•Immune checkpoint inhibitors are the most promising treatment for advanced NSCLC.•Primary and acquired resistance to immune checkpoint inhibitors is common.•We examined biopsies taken from sites of acquired resistance or mixed responses.•Transformation from NSCLC to SCLC was identified as a mechanism of resistance. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death world-wide. Immune checkpoint inhibitors (ICI) have become the most promising type of treatment in oncology in general, and significantly so in NSCLC. Limited data is available about mechanisms of primary resistance. Data is lacking about mechanisms involved in acquired resistance or mixed responses in NSCLC. We aimed to identify mechanisms of resistance by studying biopsies taken from sites of secondary progression. We identified all cases of NSCLC that have received ICI for advanced disease in our institute. Of these cases, those that have demonstrated acquired resistance or mixed responses, and have underwent a biopsy from a progressive lesion were analyzed. Selected specimens were subjected to next-generation sequencing (NGS; Oncomine™ Solid Tumour Fusion Transcript Kit). Out of 664 lung cancer cases, 249 were NSCLC that have received ICI. Of these, eight cases matched our search criteria. Two of them demonstrated transformation to small cell lung cancer (SCLC; 2/8, 25%). NGS verified a common origin to a matched pre-treatment NSCLC specimen and an on-treatment progressive SCLC specimen. In two cases no tumor cells were found and in the remaining four the pathology was similar to the initial biopsy. In one of the cases of SCLC transformation platinum-etoposide chemotherapy was administered, with short-term benefit only and further disease progression. Mechanisms of acquired resistance to ICI include SCLC transformation. Repeat biopsies of progressing lesions after initial response or in cases of mixed response can shed light on mechanisms of resistance.