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BACKGROUND Cytology specimens are the main samples used for the diagnosis of advanced lung cancer. The objective of our study was to assess anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 receptor tyrosine kinase (ROS1) genes by an amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) using cytology specimens and to then evaluate the mutation frequency of ALK and ROS1 in non-small-cell lung cancer (NSCLC) patients. METHODS A large cohort that consisted of 8180 NSCLC patients who were genetically tested us