MOL3101 Praktisk molekylærmedisin (laboratoriekurs)

Molecular methods are important for diagnosing human diseases and for the follow up of patients during treatment. In addition, molecular methods are important in medical and biological research. In this course the student will be introduced to the use of molecular methods in detection and characterization of driver mutations in human cancer, and to the consequences of such mutations at the cellular level. Methods include cell culture and methods for the measurement of cell growth/-survival., extraction and purification of nucleic acids (DNA and RNA) from human cell lines, gel electrophoresis, polymerase chain reaction (PCR and RT-qPCR), DNA sequencing (Sanger dideoxy method; guided), preparation of protein extracts, detection of epidermal growth factor receptor (EGFR) by Western blot analysis, fixation of cells and in situ immunohistochemical detection of EGFR. Prior to the practical part of the course the student will be presented to guidelines for safe laboratory practices, background theory on human cancer and the role of EGFR, principles for primer and probe design, as well as the use of bioinformatics tools in mutation detection and characterization. Methods for mutation detection and genotyping are important in molecular characterization of cancer, for selecting the most effective first-line treatment, and for detecting molecular alterations in the tumor that may occur during treatment. A range of genotyping methods developed for this purpose will be presented and discussed. Activation of the EGFR pathway (increased EGFR expression, activating EGFR mutation, EGFR gene copy gain) has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy. To identify patients who are most likely to benefit from treatment with EGFR tyrosine kinase inhibitors (TKIs), EGFR mutation testing is being increasingly utilized in clinical practice. Quite commonly, patients who are treated with TKIs will after a while become resistant to the drug. Over 50% of acquired resistance to EGFR TKIs is caused by a mutation in the ATP binding pocket of the EGFR kinase domain involving substitution of a small polar threonine (T) residue with a large nonpolar methionine (M) residue at amino acid position 790 (T790M). The effect of this mutation on cell growth in the presence of TKIs (erlotinib, gefitinib) will be demonstrated.