Background Immune Checkpoint Inhibitors (ICIs) are associated with long-term survival in ~20% of advanced NSCLC patients while biological mechanisms triggering resistance are not fully elucidated. Myeloid-Derived Suppressor Cells (MDSC) might however play a key role. The PIONeeR project (NCT03493581, ANR-17-RHUS-0007) aims to predict the response/resistance to PD1/L1 ICIs in advanced NSCLC patients through comprehensive agnostic multiparametric biomarker assessment. Here, the combination of both lymphoid and myeloid lineages infiltration in the tumor is evaluated on tissue collected at diagnosis to predict outcome.

Methods Tumor samples from 60 advanced NSCLC patients, all ECOG PS0/1, treated with standard PD1/L1 monotherapy as ≥2nd line of treatment were studied. PD1/L1 ICIs Overall Response Rate was assessed by RECIST 1.1. Multiplex IHC assay Brightplex® TCE (former Immunoscore® CR T cells exhaustion) quantifies T-lymphocytes allowing tumor stratification into 4 groups: Hot, Parenchyma Hot, Cold and tumors with Stromal TILs.¹ Brightplex® MDSC assay quantifies myeloid cells (monocytes, neutrophils and other granulocytes), Monocytic- (M-) and PolyMorphoNuclear- (PMN-) MDSC. Correlation analyses: spearman non-parametric test. Association between biomarkers: Fisher’s exact test. Samples’ classification: unsupervised neural-network-based machine learning algorithm Self-Organizing Maps (SOM). Statistical significance of overall (OS) and progression-free survival (PFS) differences between groups: log-rank test.

Results Patients were mainly male (60%), smokers (98.33%), <70yrs (71.66%), median PFS was 3.9 months. Across the 60 tumors, M-MDSC were not correlated to any other cell type while PMN-MDSC were negatively correlated to Monocytes (R=-0.28) and highly correlated to granulocytes (R=0.85), even more when removing neutrophils (R=0.93). SOM clustering on myeloid cells alone did not allow any patients stratification regarding PFS nor OS. Considering previously described patient groups through Brightplex® TCE [1], the 20 “Hot tumors”, with high T-cells infiltration initially presenting the best PFS (median PFS=8.1months) were split into 2 groups. Patients’ subgroup with high PMN-MDSC and granulocytes tumor infiltration included no responder and had poor survival: 20% PFS at 20months vs 70% (p=0.035) in patients with low-infiltrated tumors, which included the 3 responders. Interestingly, in the 17 “Cold tumors”, high infiltration of PMN-MDSC and granulocytes also identified 5 patients with worse prognostic: at 2.5 months, only 20% PFS vs 58% in the low-density group (NS) (table 1).

Conclusions Brightplex® TCE and MDSC assays in combination allowed the stratification of advanced NSCLC patients in 5 subgroups, among which two subgroups with poor outcome and no responder, representing 45% of the total population that would likely not benefit from anti-PD1/L1 monotherapy.

Acknowledgements This work is supported by French National Research Agency (ANR-17-RHUS-0007), a partnership of AMU, APHM, AstraZeneca, Centre Léon Bérard, CNRS, Veracyte, ImCheck Therapeutics, Innate Pharma, Inserm, Institut Paoli Calmettes and sponsored by APHM. Drug supply is funded by AstraZeneca. Special thanks to patients and families.

References

1. Leca V, Kassambara A, Ghezali L. Spatial distribution of infiltrating T lymphocytes with Immunoscore® CR T cells exhaustion test helps stratification of NSCLC patients treated with PD1/PDL1 inhibitors in the PIONeeR project. JITC. 2021;9

Ethics Approval The study is conducted in accordance with Good Clinical Practice and the French applicable regulatory requirements (Public Health Code, article L.1121-1/La loi n° 2012–300 du 5 mars 2012 relative aux recherches impliquant la personne humaine (dite loi Jardé), the applicable subject privacy requirements, and the ethical principles that are outlined in the Declaration of Helsinski. The study was approved by the French Ethic Committee, CPP Ouest II – Angers, ref. CPP: 2028/08, Ref ANSM (French competent authority) 2018020500208, 2018072600120, 2019083000148. Freely given written informed consent was signed and obtained from each individual participating in the study, before any study specific procedure was undertaken and after the provision of information about the study by the investigator during a physician-patient consultation and sufficient time for reflection.