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782-C Regulatory T-cell tumor infiltration is associated to better outcome in advanced NSCLC patients under ≥2nd line anti-PD1/L1 monotherapy in the PIONeeR project
  1. Marcellin Landri1,
  2. Margot Vasseur1,
  3. Florence Monville1,
  4. Vanina Leca1,
  5. Chafik Hamdad1,
  6. Margaux Mercadal1,
  7. Laurent Vanhille1,
  8. Alboukadel Kassambara1,
  9. Sebastien Benzekry2,
  10. Maryannick Le Ray3,
  11. Marie Roumieux4,
  12. Richard Malkoun3,
  13. Arnaud Boyer5,
  14. Clarisse Audigier-valette6,
  15. Stephanie Martinez7,
  16. Hervé Pegliasco8,
  17. Patrice Ray9,
  18. Lionel Falchero10,
  19. Antoine Serre11,
  20. Nicolas Cloarec12,
  21. Louisiane Lebas13,
  22. Stéphane Hominal14,
  23. Patricia Barré15,
  24. Sarah Zahi16,
  25. Ahmed Frikha17,
  26. Pierre Bory18,
  27. Lilian Laborde19,
  28. Julien Mazières20,
  29. Maurice Pérol21,
  30. Laurent Greillier22,
  31. Fabrice Barlesi23 and
  32. Jacques Fieschi1
  1. 1Veracyte SAS, Marseille, France
  2. 2Inria, Marseille, France
  3. 3APHM, Marseille, France
  4. 4Aix Marseille Université, Marseille, France
  5. 5Hôpital Saint-Joseph, Marseille, France
  6. 6CHIC Toulon, Toulon, France
  7. 7Centre Hospitalier D’Aix-en-Provence, Aix-en-Provence, France
  8. 8Hôpital Européen, Marseille, France
  9. 9CHU de Nimes, Nimes, France
  10. 10Hopital Nord-Ouest, Ville Franche-sur-Saone, France
  11. 11Institut Cancerologie du Gard, Oncogard, Names, France
  12. 12Centre Hospitalier Henri Duffaut, Avignon, France
  13. 13Centre Hospitalier Du Val D’Ariège, St Jean de Verges, France
  14. 14Centre Hospitalier Annecy Genevois, Epagny-Metz Tessy, France
  15. 15Centre Hospitalier Jean Rougier, Cahors, France
  16. 16Centre Hospitalier de Montauban, Montauban, France
  17. 17Polyclinique Maymard, Bastia, France
  18. 18Centre Hospitalier de Bastia, Bastia, France
  19. 19Institut Paoli-Calmettes, Marseille, France
  20. 20Toulouse Universitary Hospital, Toulouse, France
  21. 21Centre Leon Berard, Lyon, France
  22. 22Aix Marseille Université, APHM, Marseille, France
  23. 23Paris Saclay University, Gustave Roussy, Paris, France
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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. 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 biomarkers assessment. The immune system is crucial for tumor evolution and is composed of different subsets of immune cells that can be activating (T-, B-lymphocytes, ...) or regulating such as regulatory T-cells (Treg).

Methods Tumor was sampled at diagnosis from 101 advanced pretreated NSCLC patients, ECOG PS0/1, treated with standard PD1/L1 ICIs monotherapy. Complete database of ≥2nd line PIONeeR patients was released in July 2023. Overall Response Rate was assessed by RECIST 1.1. Multiplex IHC Brightplex® T-cells exhaustion quantifies cytotoxic (Tc) (CD3+CD8+) and helper (Th) (CD3+CD8-) T-lymphocytes in both tumor parenchyma and stroma. This quantification allows stratification into 4 tumor groups: Hot, Parenchyma Hot, Cold and Stroma Tumor Infiltrating Lymphocytes (TILs).1 2 Dual staining CD4 FOXP3 quantifies Treg density in parenchyma and stroma. Correlation analyses: spearman non-parametric test. Samples’ classification: unsupervised neural-network-based machine learning algorithm Self-Organizing Maps (SOM). Statistical significance of progression-free/overall survival (PFS/OS) differences: log-rank test. Response distribution differences: Fisher’s test.

Results Patients were mainly male (65%), current/previous smoker (92%), <70yrs (68%) with median PFS=4.4months. Across the 101 tumors, Treg were not strongly correlated to any other cell type (Tc: R=0.54, Th: R=0.59). As expected, Brightplex® TCE identified 4 patient groups based on Th/Tc infiltration revealing outcome differences: Hot (N=32), Cold (N=19), Parenchyma Hot (N=15) and Stroma TILs (N=35). Each group was stratified according to Th/Treg infiltration. The 35 Stroma TILs patients (median PFS=6.4) were split into 4 groups (SOM): low Th/Treg-infiltration (N=10); Th-only parenchyma-infiltration (N=7); intermediate Treg+Th infiltration in both compartments (N=9); Treg+Th high infiltration (N=9). The two lowest infiltrated groups had poorer outcome (median PFS=1.6/1.8; median OS=6.9/7.4 respectively) than both infiltrated groups (median PFS=17.3/14.1; median OS=17.3/not reached respectively), p=4.1e-4. 10/11 responders were part of both infiltrated groups (p=2.6e-3) regardless of PDL1 status.

Conclusions Treg infiltration evaluation improved previous lymphocyte-associated NSCLC classification regarding response to anti-PD1/L1 ICIs.1 2 Absence of Treg, regardless of Th cells infiltration, in the Stroma TILs patient subset, was associated with early progression and poor survival. These unexpected results were already described in some cancers and could be linked to Tregs’ ability to suppress general inflammation that itself triggers cell proliferation and metastasis.

Acknowledgements This work benefited from a government grant handled by the French National Research Agency (ANR) as part of the France 2030 investment plan, under the reference ANR-17-RHUS-0007. A partnership of AMU, AP-HM, CNRS, Inserm, Centre Léon Bérard, Institut Paoli-Calmettes, AstraZeneca, Veracyte, Innate Pharma & ImCheck Therapeutics, sponsored by AP-HM and initiated by Marseille Immunopole. Drug supply is funded by AstraZeneca.

Trial Registration NCT03493581


  1. Ghezali L, Landri M, Monville F, et al. Brightplex® TCE and Brightplex® MDSC assays combination improves NSCLC patients’ stratification under anti-PD1/L1 immunotherapy in the PIONeeR project. Journal for ImmunoTherapy of Cancer 2022;10.

  2. Leca V, Kassambara A, Ghezali L, et al. 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. Journal for ImmunoTherapy of Cancer 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.

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