Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ macrophages as a predictor of improved survival in triple negative breast cancer

Sci Rep. 2021 Nov 3;11(1):21608. doi: 10.1038/s41598-021-01116-6.


Triple negative breast cancer (TNBC) comprises 10-15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33-0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25-0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Antigens, CD / metabolism*
  • Antigens, Differentiation, Myelomonocytic / metabolism*
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • B7-H1 Antigen / metabolism*
  • Biomarkers, Tumor / analysis
  • Female
  • Fluorescent Antibody Technique / methods*
  • Follow-Up Studies
  • Humans
  • Lymphocytes, Tumor-Infiltrating / immunology*
  • Macrophages / immunology*
  • Middle Aged
  • Prognosis
  • Retrospective Studies
  • Stromal Cells / immunology*
  • Survival Rate
  • Triple Negative Breast Neoplasms / drug therapy
  • Triple Negative Breast Neoplasms / immunology
  • Triple Negative Breast Neoplasms / mortality*
  • Triple Negative Breast Neoplasms / pathology
  • Tumor Microenvironment


  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • B7-H1 Antigen
  • Biomarkers, Tumor
  • CD274 protein, human
  • CD68 antigen, human