Doc. S1
Mutational analyses and MLH1 expression analysis
Mutations in KRAS codons 12, 13, and 61, BRAF V600E, PIK3CA exons 9 and 20, and TP53 exons 5, 6, 7, and 8 were analyzed by direct DNA sequencing. Primer sequences and PCR conditions were presented in Table
S4.
MLH1 protein expression was analyzed by immunohistochemistry (IHC) at the Department of Pathology, Tohoku University Hospital using a mouse anti-MLH1 monoclonal antibody (BD Biosciences, San Diego, CA, USA). The complete absence of nuclear staining of tumor cells was defined as negative expression. Intact nuclear staining of normal tissue adjacent to the tumor was used as an internal positive control.
Gene expression analysis using microarrays
Total RNA was extracted using an Absolutely RNA FFPE Kit (Agilent,
Santa Clara, CA, USA). cDNA was amplified from total RNA (300 ng) using a
TransPlex Complete Whole Transcriptome Amplification Kit (Sigma, St

Louis, MO, USA). The amplified cDNA was then purified using a QIAquick
PCR Purification Kit (Qiagen). The purified cDNA (1.5 μg) was labeled with
Cy3 using a Genomic DNA ULS Labeling Kit (Agilent). The labeled cDNA was then hybridized to the Whole Human Genome 4 × 44 K Microarray
(Agilent). Scanning and image analysis of the microarray were performed using a DNA microarray scanner (Agilent). Raw fluorescence intensities were quantified and normalized using Feature Extraction Software (Agilent).
Analysis and visualization of the microarray expression data were performed using GeneSpring GX ver. 11.5.1 (Agilent). The data were normalized by percentile shift (all samples were normalized to the signal value of the 75 th percentile of all probes on the microarray). Genes with low signals that could not be detected in all samples were ruled out. To exclude genes with low variance of signal intensity across all samples, 25,454 genes with standard deviations of signal intensity ≥1.0 were selected for further analysis.
Validation analysis using microarray data from another cohort
To validate sensitivity to anti-EGFR antibodies, we used microarray data for a cohort (GSE5851) registered at GEO. GSE5851 included 80 CRC

samples treated with the anti-EGFR antibody C-mab. FF tissues were used for microarray analysis. The data were normalized by MAS5 and scaling (all genes were normalized to the median of all samples). Genes with low signals that could not be detected were ruled out. To exclude genes with low variation of signal intensity across samples, 2,250 genes with standard deviations of signal intensity ≥1.0 were selected for further analysis.