Patients and samples
One hundred and twenty cases of primary colorectal cancer with the corresponding liver metastases were analyzed in this study (70 men and 50 women; median age, 63.5 (range, 32- 91). The metastases in 100 patients were synchronous, and in 20 were metachronous. These patients had undergone surgical resection of primary colorectal adenocarcinoma and liver metastasis between 1998 and 2008 at the Department of Gastroenterology, Tokyo Women's Medical University, Tokyo, Japan. One hundred two patients had received fluoropyrimidinebased chemotherapy after surgery, 6 had never received chemotherapy and 12 were unknown. No patients had received neoadjuvant/adjuvant radiotherapy. No patients had ever received anti-EGFR antibody agents. All of the patients were Japanese, and all gave their written informed consent according to the institutional regulations. The characteristics of the 120 patients are shown in Table .
Demographic and clinical parameters of patients with metastatic colorectal cancer
Formalin-fixed, paraffin-embedded tumor specimens were cut into serial sections with a thickness of 10 μm. For pathological diagnosis, one slide was stained with H&E and evaluated by a pathologist. Manual microdissection using a scalpel was performed if the histology was homogeneous and contained more than 90% of cancer cell tissue. For all other samples, laser-capture microdissection (P.A.L.M. Microlaser Technologies AG, Munich, Germany) was performed to ensure that only tumor cells were dissected.
RNA isolation and cDNA synthesis
RNA isolation from formalin-fixed paraffin-embedded (FFPE) specimens was performed using an RNeasy FFPE Kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. From the total RNA yielded, cDNA was converted using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan).
cDNA was pre-amplified using a Taqman PreAmp Master Mix Kit (Applied Biosystems, Tokyo, Japan) according to the manufacturer's instructions. Quantification of AREG and EREG and internal reference gene (beta-2-microglobulin) was done using a fluorescence-based real-time detection method (StepOne real-time PCR system, Applied Biosystems Inc., Tokyo, Japan). Single internal reference gene (Beta-2-microglobulin) was used in this study. The primers and probes used were from Taqman Gene Expression Assays (Applied Biosystem Inc.), Assay IDs were Hs00950669_m1 for AREG, Hs00914312_m1 for EREG, and Hs99999907_m1 for beta-2-micoroglobulin. The PCR reaction mixture consisted of 10 μl of Taqman Fast Universal PCR Master Mix, No UNG (Applied Biosystem Inc.), 5 μl of preamplified cDNA sample, 1 μl of Taqman Gene Expression Assays primers and probe (20×), and 3 μl of Nuclease-Free Water. Cycling conditions were 95°C for 20 seconds, followed by 40 cycles at 95°C for 1 second and 60°C for 20 seconds. The threshold cycle (CT) value for each gene was determined by SDS software v1.2 (Applied Biosystems). Delta-CT(ΔCT) value, which is the difference between the CT value of the target gene and the CT value of the endogenous control gene was also calculated by the same software. Delta-ΔCT (ΔΔCT), which is the difference in the ΔCT value for each sample and the highest ΔCT value as a calibrator, was calculated. The 2-ΔΔCT number was used for relative mRNA quantification.
Median values were used as the cut-off values to divide high and low expression.
KRAS mutation screening
DNA was extracted from FFPE specimens using the Qiamp DNA FFPE tissue Kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. Concentrations were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and 500 μg of DNA was added to a 6 μl of forward and reverse primer and 25 μl of Quick Tag HS DyeMix (Toyobo, Osaka, Japan). Primers that spanned codons 12 and 13 of the KRAS gene were: forward, 5'--GAATGGTCCTGCACCAGTAA-3'; and reverse, 5'-GTGTGACATGTTCTAATATAGTCA-3'. PCR cycling was run according to the followingconditions: one cycle of 94°C for 3 minutes, 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45 seconds; and one cycle of 72°C for 10 minutes.
PCR products were purified using the MinElute PCR Purification Kit (Qiagen). The purified PCR product was then used as a template in cycle sequencing with the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter, Tokyo, Japan) according to the manufacturer's instructions. Nested PCR primer sequences were Forward: 5'-GTCCTGCACCAGTAATATGC; reverse: 5'-ATGTTCTAATATAGTCACATTTTC-3'. Sequencing reactions were precipitated with ethanol, and run on a CEQ-8800 Genetic Analyzer (Beckman Coulter). Direct sequencing was performed in duplicate for each sample.
The comparisons between the median mRNA levels of the primary tumor and the corresponding liver metastases, median mRNA levels of KRAS mutant and wild-type were assessed using the Wilcoxon signed-rank test. The correlation between the mRNA levels of primary tumor and of liver metastases was assessed using Spearman's rank correlation. The Kaplan-Meier method was used for survival curves, and the log-rank test was used for statistical analysis. Overall survival was defined as the time from the day of primary tumor resection to death from any cause. Multivariate analyses were performed by Cox proportional hazard model. Statistical significance was recognized at P-values of less than 0.05. All values were two-sided.