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Preclinical Studies and Early Drug Development: Predicting Tumor Response to Cytotoxic Therapy

Detection of tumor-derived DNA with EGFR mutations in different biological samples from cancer patients: feasibility and optimization of the method

Russian Clinical Research Center for Pediatric Hematology/Oncology and Immunology, Moscow, Russian Federation

Abstract

B19

The aim of the present study is to develop an assay for quick and reliable detection of tumor-derived point mutations in EGFR gene in different biological samples from cancer patients.Epidermal growth factor receptor is one of the most attractive targets for molecular therapy of cancer. Among different types of EGFR modulators that have already been successfully introduced into clinic there are synthetic small molecules designed to inhibit the intracellular tyrosine kinase domain of EGFR. Reliable markers that can be used to predict the effectiveness of these drugs would be of great value. The whole spectrum of predictive markers for such drugs as gefitinib and erlotinib is not yet known; nevertheless, some point mutations in the tyrosine kinase domain of EGFR are known to be connected with extreme responsiveness to these drugs and other point mutations in the same domain strongly predict tumor resistance to both Iressa and Tarceva.Several assays for detection of mutant EGFR in biopsy specimens or surgically removed tumor tissue have been developed. However, the early detection of newly-established resistance to EGFR inhibitors during the course of treatment with such drugs is also of crucial importance since it gives an opportunity to circumvent this resistance by means of polychemotherapy. We tried to establish an assay which can be used not only for initial determination of EGFR status, but also for its monitoring in dynamic.43 samples from 14 patients with different solid tumors were analyzed. DNA from tumor tissue, saliva, blood cells, blood plasma and urine cell sediment was extracted using the three-stage protocol (proteinase K treatment, phenol deproteinization and ethanol precipitation) with appropriate modifications. Exons 18 to 21 of EGFR gene were amplified by PCR and amplicons were analyzed by SSCP electrophoresis. All major bands were also analyzed by direct sequencing. Results of the sequencing were interpreted with the help of main genetic databases and literature search.All four techniques were found feasible when applied to every type of samples. Results of repeated SSCP experiments appeared to be reproducible. 5 germline heterozygous mutations in exon 20 and one somatic mutation in exon 19 were detected. All of them appeared to be silent.PCR-SSCP analysis of exons 18-21 of EGFR gene combined with direct sequencing of non-"wild-type" bands seems to be a reliable tool for establishing EGFR status in different biological samples and may be used in clinical trials of small-molecule EGFR inhibitors.