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Updated: Nov 22, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Differential PD-1/LAG-3 expression and immune phenotypes in metastatic sites of breast cancer
Bettina Sobottka1, Holger Moch2, Zsuzsanna Varga2
1Department of Pathology and Molecular Pathology, University and University Hospital Zurich, Schmelzbergstrasse 12, CH-8091, Zurich, Switzerland. annabettina.sobottka-brillout@usz.ch.
Background:
A dual blockade against the novel immune checkpoint inhibitor lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1) is currently considered in advanced breast cancer. Nevertheless, PD-1 or LAG-3 expression within distant metastatic breast cancer tissue remains understudied.
Methods:
To address this knowledge gap, we investigated the PD-1 and LAG-3 expression in combination with the CD8-based immune phenotype in intrapatient matched primary tumor distant metastases, representing 95 breast cancer patients with metastases occurring at four different anatomical locations. The immune phenotype was categorized into 2 categories: inflamed corresponding to the clinical category "hot" and exhausted or desert consistent with clinically "cold" tumors.
Results:
Metastases of "cold" primary tumors always remained "cold" at their matched metastatic site. Expression of PD-1/LAG-3 was associated with a "hot" immune phenotype in both the primary tumors and metastases. We could not observe any association between the immune phenotype and the breast cancer molecular subtype. Brain and soft tissue metastases were more commonly inflamed with signs of exhaustion than other anatomical sites of metastases. Taken together, (i) the immune phenotype varied between sites of distant metastases, and (ii) PD-1+/LAG-3+ was strongly associated with a "hot" immune phenotype and (iii) was most prevalent in brain and soft tissue metastases among distant metastases.
Conclusions:
Our data strongly support an integrated analysis of the immune phenotype together with the PD-1/LAG-3 expression in distant metastases to identify patients with inflamed but exhausted tumors. This may eventually improve the stratification and likelihood for advanced breast cancer patients to profit from immunotherapy.
Insights
Dual blockade of lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1) is promising for advanced breast cancer. PD-1/LAG-3 expression is linked to inflamed tumors, especially in brain and soft tissue metastases, aiding patient stratification for immunotherapy.
Area of Science:
- Immunotherapy
- Oncology
- Translational Research
Background:
- Advanced breast cancer treatment considers dual blockade of immune checkpoints lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1).
- Expression of PD-1 and LAG-3 in distant metastatic breast cancer tissue is understudied.
Purpose of the Study:
- Investigate PD-1 and LAG-3 expression in matched primary tumors and distant metastases.
- Analyze the combination of PD-1/LAG-3 expression with CD8-based immune phenotypes ('hot' vs. 'cold' tumors).
- Determine the prevalence of PD-1/LAG-3 expression in different metastatic sites.
Main Methods:
- Studied 95 breast cancer patients with matched primary tumors and distant metastases across four anatomical locations.
- Assessed PD-1 and LAG-3 expression and CD8-based immune phenotype (inflamed/hot vs. exhausted/cold).
- Categorized immune phenotypes and correlated them with PD-1/LAG-3 expression and metastatic site.
Main Results:
- Metastases from 'cold' primary tumors remained 'cold'.
- PD-1/LAG-3 expression correlated with a 'hot' immune phenotype in both primary tumors and metastases.
- Brain and soft tissue metastases showed a higher prevalence of inflamed phenotypes with exhaustion signs compared to other sites.
- PD-1+/LAG-3+ expression was most prevalent in brain and soft tissue metastases.
Conclusions:
- The immune phenotype varies across metastatic sites.
- PD-1+/LAG-3+ expression is strongly associated with a 'hot' immune phenotype.
- Integrated analysis of immune phenotype and PD-1/LAG-3 expression in metastases can identify patients likely to benefit from immunotherapy.
- This approach may improve patient stratification for advanced breast cancer immunotherapy.
