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Background More than 60% of patients with lung cancer are diagnosed at advanced stages. The introduction of targeted therapies requires molecular diagnosis to guide treatment. The aim of this study was to evaluate the feasibility of performing mutational analysis with brushing specimens obtained by radial-miniprobe endobronchial ultrasound (R-EBUS) plus fluoroscopy-guided bronchoscopy in patients with peripheral pulmonary adenocarcinoma. Methods Brushing specimens were deposited on cytological slides and were conserved in Roswell Park Memorial Institute (RPMI) culture medium. DNA was isolated to perform a mutational analysis with real-time quantitative polymerase chain reaction and Sanger sequencing for epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS). Results Thirty patients with adenocarcinoma were prospectively included. In 100% of the patients, the molecular study was viable with brushing specimens. In 16 (53.3%), KRAS or EGFR mutations were detected: 10 KRAS mutations (33.3%) and 6 EGFR mutations (20%). In a comparison with histological samples, a correlation of 86.6% was detected, and only 2 patients with wild-type results from brushing specimens presented with an EGFR mutation in histological samples. Conclusions Brushing specimens conserved in RPMI medium and obtained by R-EBUS plus fluoroscopy-guided bronchoscopy are valid for molecular studies. They allow the detection of EGFR/KRAS mutations in patients with peripheral adenocarcinoma. In addition, invasive techniques are avoided, the risk of complications is reduced, and the obtained samples are optimized.

Introduction Chemotherapy is the principal treatment in metastatic colorectal cancer (mCRC) patients. RAC1b, a RAC1 spliced variant, is over-expressed in colorectal cancer (CRC), and impairs apoptosis by activation of nuclear-factor-KB. Since RAC1b has been associated with the BRAF V600E mutation, associated with poor prognosis in CRC, we evaluated the role of RAC1b expression as a predictor of chemotherapy efficacy in mCRC. Methods We analysed KRAS and BRAF mutation, microsatellite instability and RAC1b expression in 157 mCRC patients treated with FOLFOX/XELOX in first-line therapy. Results KRAS mutations were detected in 46 patients (34%), 10 patients were BRAF mutant (7%) and 79 were WT for both, KRAS and BRAF (59%). RAC1b overexpression was found in 30 patients (19%). In the multivariate analysis, BRAF mutational status was a poor prognostic factor for overall survival (OS); hazard ratio (HR), 2.78 (95% confidence interval (CI), 1.35–5.72; p =0.0057). RAC1b overexpression was a poor survival factor for OS (HR, 2.35; 95% CI, 1.2–4.59; p =0.01) and progression-free survival (PFS) (HR, 2.4; 95% CI, 1.2–4.78; p =0.01) in KRAS/BRAF WT mCRC patients. Conclusions RAC1b overexpression constitutes a marker of poor prognosis in KRAS/BRAF WT mCRC patients treated with first-line FOLFOX/XELOX therapy.

Although receiving adjuvant chemotherapy after radical surgery, a disappointing proportion of patients with colorectal cancer will develop tumor recurrence. Probability of relapse is currently predicted from pathological staging, there being a need for additional markers to further select high-risk patients. This study was aimed to identify a gene-expression signature to predict tumor recurrence in patients with Stages II and III colon cancer treated with 5'fluoruracil (5FU)-based adjuvant chemotherapy. Two-hundred and twenty-eight patients diagnosed with Stages II-III colon cancer and treated with surgical resection and 5FU-based adjuvant chemotherapy were included. RNA was extracted from formalin-fixed, paraffin-embedded tissue samples and expression of 27 selected candidate genes was analyzed by RT-qPCR. A tumor recurrence predicting model, including clinico-pathological variables and gene-expression profiling, was developed by Cox regression analysis and validated by bootstrapping. The regression analysis identified tumor stage and S100A2 and S100A10 gene expression as independently associated with tumor recurrence. The risk score derived from this model was able to discriminate two groups with a highly significant different probability of tumor recurrence (HR, 2.75; 95%CI, 1.71-4.39; p = 0.0001), which it was maintained when patients were stratified according to tumor stage. The algorithm was also able to distinguish two groups with different overall survival (HR, 2.68; 95%CI, 1.12-6.42; p = 0.03). Identification of a new gene-expression signature associated with a high probability of tumor recurrence in patients with Stages II and III colon cancer receiving adjuvant 5FU-based chemotherapy, and its combination in a robust, easy-to-use and reliable algorithm may contribute to tailor treatment and surveillance strategies.

3553 Background: Metastatic colorectal cancer (mCRC) patients (pts), with BRAF tumor mutation (V600E) present poor overall survival (OS). RAC1b, a RAC1 spliced variant, is overexpressed in CRC, and impairs apoptosis by activation of nuclear-factor-KB. Because RAC1b and BRAF (V600E) are significantly associated in CRC (Matos et al, 2008), we evaluated if RAC1b/RAC1 mRNA expression ratio (RNA ER) was an independent prognostic factor in mCRC. Methods: We analyzed tumor samples from 186 mCRC pts, treated in first-line therapy with FOLFOX or CAPOX in three Spanish hospitals. We examined BRAF (V600E) mutational status by allelic discrimination, RAC1b/RAC1 expression ratio by mRNA RT-PCR (quantitative real time polymerase chain reaction) and mismatch repair (MMR) deficiency by microsatellite instability (MSI) analysis. We assessed whether these biomarkers were independently predictive of response rate (RR), progression free survival (PFS) or OS. Results: 88% of samples were informative for BRAF, 75% for MMR status and 85% for RAC1b/RAC1 (RNA ER). BRAF (V600E) was found in 7%, MSI-H in 5% and high >0.9-fold RAC1b/RAC1 (RNA ER) in 20% of pts. Five of 11 pts (46%) with BRAF mutation had high RAC1b/RAC1 (RNA ER) compared with 25/144 (18%) without BRAF mutation (p=0.036). All pts with BRAF mutation were MMR and none of the MSI-H pts, showed high RAC1b/RAC1 (RNA ER). Patients with low RAC1b/RAC1 (RNA ER) and BRAF WT had higher response rate (68% vs 43%; p=0.035). Response rate were not different according BRAF mutation (64% WT vs 63% mutant) (p=NS). The multivariate regression analysis identified ECOG PS (HR: 4.2 (CI 1.4-12.7) as a significant variable for PFS and LDH levels (HR: 2.26 (CI 1.3-3.8), PS (HR: 3.85 (CI 1.5-9.8) and high RAC1b/RAC1 (RNA ER) (HR: 2.6 (CI 1.1-5.9) for OS in WT BRAF pts. Conclusions: High RAC1b/RAC1 (RNA ER) constitutes a marker of poor OS and a potential marker of acquired chemo-resistance in WT BRAF mCRC pts treated with first-line oxaliplatin-based therapy.

10639 Background: To prospectively explore the role of serum MMP-7 as a predictive and prognostic marker of anti-EGFR therapy and irinotecan efficacy in third-line advanced colorectal cancer therap...

4060 Background: Single nucleotide polymorphism (SNP) in codon R497K of EGFR by reducing EGFR activity has been associated to resistance to anti-EGFR monoclonal antibodies in ACRC (Carcereny, ASCO2008). Methods: We retrospectively investigated the role of EGFR R497K and KRAS mutational status in primary CRC or metastases, in predicting response rate (RR), progression free survival (PFS) and overall survival (OS) of cetuximab or panitumumab in second or third line therapy in patients (pts) with ACRC. EGFR R497K was detected by real-time PCR using the TaqMan technology and KRAS mutation status in codons 12/13 was determined by sequencing. CT scans were done every 6–8 weeks (w) until progressive disease. Results: A total of 117 pts with available tissue out of 168 pts treated with anti-EGFR therapy in 6 Spanish Institutions, were analysed for EGFR R497K and KRAS mutational status. There were no differences in RR (18.8 vs.16.8%), PFS (13.2 vs. 12 w) and OS (31 vs. 28 w) between the whole and the selected cohort. We found no significant differences on RR (9/59;15.2 vs. 9/52; 17.3%), PFS (13.5 vs. 13.2 w) and OS (33 vs. 26.8 w) according to EGFR R497K (GG vs. GA/AA). Pts with wild-type (WT) KRAS had better response (20.7% vs. 8.1%;p=0.07) and PFS (14.4 vs. 11.7 w; p=0.006) compared with mutant KRAS. Interestingly, a significant increment on RR (30 vs. 0%, p=0.003), PFS (14.4 vs. 7.4 w, p=0.002) and OS (34.1 vs. 20 w, p=0.03) was observed only in those patients with WT KRAS and >1x upper limit of normal (ULN) lactate dehydrogenase (LDH) levels compared with mutant KRAS, but these differences were not found in pts with WT KRAS and 1xULN levels of LDH, have major benefit to anti-EGFR therapy in second-third line therapy. No significant financial relationships to disclose.