Comprehensive SCLC Genomic Profiling Protocol

The institutional review board of the University of Cologne approved this study. We collected and analysed fresh-frozen tumour samples of 152 SCLC patients, which were provided by multiple collaborating institutions as fresh-frozen tissue specimen, frozen sections or as genomic DNA extracted from fresh-frozen material (Extended Data Fig. 1). Human tumour samples were obtained from patients under IRB-approved protocols following written informed consent.
The fresh-frozen SCLC samples were primary tumours diagnosed as stage I–IV tumours, and snap-frozen after tissue sampling. All tumour samples were pathologically assessed to have a purity of at least 60% and no extensive signs of necrosis. Additionally, these tumour samples were reviewed by at least two independent expert pathologists and the diagnosis of SCLC was histomorphologically confirmed by H&E staining and immunohistochemistry for chromogranin A, synaptophysin, CD56 and Ki67. Matching normal material was provided in the form of EDTA-anticoagulated blood or adjacent non-tumorigenic lung tissue (Supplementary Table 1). The matched normal tissue was confirmed to be free of tumour contaminants by pathological assessment. Furthermore, tumour and matching normal material were confirmed to be acquired from the same patient by short tandem repeat (STR) analysis conducted at the Institute of Legal Medicine at the University of Cologne (Germany), or confirmed by subsequent SNP 6.0 array and sequencing analyses. Patient material was stored at −80 °C.
Whole-genome sequencing was performed on 110 SCLC fresh-frozen tumour samples and matched normal material. Additionally, we analysed RNA-seq data of 81 SCLC primary tumours (Extended Data Fig. 1 and Supplementary Table 1), among which 20 cases were previously published^{2 (link),43} . Furthermore, we studied the copy-number alterations of a total of 142 fresh-frozen tumour specimen by Affymetrix SNP 6.0, among which 74 cases were described before⁴⁴ .
Clinical correlation studies were performed with the study cohort of 110 SCLC patients considering age of diagnosis, gender, tumour stage, surgery, treatment with chemotherapeutics, smoking status, smoking history and overall survival (Extended Data Figs 2 and 4 and Supplementary Table 1). The median follow-up time for this cohort of 110 SCLC patients was 69 months, and 31% of the patients were alive at the time of last follow-up (Extended Data Fig. 2a and Supplementary Table 1). Smoking status was available for 88% (n = 97) of the patients; 63% (n = 69) reported a smoking history amounting to a median of 45 pack-years. Patients with a known smoking history were further subcategorized to heavy smokers (>30 pack-years), average smokers (10–30 pack-years) and light/never smokers (<10 pack-years).
Primary findings on somatic mutations were further studied in a second independent cohort consisting of 112 SCLC cases. This validation cohort refers to the exome sequencing data of 28 fresh-frozen SCLC primary tumours and 9 SCLC cell lines^{2 (link),3 (link)} which were re-analysed in this present study (Supplementary Table 7). Additionally, we performed targeted sequencing on 8 fresh-frozen and 67 formalin fixed paraffin embedded (FFPE) samples from SCLC patients (Supplementary Table 1).