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Background Detection of epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients has relied on DNA purification from biopsies, amplification, and sequencing. However, the number of tumor cells in a sample is often insufficient for EGFR assessment. Methods We prospectively screened 1380 NSCLC patients for EGFR mutations but found that 268 were not evaluable because of insufficient tumor tissue. We therefore developed and validated a method of detecting EGFR mutations in these samples. Tumor cells were microdissected into polymerase chain reaction buffer and amplified. EGFR mutations were detected by length analysis of fluorescently labeled polymerase chain reaction products and TaqMan assay. Results We determined EGFR status in 217 (81%) of the 268 primary NSCLC samples not evaluable in our original study—fresh and paraffin-embedded with less than 150 cells. Exon 19 deletions were detected in 11.5% of patients and exon 21 L858R mutations in 5.5%. In addition, the exon 20 T790M mutation was detected in 6 of 15 (40%) patients at the time of progression to erlotinib. The primary, sensitive mutation was present in all tumor cells, whereas the T790M mutation was absent in some groups. Conclusions The method presented here eliminates the need for DNA purification and allows for detection of EGFR mutations in samples containing as few as eight cancer cells.

Background: c-Met mutations play a critical role in the development and progression of primary tumors and metastases. Activation of the HGF/SF-c-Met pathway determines a poor prognosis in non-small-cell and small-cell lung cancer (SCLC) patients. Missense mutations of c-Met have been identified in SCLC patients located in the juxtamembrane (JM) and in the Sema domain. To determine the role of the c-Met pathway in SCLC, we have investigated the presence of c-Met mutations in SCLC patients. Patients and methods: Forty-four tumor tissue samples from SCLC patients were obtained with bronchoscopy before beginning treatment. Analysis of c-Met mutations was performed in exon 2 and exon 14. Results: Of the 44 patients included in this study, 23 were classified as limited disease and were treated with sequential or concurrent chemotherapy and thoracic radiotherapy. Twenty-one patients with extensive disease received chemotherapy alone, the majority with cisplatin or carboplatin plus etoposide. The median survival was 14 months (95% CI: 9.4 to 18.5 months) and the 2- and 5-year survival rates were 24% and 15%, respectively. Previously identified missense mutations E168D, R988C and T1010I in c-Met were not found in our study. However, novel mutations were identified, including T995I in the juxtamembrane domain (T995I) and a mutation which does not change amino acid in codon 178 in the Sema domain. Conclusion: In SCLC patients, the presence of mutations in c-Met gene is a rare event. Other genetic alterations involved in the HGF/SF-c-Met pathway should be assessed to define the role of this signaling pathway in SCLC.

e22110 Background: Identification of distinct oncogenic driver mutations is routinely used to guide clinical care in NSCLC patients (p). Tumor tissue availability is one of the major challenges for...

11025 Background: SOX2 is a key transcription factor that is amplified in lung SCC. FGFR1 is a receptor tyrosine kinase that promotes cell proliferation, survival and apoptotic resistance through the PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is amplified in 15-25% of lung SCC. Pre-clinical studies of targeted inhibitors showed a growth dependency on FGFR1 amplification both in vitro and in vivo. A European, multicenter clinical trial of second-line treatment with BIBF1120, an FGFR1 inhibitor, will be performed in p with lung SCC with FGFR1 amplification. Methods: We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC p by multiplex ligation-dependent probe amplification (MLPA). Genomic DNA (gDNA) was isolated from enriched tumor cells by laser capture microdissection from formalin-fixed paraffin embedded (FFPE) tumor tissue. 50-100 ng of gDNA was analyzed in each of three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN comparisons, the results from each patient were normalized against the GCN values derived from FFPE peripheral blood leukocytes. In order to study intra-tumor heterogeneity (TH), we examined FGFR1 and SOX2 GCN in different areas of 4 tumors. In 2 p, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression. Results: High FGFR1 amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%) p. High SOX2 amplification was observed in 38/63 (60.32%) and low amplification in 9/63 (14.28%) p. 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. Intra-TH was observed in 2/4 tumors. Survival according to FGFR1 and SOX2 GCN will be presented. In addition, GCN changes in FGFR1, SOX2, PIK3CA, PDGFRA, KDR, EGFR and MET over 10 years of follow-up will be presented for one surgically resected SCC lung p. Conclusions: FGFR1 and SOX2 co-amplification could represent a novel therapeutic target and warrants further research.

SRY-related HMG-box (SOX2) gene is a key transcription factor that coordinates development, differentiation and self-renewal of normal non-alveolar epithelium of the airway. SOX2 amplification has been reported in lung squamous cell carcinoma (SCC). Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that, upon activation, promotes cell proliferation, survival and apoptotic resistance through PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is also amplified in 15-25% of lung SCC and pre-clinical data with targeted inhibitors showed a growth dependency on FGFR1 amplification either in vitro and in vivo. A clinical trial with BIBF1120 (which also inhibits FGFR1), will be developed in the Netherlands and Spain in second line SCC of the lung with FGFR1 amplified by FISH. Genetic heterogeneity in solid tumors is a major research area and therefore its assessment in SCC of the lung is of great relevance. We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC by multiplex ligation-dependent probe amplification (MLPA). Analysis of mutations in DDR2, PIK3CA, BRAF, EGFR, KRAS, and TP53 is ongoing. Genomic DNA (gDNA) was isolated from enriched tumor cells by laser captured microdisection from formalin-fixed paraffin embedded (FFPE) tumor tissue sections. 50-100 ng of gDNA was analyzed in each of the three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN value comparisons, the tumor results from each patient was normalized against the GCN values derived from FFPE peripheral blood leukocytes control. We also used MLPA technique to study intra-tumor heterogeneity (TH) by analyzing FGFR1 and SOX2 in different tumor areas. In particular, in 4 cases FGFR1 and SOX2 were co-amplified. Also, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression in two patients. Of the 76 patients evaluable for FGFR1 and SOX2 GCN, FGFR1 high amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%). SOX2 presented high amplification in 38/63 (60.32%) and low amplification in 9/63 (14.28%). Interestingly, 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. An important degree of TH was found in 2 of the 4 tumors analyzed. GCN changes in FGFR1, SOX2, PIK3CA, PDGFRa, KDR, EGFR and MET over 10 years follow-up will be presented for one surgically resected SCC lung cancer patient. Frequencies of FGFR1 and SOX2 gene amplifications detected are in agreement with previously published data. Survival according to FGFR1 and SOX2 status will also be presented. FGFR1 and SOX2 co-amplification, together with the TH, warrants further research and could represent a novel therapeutic target. Citation Format: Pedro Méndez, José L. Ramirez, Amaya Gascó, Teresa Morán, Enric Carcereny, Miquel Taron, José Luís Mate, Irene Sansano, María Pérez, Mónica Botia, Montserrat Tierno, Harry J.M Groen, Rafael Rosell. FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2013-53

7593 Background: DAB2IP loss promotes primary tumor growth by activating Ras and drives metastasis through NFkB, serving as a signaling scaffold to coordinately regulate these pathways. DAB2IP is f...

7545 Background: DNA repair genes such as BRCA1 are induced by c-Myc (Menssen and Hermeking, PNAS 2002), and high BRCA1 mRNA levels have been related to poor survival in resected NSCLC (Rosell et a...

7687 Background: The primary aim of this trial was response. In both the control arm and in the genotypic arm with low tumor ERCC1 mRNA levels, pts received docetaxel (doc)/cis; in the genotypic arm with high tumor ERCC1 mRNA levels, pts received doc/gemcitabine. Response was significantly higher in the genotypic arms. We examined 324 pts for genetic markers that could influence response, including ERCC1 118 C/T, ERCC1 C8092A, XRCC3 241 (Thr to Met), Aurora A 91 T>A, Aurora A 169G>A, a SNP within intron 7 of the TGFBR1 gene (Int7G24A), and an in-frame germline deletion (TGFBR1*6A). Methylation of 14–3-3s and CHFR were also analyzed. Methods: DNA from peripheral lymphocytes was used for genotyping (Taqman assay) and methylation-specific PCR was used for 14–3- 3s and CHFR in pretreatment serum DNA. Results: There were no differences in clinical characteristics among the different SNP types, except that pts with Aurora A 91 AA had higher tumor ERCC1 mRNA levels (P=0.005). No relationship was found between ERCC1 SNPs and tumor ERCC1 mRNA levels. A strong correlation was found between the Int7G24A and XRCC3 241 SNPs (P=0.03). The Int7G24A GA type had a higher odds ratio (OR) of response (OR 2.32) than the AA type (OR 3.15) (P=0.02). XRCC3 241 MetMet had a lower probability of response (OR 0.23) (P=0.04). No other differences in response were observed according to any of the other SNPs or methylation. In the multivariate model, the best response was observed in pts with performance status (PS) 0, low ERCC1 levels, and XRCC3 241 SNP ( Table ). Conclusions: Further research is warranted to define the role of theTGFBR1 Int7G24A gene in customized treatments. No significant financial relationships to disclose. [Table: see text]

4043 Background: The purpose of this study was to determine the maximum tolerated dose (MTD) of CDDP/TXT/CPT-11 in p with advanced esophagogastric cancer. XPD and UGT1A1 polymorphisms were also ana...