br We hypothesized that TP
We hypothesized that TP53 mutational status determined by routine targeted next-generation sequencing (NGS) diagnosis may correlate with response to ICI (either as monotherapy or double PD-1 and CTLA-4 blockade). We focused on aNSCLC patients treated in a University center, either in registered indications or in clinical trials. We also hypothesized that TP53 mutational status could be used as a prognostic biomarker in NSCLC patients treated with ICI in routine clinical prac-tice.
2. Materials and methods
2.1. Patients and methods
We conducted a single-center retrospective study on consecutive aNSCLC patients treated with ICI in our university thoracic oncology department from September 2015 to November 2017. All patients were selected using our drug prescription software and treated with nivo-lumab, with or without CTLA-4 blocker ipilimumab, or pembrolizumab. Out of the 122 initially-identified consecutive patients, we only in-cluded those with NGS diagnosis available, performed on tumor tissue sampled before initiating ICI. Patients were included whether they sybr safe re-ceived ICI therapy as a standard of care for registered drugs, as parti-cipants in a clinical trial for other drugs or combinations or as a no-minative temporary authorization for use (ATU) for drugs awaiting French registration after obtaining European Medicines Agency (EMA) registration, due to positive Phase 3 clinical trials, or as compassionate use after validation by our multidisciplinary tumor board (MTB), based on the lack of any other recognized therapeutic option. Clinical, bio-logical, pathological, and molecular data were retrospectively collected from digital patient files. Responses were systematically evaluated by an MTB comprising an expert thoracic radiologist (AK), according to Response Evaluation Criteria In Solid Tumors (RECIST) rules. Immune RECIST criteria were not used in this study.
All patients were informed that their tumor samples were routinely analyzed at a molecular level for diagnosis purposes, and that they had the possibility to express their opposition to such routine analyses, according French law. This study was registered at the National Commission for Computing Liberties (CNIL registration number # 2,161,770) and received approval from the Institutional Review Board of the French-learned Society for Respiratory medicine-Société de Pneumologie de Langue Française (CEPRO number #2018-008), ac-cording to French regulatory rules for purely observational retro-spective studies.
Tumor genomic profiling was performed as a routine reimbursed diagnostic procedure using targeted somatic NGS at the Bichat University Hospital genetics department, for advanced NSCLC. The process involved extraction of DNA, performed using Maxwell auto-maton (Promega, Madison, Wisconsin, USA) with the FFPE Plus LEV DNA kit, then search for hotspots and targeted regions 25 known genes using NGS (S5XL - Life Technologies, USA), data analysis was carried out on Torrent Suite and Ion reporter (Life Technologies). The genes amplified using the NGS panel CEIVD targeted kit, Oncomine tumor solid DNA (OST) and complementary panel OST+ (Life Technologies) are described in Table 1. The sequenced TP53 gene regions are depicted in Fig. 1S. The structural and functional consequences of each TP53 mutation were verified on the International Agency for Research on Cancer (IARC) database [16,17]. Patients harboring synonymous TP53 mutations or TP53 mutations considered neutral according to the IARC database (especially c.217 G > C p.V73lL in exon 4) were included in
Genes Analyzed Using Next-Generation Sequencing in Patients with Advanced Non-Small-Cell Lung Cancer in routine care.
Baseline Characteristics of Patients with Non-Small-Cell Lung Cancer Treated with Immune Checkpoint Inhibitors.
Smoking status (%)
Performance status* (%)
ICI, immune checkpoint inhibitor; PD-L1, programmed cell death-ligand 1 expression.
* at ICI initiation. a comparison of nivolumab alone versus other treatments.
the TP53 non mutated group. Data for PD-L1 expression was analyzed on tumor cells using immunohistochemistry with E1L3N antibody (Cell Signaling Technology, Danvers, MA, USA), as previously described, with a published laboratory-developed test (LDT) on a Leica Bond III platform [18,19]. We applied a cut-oﬀ for PD-L1 expression positivity of at least 1% of tumor cells, in samples containing at least 200 tumor cells, evaluated by two expert thoracic pathologists from the multi-centric French panel for PD-L1 IHC testing (AC & CD).
The primary endpoint was OS, defined as time from ICI initiation to death due to any cause. Patients who did not die were censored at the date of last follow-up. Secondary endpoints included PFS, defined by the time from ICI initiation to disease progression (as assessed by MTB) or death due to any cause, objective response rate (ORR), and disease control rate (DCR). Response was evaluated according to RECIST Version 1.1 criteria by a MTB including a thoracic radiologist expert (AK) at 8 weeks and then every 3 months until progression. Patients who exhibited no progression were censored at the date of last follow-up.