The retrospective identification cohort consists of patients with metastatic disease treated with front line immunotherapy as per clinical practice from 2017 to 2021 at the IRCCS National Cancer Institute "Regina Elena".

The optimal parameters for identifying KEAPness and obtaining information on the interactions between pairs of genes fundamental for the activation of KEAPness itself.

The validation court will collect NSCLC patients (metastatic setting, treated with first-line immunotherapy possibly in association with chemotherapy as per clinical practice) and also pancancer patients (patients with metastatic cancer other than NSCLC and who have received an ICI, either alone or in association with other medical treatments as per clinical practice), from which patients with NSCLC are excluded (PCC cohort). These patients will be recruited from the collaborating institute IRCCS Pascale of Naples. To ensure reproducibility of the study, the same approach of the first court will be applied to this one, i.e. the optimal parameters will be set for the identification of KEAPness and to obtain information on the interactions between pairs of genes fundamental for the activation of KEAPness.

To generate an innovative molecular tool for prediction DNA/RNA will be extracted from 5μm FFPE tissue sections using the AllPrep DNA/RNA FFPE kit (Qiagen). The quality of the RNA will be evaluated with the Bioanalyzer, the integrity of genomic DNA through the Agilent NGS FFPE qPCR QC Kit (Agilent). The libraries for RNA-Seq will be prepared using the RNA TruSeq Exome Kit (Illumina). The quality of the libraries resulting will be checked using Bioanalyzer (high sensitivity DNA kit). The intermediate library before exon enrichment will be quantified with Qubit, the final library with qPCR. The samples will be sequenced in paired-end mode, sequencing 76 bp on each side. The Exome DNA sequencing will be performed using SureSelectXT Low Input Kit reagents for the initial preparation of the library followed by enrichment with Clinical Research Exome Kit (Agilent).

Tracing the evolutionary path of NRF-2 dependent tumors, in his association with the microenvironment, and in particular the related immune subtypes, and characterize the interactions between pairs of genes (epistatic) underlying the KEAPness.i phenotype, the multi-region sequencing technology that will be used involves the use of surgical samples, taken in a single session. This allows you to isolate different regions of the tumor, corresponding to different time points (central region, R1; expansion zone, R2; locoregional metastases, R3).

Through this methodology it is possible to trace the evolution of the tumor over time, and in particular the interactions between pairs of mutant genes linked to the KEAPness phenotype.