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Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies (Pasteur Hospital). MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections (Pasteur Hospital). CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs (Spearman correlation, 0.93). MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials.

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Clinical responses to immune checkpoint blockade by anti-PD-1/PD-L1 monoclonal antibodies in non-small-cell lung cancer (NSCLC) may be associated with PD-L1 expression. This study was undertaken to determine the expression profile of PD-L1 in patients with Kras-mutant lung adenocarcinoma (LUAD) and to investigate the activation of Kras codon subtypes as a mechanism of PD-L1 regulation. Immunohistochemistry analysis of PD-L1 expression (SP142 clone) on 117 LUAD (KrasWT, n = 51; KrasG12D, n = 25; KrasG12V, n = 14; KrasG12C, n = 27), showed significantly greater expression of PD-L1 in both tumor and immune cells compartments in Kras mutant LUAD compared with KrasWT wild-type tumors (37% vs. 18%; P = 0.005). PD-L1+ tumors had greater CD66b+ neutrophil infiltrate and lower CD8+ T-cell infiltrate than PD-L1− tumors, notably in KrasG12D tumors. To determine the effect of Kras codon subtypes on PD-L1 expression, stable cell lines were generated by transfection of KrasG12D, KrasG12V, KrasG12C and KrasWT plasmids into Beas2B bronchial cells. At basal level, mutant Kras led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, notably in KrasG12C and KrasG12V cells, suggesting transcriptional regulation. Moreover, there was differential activation of NF-kB, ERK and Pi3k/Akt pathways between Kras mutant subtypes. In addition, PD-L1 was upregulated 3-fold by stimulation with IFNγ, independently of the Kras codon subtypes. Instead, hypoxia significantly increased PD-L1 expression in KrasG12C and KrasG12D cells. Co-culture experiments with human PBMCs from healthy patients were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 expression by tumor cells induced by Kras mutations led to decreased PBMCs proliferation and increased apoptosis. PD-L1 is expressed in 37% of Kras mutant LUAD, suggesting PD-L1 as a therapeutic target in this subset. According to the Kras mutation subtype, potential drugs targeting the NF-kB, ERK or Pi3k/Akt pathways may increase the antitumor adaptive immune responses. Citation Format: Marius Ilie, Laetitia Fazzalari, Nicolas Guibert, Nathalie Yazbeck, Laurie Signetti, Véronique Hofman, Elodie Long, Katia Zahaf, Julien Fayada, Virginie Lespinet, Olivier Bordone, Virginie Tanga, Kevin Washetine, Nicolas Vénissac, Jérôme Mouroux, Charles Hugo Marquette, Patrick Brest, Paul Hofman. Impact of Kras mutant subtypes on PD-L1 expression in lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4025.

Background: Detection of genomic rearrangements, like anaplastic lymphoma kinase (ALK) fusions, is a pivotal requirement in non-small cell lung cancer (NSCLC) for the initiation of a targeted treatment. While tissue testing remains the gold standard, detection of these alterations using liquid biopsies is an unmet need.To enable the detection of ALK rearrangements from circulating-free RNA (cfRNA) from NSCLC patients, we have evaluated a novel reverse transcription PCR (RT-PCR) based assay.Methods: Sixty-six patients with advanced stage NSCLC were included in the study. ALK status was determined by immunohistochemistry (IHC) and/or FISH on tissue sections. For the detection of ALK rearrangements from 2ml plasma collected in EDTA or Streck BCT DNA tubes, cfRNA was extracted using a prototype cfRNA sample preparation method and tested by a novel multiplex ALK/RET RT-PCR assay (Roche).Results: Of the forty-two patients with an ALK rearrangement, 30 (71%) were included at baseline. In 10 of the baseline patients, an ALK rearrangement was detected by RT-PCR [baseline sensitivity 33.33% (95% CI: 17.29-52.81%)]. All 24 negative ALK IHC/FISH-negative patients were negative using the RT-PCR based assay (specificity =100%). Conclusions:The prototype Roche ALK/RET RT-PCR assay was able to detect ALK fusion transcripts in the plasma of NSCLC patients at baseline as well as at disease progression with limited sensitivity but high

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