2.6. Pathological Analysis

The histological, immunohistological and immunohistochemical evaluation of extracted solid tissues by FB or CB, and the cytological, immunocytological and immuncytochemical evaluation of TBNA extracted cytoblock or smear, are performed at the local pathological institute (including ALK, ROS 1 and PD-L1 expression level on tumor cells). Histological and immunohistological tissue evaluation, or cytological and immunocytological evaluation, assigns the tumor tissue to either NSCLC or small cell lung cancer (SCLC). Any cases of SCLC will be excluded from further analyses and classified as a ‘drop out’. For any NSCLC, further molecular genetic evaluation will be undertaken, as described below.

Harmonization of the analytical processes is guaranteed in the study setting by the choice of the individual institutes of pathology, which are partners in the German national network of genomic medicine lung cancer (NGM lung cancer) [47], or which have participated in national collaborative ring trials. Additional collaborative ring trials for the analytical process will be performed for a selection of samples prior to the planned analytical process in this study.

Molecular genetic testing will be performed for solid bronchoscopically extracted material, for the cytological specimen extracted by TBNA (if applicable), and for the liquid biopsy samples. All currently targetable and non-targetable genetic aberrations, as suggested by the guidelines [10,35,36,37] (mutations, fusions, copy number variation (CNV)), as well as other important prognostic markers, are analyzed in accordance with the guidelines for standardized evaluation from the nNGM lung cancer consortium [48]. Targeted multigene mutation screening will be performed using different next generation sequencing techniques and platforms, depending on the participating site:

Tübingen: Amplicon library preparation will be done using the nNGM Panel 1.0 and 2.0. Semiconductor sequencing will be performed according to the manufacturers’ manuals using the Ion AmpliSeq Library Kit v2.0, the Ion 510 and Ion 520 and Ion 530 Kit—Chef and the Ion 520 Chip Kit on the Ion GeneStudio S5 (Ion Reporter software) (Thermo Fisher Scientific, Waltham, MA, USA). Fusion and CNV detection will be undertaken with immunohistochemistry following fluorescent in situ hybridization, or with the Archer^® FusionPlex Lung panel (Archer Analysis software, ArcherDX, Boulder, CO, USA).

Hamburg: The NEOselect assay (NEO New Oncology, Cologne, Germany) is used to detect single nucleotide mutations, copy number variations and fusions on the Illumina NextSeq system (Illumina, San Diego, CA, USA), according to the manufacturers’ manual. In the case of very limited material, CNVs will be tested using PCR technologies such as cobas^®; fusion and CNV detection will be undertaken with immunohistochemistry.

Köln: The amplification of DNA will be performed using the customized GeneRead DNAseq custom Panel V2 with primers for the nNGM Panel 1.0 and 2.0 (Qiagen, Hilden, Germany), following the manufacturer’s instructions. For library preparation, the Gene Read DNA Library I Core Kit and the Gene Read DNA I Amp Kit (Qiagen) will be used. After end-repair and adenylation, libraries were ligated to NEXTflex DNA Barcodes (Bio Scientific, Austin, TX, USA) and sequenced on the MiSeq (Illumina, San Diego, CA, USA) with a MiSeq reagent kit V2 (300 cycles) (Illumina), following the manufacturer’s recommendations. Data will be exported as FASTQ files. Alignment, variant calling and annotation will be done using an in-house bioinformatic pipeline. A 5% cutoff for variant calls will be used, and results will only be interpreted if the coverage was >200×. The detection of gene fusions will either be done by a combination of immunohistochemistry and fluorescence in situ hybridization (FISH), or by using the Archer^® FusionPlex Lung panel with the Archer Analysis software (ArcherDX, Boulder, CO, USA). CNVs will be analyzed by FISH.

Essen: The QIASeq Targeted DNA Panels (Qiagen, Hilden, Germany) nNGML1 and/or nNGML1.2 [48] will be run on an Illumina MiSeq or Illumina NextSeq platform, and the CLC Genomics Workbench versions 5.0.1, 12.0.3 or 20 will be used for data acquisition. Fusion and CNV detection will be carried out by immunohistochemistry, FISH, or by using Archer^® FusionPlex CTL panel (ArcherDX, Boulder, CO, USA).