Genomic analyses
tumor
BLOOD, TUMOR
The tumor samples were qualified for WES if the sample contained ≥30% tumor cells. In total, 89 frozen tumor biopsies at baseline (38 cohort 1, 37 cohort 2, 14 cohort 3) and 21 (5 cohort 1, 11 cohort 2, 5 cohort 3) at resistance were analyzed. Eighty-four blood samples were used as germline control. Genomic DNA was isolated from biopsy and blood of patients using the QIAamp DNA Mini Kit and DNeasy Blood and Tissue Kit (Qiagen), respectively, according to the manufacturer’s guidelines. DNA concentration was measured using QubitTM dsDNA Broad Range Assay (Invitrogen). A quantity of 30–100 ng of DNA was used for preparing the WES libraries. For the WES, the DNA was sheared with the Covaris E220 system (LGC Genomics/KBioscience). SureSelect Low Input Target Enrichment was used. In brief, DNA fragments were end-repaired, extended with an ‘A’ base on the 3′ end, ligated with paired-end adaptors with the Bravo Platform (Agilent Technologies) and amplified to generate libraries (10 cycles). Hybridization-based exome enrichment was performed using the Agilent SureSelect Low Input Clinical Research Exome V2 target enrichment system (Agilent Technologies). The final libraries were indexed, pooled and sequenced using the onboard cluster method, as paired-end sequencing (2 × 100-bp reads) on an Illumina NovaSeq 6000 sequencer at Gustave Roussy.Statistical analyses of association with efficacy were done with the confirmed objective response.