a spline quantile normalization method. 128 of the 130 microarrays gave good data (% present > 40, scaling factor < 10) while the remaining 2 samples (LS78, LS82) gave acceptable results (% present > 30, scaling factor < 15). All samples were included in the analyses. The CEL files of the external validation datasets were downloaded from http://data.cgt.duke.edu/oncogene.php (2) and the data were extracted using Affymetrix MAS 5 software and were globally scaled to an average intensity of 600 units. Since the validation datasets were from external sources on different platforms (U133Plus 2.0), analysis of variance (ANOVA) was used to normalize the batch effect such as different sample preparation methods, different RNA extraction methods, different hybridization protocols, and different scanners. Array data from the duplicate samples LS-71 and LS136 was averaged for unsupervised and supervised analyses. The SCC data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE4573. The 50 probe sets from the adenocarcinoma classifier were identified on the Affymetrix HU6800 GeneChip (1). For validation of this classifier using the Duke data set it was necessary to map the HU6800 probe sets to the U133Plus GeneChip. To this end, probe sets on HU6800 were mapped to U133Plus through the U95 GeneChip based

on the Affymetrix Array Comparison Spreadsheets (Best Match). For those probe sets that could not be mapped by the Affymetrix documentation, a BLAST was performed on the target sequence from HU6800 to identify the corresponding probe sets on U133Plus. Using this approach 47 of the 50 probe sets were mapped to the U133Plus GeneChip.

Real-Time Quantitative RTPCR. Total RNA samples were normalized by OD260. Quality testing included analysis by capillary electrophoresis using a Bioanalyzer (Agilent). For aRNA, the RibobeastTM 1- Round Aminoallyl-aRNA amplification kit (Epicentre) was used. All first-strand cDNA synthesis, second-strand cDNA synthesis, in vitro transcription of aRNA, DNase treatment, purification and other steps were performed according to the manufactures protocol. For each sample aRNA was reverse transcribed into first-stand cDNA and used for real-time quantitative RT-PCR. The firststrand cDNA synthesis reaction contained, 100 ng of aRNA, 1 L of 50 ng/L T7Oligo(dT) primer, 0.25 L of 10 mM dNTPs, 1 L of 5 X SuperscriptTM III Reverse Transcriptase Buffer, 0.25 L of 200 U/L SuperscriptTM III Reverse Transcriptase (Invitrogen Corp), 0.25 L of 100 mM DTT and 0.25 L of 0.3 U/ul RNase Inhibitor (Epicentre) in a total reaction volume of 5 L. Real-time quantitative RTPCR analyses were performed on the ABI Prism 7900HT sequence detection system (Applied Biosystems). Each reaction contained 10 L of 2X TaqMan Universal PCR Master Mix (Applied Biosystems), 5 L of cDNA template, and 1ul of 20 X Assays-on-Demand Gene Expression Assay Mix (Applied Biosystems) in a total reaction volume of 20 L. The PCR consisted of an UNG activation step at 50C for 2 min and initial enzyme

activation step at 95C for 10 min, followed by 40 cycles of 95C for 15 sec, 60C for 1 min.

Statistical Analyses Univariate Cox proportional hazards regression was used to identify gene transcripts whose expression levels were correlated to patient disease-free time. To reduce the effect of multiple testing and to test the robustness of the selected gene transcripts, the Cox model was performed with bootstrapping of the patients in the training set. Briefly, 400 bootstrap samples of the training set were constructed, each containing the same number of patients as the training set but randomly chosen with replacement. Cox modeling was run on each of the bootstrap samples. A bootstrap score was created for each gene transcript by averaging the inverses of the bootstrap p-values with the top and bottom 5% of p-values removed. This score was used to rank the gene transcripts. To determine the minimum number of gene transcripts used to construct the signature, combinations of gene expression markers were tested by adding one gene at a time according to the rank order. For each signature with increasing number of genes, Receiver Operating Characteristic (ROC) analysis using death within 3 years as the defining point was performed, in 100 5-fold cross validations, to calculate the average area under the curve (AUC). The optimal number of gene transcripts was determined when the average AUC reached plateau. A maximum 3 years follow-up was employed since the majority of patients who will relapse in this population will do so within 3 years (4). Also many of these patients

were aged and death due to non-cancer related illnesses would likely increase after 3 years (4). This rationale was also employed when performing Cox modeling. Cox score was used to determine each patients risk of survival. For lung SCC, it was defined as the sum of the linear combination of weighted log2 expression intensity with the average standardized Cox regression coefficient from 400 bootstrapping samples as the weight. For lung adenocarcinoma, the Cox score was defined in the previous study (2). The threshold for risk stratification was determined in another 100 5-fold cross validations to test a series of cutoffs (percentile of risk index for the patients in the training set) and the optimal cutoff was chosen to give the minimum overall error rate. Because the Cox scores were from the two tumor types (i.e. adenocarcinoma and SCC), they were normalized to have the their corresponding thresholds set at zero and to have the same variance in order to combine them together to create the final score for each patient. Patients whose scores were equal to or greater than 0 were classified in the high risk of survival group while patients whose scores were less than 0 were predicted as the low risk of survival group. The gene expression signature and the cutoff were validated in the independent testing set (2). Kaplan-Meier survival plots and log-rank tests were used to assess the differences in survival of the predicted high and low risk groups. Patients were censored from statistical analysis if they were alive but had less than three years of clinical follow up. Sensitivity was defined as the percent of the patients who died within 3 years that were predicted correctly by the gene expression signature, and specificity was defined as the percent of the patients who survived for at least 3 years that were predicted correctly by the classifier. All the statistical analyses were performed using SPlus 6 software (Insightful, VA).

Hierarchical clustering was performed with GeneSpring 7.0 (Silicon Genetics) to identify major clusters of patients and to investigate their association with patient covariates. Prior to clustering, genes that had a coefficient of variation (CV) smaller than 0.3 (arbitrarily chosen) were removed to reduce the impact of genes that displayed minimal change in expression across the dataset. Thus a dataset with 11,101 gene transcripts was created for this clustering analysis. The signal intensity of each gene transcript was divided by the median expression level of that gene from all patients. Samples were clustered using Pearson correlation as measurement of similarity. Genes were clustered in the same way. Pathway analysis was performed by first mapping the gene transcripts on the Affymetrix U133A GeneChip to the Biological Process categories of Gene Ontology (GO). The categories that had at least 10 genes on the U133A chip were used for subsequent pathway analyses. Genes that were selected from data analysis were mapped to the GO Biological Process categories. Then the hypergeometric distribution probability of the genes was calculated for each category. A category that had a p-value less than 0.05 and contained at least two genes was considered over-represented in the selected gene list.

Supplementary Table 1. Patient sample data.

Supplementary Table 2. 121 probe sets differentially expressed between two major SCC clusters.

Supplementary Table 3. Significantly represented Gene Ontology groups between SCC subgroups.
GO ID 8544 6325 7586 7156 7148 7565 165 6805 7169 6832 Gene Count 17 3 3 4 3 3 2 2 3 2 GO Class epidermal differentiation chromatin architecture digestion homophilic cell adhesion cell shape and cell size control pregnancy MAPKKK cascade xenobiotic metabolism receptor tyrosine kinase signaling small molecule transport Gene Number on U133A 56 12 15 39 28 28 15 15 41 29 p-value 7.31E-12 2.75E-04 7.08E-04 0.0049 0.0079 0.0079 0.0082 0.0082 0.0293 0.0493

Supplementary Table 4. 50 lung SCC prognostic probe sets.

Supplementary Table 5. 47 of 50 lung adenocarcinoma prognostic probe sets that were used in this study.

0.4

0.6

0.8

1.0

0.2

Stage I Stage II Stage III 0 10 20 months P = 0.027

0.0

30

Supplementary Figure 1. Overall survival of patients with SCC lung cancer as stratified by stage.

NTRK2 - Affy vs TaqMan

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 Affy log expression

FGFR2 - Affy vs TaqMan

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0 Affy log expression

R = 0.96

R = 0.83

-5 0 5 10 Taqman dCT (NTRK2-ACTB)

15

5 10 15 Taqman dCT (FGFR2-ACTB)

VEGF - Affy vs TaqMan

4.1 3.9 3.7 3.5 3.3 3.1 2.9 2.7 2.5 0 5 Taqman dCT (VEGF-ACTB)

KRT13 - Affy vs TaqMan

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 -10

R = 0.71
Affy log expression

Affy log expression

R = 0.92

10

-5

10

15

Taqman dCT (KRT13-ACTB)

Supplementary Figure 2. Validation of Affymetrix gene expression using Taqman RTPCR of 4 genes. NTRK2, FGFR2, KRT13 were randomly chosen among the 121 genes that significantly differentiate the unsupervised clusters of lung SCC cancers.

FGFR2

KRT5

Expression High

low

KRT19

KRT17 High

low

Supplementary Figure 3. IHC of select proteins using SCC lung tissue microarrays.

A
mean of 5-fold cross-validation error rate 0.44 0.46 0.48 0.50 0.52 0.54 0.56

B
mean of 5-fold cross-validation error rate 0.0 0.2 0.4 0.6 0.8 1.0 0.55 0.45 0.0 0.50

0.2

0.4

0.6

0.8

1.0

percentile of scores

percentile of scores

Supplementary Figure 4. Identification of a cutoff from the percentile of the risk index in the training set. Panel A shows the results for the SCC training set and Panel B shows the results for the adenocarcinoma training set.

1.0

1.0

Good (n=21) 0.8 0.8 Overall Survival 0.4 0.6

sensitivity 0.4 0.6

Poor (n=15)

0.2

AUC = 0.83 0.0 0.0 HR (95% CI): 8.33 (1.89-36.6) 0 1 Years log rank P = 0.0008 2 3

0.0

0.2

0.4

0.6

0.8

1.0

1-specificity 1.0

1.0

0.2

Good (n=12)

0.8

Overall Survival 0.4 0.6

sensitivity 0.4 0.6

0.8

Poor (n=15)

0.2

AUC = 0.85 0.0 0.0 HR (95% CI): 12.2 (1.55-95.7) 0 1 Years log rank P = 0.0025 2 3

0.0

0.2

0.4

0.6

0.8

1.0

1-specificity

Supplementary Figure 5. Performance of 50-gene adenocarcinoma classifier in Duke samples. Panel A shows ROC analysis in 36 lung adenocarcinoma samples from Duke University. Panel B shows the Kaplan-Meier analysis using a cutoff defined in the original training set (1). Panel C shows ROC analysis in 27 stage I samples only and Panel D shows the associated Kaplan-Meier analysis.

0.2

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