P114: Intratumor heterogeneity of driver mutations in lung adenocarcinoma

Abstract

Background Lung cancers are characterized by the genetic alterations that often affect a common group of oncogenic signaling pathways. Identification of biologically significant genetic alterations in lung cancer that lead to activation of oncogenes and inactivation of tumor suppressor genes has the potential to provide further therapeutic opportunities (Cooper, 2013). In this context, it is important that molecular aberrations may act as negative predictors of sensitivity to treatment. The discovery of driver oncogenes, such as activating mutations in the epidermal growth factor receptor gene (EGFR), has made personalized medicine for lung cancer a reality. Despite the high initial results, NSCLC patients acquire resistance to tyrosine kinase inhibitors in the worldwide. There are several findings suggesting that the effect of tyrosine kinase inhibitors could be limited to patients harboring KRAS mutation. To date, a limited information regarding link of morphological portrait of adenocarcinomas with bearing of simultaneously concurrent mutations of KRAS and EGFR genes. The detection of concurrent gene mutation is usually carried out in a mixture of tumor cells. We plan to analyze EGFR and KRAS mutations in all clonal components (or morphological structures) of NSCLC tumors isolated by laser microdissection. So, we suggest that NSCLC clonal components might bear two concurrent EGFR and KRAS mutations simultaneously or different clonal components might bear either EGFR or KRAS mutations. The data obtained can predict resistance to EGFR-TKIs and affect therapeutic decision making. Materials and methods The tumor samples were obtained from archived formalin-fixed paraffin-embedded tissue blocks. Paraffin was removed by xylene extraction, and the sample was subsequently lysed by proteinase K. The region containing the highest percentage of tumor cells (at least 50%) was dissected. Microscopes Axio Scope A1 and Axio Star plus (Carl Zeiss, Germany) was used to perform histological analysis. If necessary, tumour cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (PALM AdhesiveCaps, P.A.L.M., Bernried, Germany). EGFR (exon 19 deletion and 21 L858R) and KRAS (Gly12Cys, Gly12Ser, Gly12Arg, Gly12Val, Gly12Asp, Gly12Ala, Gly13Asp) gene amplification was based on RT-PCR on CFX96 Real-Time System (Bio-Rad). Results A total of 115 patients with stage (IIIB–IV) of NSCLC with EGFR-TKI- sensitive mutations were eligible for the analysis. A rare coexistence of KRAS and EGFR mutations were observed in 3 patients (2.6%). The first sample was described as nonmucinous solid adenocarcinoma and has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. The second sample was described as mucinous lepidic adenocarcinoma and has deletion of 19 exon of EGFR gene and G12D mutation of KRAS gene. The third sample was described as mucinous adenocarcinoma with solid and acinar monoclonal components and also has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. Of all the samples, only one had two different monoclonal components. Unfortunately, this sample was not available due to biopsy and few numbers of cells for microdissection. Fiala et al. (2013) reported that KRAS mutations were not only as a negative prognostic factor and also as a biomarker predicting resistance to EGFR-TKIs, especially G12C. Patient with nonmucinous solid adenocarcinoma with deletion of 19 exon and G12C (EGFR and KRAS genes, respectively) is unlikely to benefit from receiving gefitinib or erlotinib. Conclusion The data obtained show that further studies are required to improve personalized therapy for NSCLC patients. The frequency and type of KRAS and EGFR mutations were first assessed in NSCLC patients from West Siberia. This work was supported by a Grant from the OPTEK Company (No 122/2014/51/Nvs ).