title: An immune stratification reveals a subset of PD-1/LAG-3 double-positive triple-negative breast cancers.
abstract: Stromal tumor-infiltrating lymphocytes (TILs) are a robust prognostic factor in triple-negative breast cancer (TNBC). However, the clinical significance of TILs may be influenced by the complex landscape of the tumor immune microenvironment. In this study, we aimed to evaluate the composition and the functionality of lymphocytic infiltration and checkpoint receptors in TNBC.
  
  
  
  Formalin-fixed, paraffin-embedded tissues were retrospectively collected from a cohort of patients with early-stage TNBC treated with adjuvant anthracycline-based chemotherapy (n = 259). Results were validated in an independent cohort of patients with TNBC (n = 104). Stromal TILs were evaluated on hematoxylin-and-eosin-stained sections. The density of CD4+, CD8+, and FOXP3+ lymphocytes, and the expression of the immune checkpoints PD-1 and LAG-3, were assessed by immunohistochemical analysis.
  
  
  
  The presence of elevated TILs positively correlated with the density of all T cell subtypes, especially cytotoxic CD8+ lymphocytes. We showed that increasing stromal TILs assessed as a continuous variable is an independent prognostic marker of prolonged relapse-free survival and overall survival in TNBC. Among immune subpopulations, CD8+ lymphocytes are the main effectors of anti-tumor immune responses. In two independent cohorts, we found that PD-1 and LAG-3 were concurrently expressed in approximately 15% of patients with TNBC. The expression of both checkpoint receptors positively correlated with the presence of TILs, but was not significantly associated with patient outcome.
  
  
  
  Overall, our data indicate that the evaluation of stromal TILs remains the most reliable immune prognostic marker in TNBC, and support the clinical evaluation of anti-PD-1/PD-L1 and anti-LAG-3 in a subset of patients with TNBC who have concurrent expression of both checkpoint receptors.
authors.affiliation: Oncology Experimental Therapeutics, IRCCS Clinical and Research Institute Humanitas, Via Manzoni 113, 20089, Rozzano-Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bottai
authors.affiliation: Oncology Experimental Therapeutics, IRCCS Clinical and Research Institute Humanitas, Via Manzoni 113, 20089, Rozzano-Milan, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Raschioni
authors.affiliation: Department of Oncology, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.
authors.forename: Agnese
authors.initials: A
authors.lastname: Losurdo
authors.affiliation: Department of Pathology, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Di Tommaso
authors.affiliation: Department of Surgery, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.
authors.forename: Corrado
authors.initials: C
authors.lastname: Tinterri
authors.affiliation: Department of Oncology, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.
authors.forename: Rosalba
authors.initials: R
authors.lastname: Torrisi
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Reis-Filho
authors.affiliation: Department of Pathology, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.|Humanitas University, Rozzano-Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Roncalli
authors.affiliation: Breast Cancer Translational Research Laboratory, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Christos
authors.initials: C
authors.lastname: Sotiriou
authors.affiliation: Department of Oncology, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.|Humanitas University, Rozzano-Milan, Italy.
authors.forename: Armando
authors.initials: A
authors.lastname: Santoro
authors.affiliation: Humanitas University, Rozzano-Milan, Italy.|Department of Immunology and Inflammation, IRCCS Clinical and Research Institute Humanitas, Rozzano-Milan, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Mantovani
authors.affiliation: Division of Cancer Medicine and Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors.forename: Sherene
authors.initials: S
authors.lastname: Loi
authors.affiliation: Oncology Experimental Therapeutics, IRCCS Clinical and Research Institute Humanitas, Via Manzoni 113, 20089, Rozzano-Milan, Italy. liberosantarpia@yahoo.it.
authors.forename: Libero
authors.initials: L
authors.lastname: Santarpia
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D014408:Biomarkers, Tumor; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D056747:Immunomodulation; D016130:Immunophenotyping; D053208:Kaplan-Meier Estimate; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D011379:Prognosis; D061026:Programmed Cell Death 1 Receptor / Q000378:metabolism*; D016016:Proportional Hazards Models; D064726:Triple Negative Breast Neoplasms / Q000276:immunology* / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D000093322:Lymphocyte Activation Gene 3 Protein
chemical_list: D015703:Antigens, CD; D014408:Biomarkers, Tumor; D061026:Programmed Cell Death 1 Receptor; D000093322:Lymphocyte Activation Gene 3 Protein; C000720326:Lag3 protein, human
keywords: Immune checkpoints; LAG-3; Prognosis; Triple-negative breast cancer; Tumor-infiltrating lymphocytes