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Observational Study
. 2020 Dec 2;22(1):134.
doi: 10.1186/s13058-020-01371-x.

Immune phenotype of patients with stage IV metastatic inflammatory breast cancer

Sandra V Fernandez  1 , Alexander W MacFarlane 4th  2 , Mowafaq Jillab  2 , Maria F Arisi  1   3 , Jennifer Yearley  4 , Lakshmanan Annamalai  4 , Yulan Gong  5 , Kathy Q Cai  6 , R Katherine Alpaugh  1   7 , Massimo Cristofanilli  1   8 , Kerry S Campbell  9
Affiliations
Observational Study

Immune phenotype of patients with stage IV metastatic inflammatory breast cancer

Sandra V Fernandez et al. Breast Cancer Res. .

Abstract

Background: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The "inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies.

Methods: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients.

Results: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4+ T cells, whereas reductions in CD8+ T cells were more concentrated in memory subsets. Immature cytokine-producing CD56bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4+ T cells and CD56dim NK cells, and higher numbers of CD8+ effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20+ B cells in the tumor.

Conclusions: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

Keywords: Checkpoint inhibitors; Immunotherapy; Inflammatory breast cancer (IBC); Lymphopenia; Metastatic IBC; NK cells; PD-1; PD-L1; Stage IV IBC; T cells; Tumor microenvironment; Tumor-infiltrating lymphocytes.

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Conflict of interest statement

This work was partially supported by funds from Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD), and JY, LA, and CM are employees of MSD. MC has advised Lilly, Cytodyn, Foundation Medicine, G1 Therapeutics, Amarex, and Sermonix and received honoraria from Pfizer and Foundation Medicine. KSC has received research funding from Janssen, Immune Oncology Biosciences, Genentech, BMS, Horizon Pharma, and NantKwest.

Figures

Fig. 1
Fig. 1
Frequencies of major lymphocyte types in peripheral blood of IBC patients compared to healthy controls. Absolute counts per μl of peripheral blood were determined for a total lymphocytes, b CD3−CD56+ NK cells, c CD19+ B cells, d CD3+ T cells, e CD3+CD4+ T cells, f CD3+CD8+ T cells, g CD4+ to CD8+ ratio of CD3+ T cells, and h CD3+CD4+CD8+ T cells. Healthy donors are shown as open circles, triple-negative IBC patients as shaded triangles, untreated IBC patients as filled squares, and chemotherapy-treated IBC patients as open squares. Horizontal lines indicate median values and statistical significance was determined by a Wilcoxon rank-sum test
Fig. 2
Fig. 2
Frequencies of T cell subsets in peripheral blood of IBC patients compared to healthy controls. Absolute counts per μl of peripheral blood were determined for a CD4+ naive (CD62L+ CD45RA+) T cells, b CD4+ effector (CD62L− CD45RA+) T cells, c CD4+ effector memory (CD62L− CD45RA−) T cells, d CD4+ central memory (CD62L+ CD45RA-) T cells, e CD8+ naive T cells, f CD8+ effector T cells, g CD8+ effector memory T cells, and h CD8+ central memory T cells. Healthy donors are shown as open circles, triple-negative IBC patients as shaded triangles, untreated IBC patients as filled squares, and chemotherapy-treated IBC patients as open squares. Horizontal lines indicate median values and statistical significance was determined by a Wilcoxon rank-sum test
Fig. 3
Fig. 3
Expression levels of CD16, granzyme B, and perforin in NK cell subsets from IBC patients compared to healthy controls. Expression levels CD56bright NK cells as measured by geometric mean fluorescence intensity (GMFI) for expression levels of a CD16 (FcγRIIIA), b granzyme B, and c perforin. Healthy donors are shown as open circles, triple-negative IBC patients as shaded triangles, untreated IBC patients as filled squares, and chemotherapy-treated IBC patients as open squares. Horizontal lines indicate median values and statistical significance was determined by a Wilcoxon rank-sum test
Fig. 4
Fig. 4
Analysis of PD-1 and PD-L1 expression in tumor biopsies from stage IV IBC patients. Sixteen tumor biopsy samples from 11 patients were stained by immunohistochemistry for PD-1 and PD-L1. Biopsies from patients #1, 5, and 8 were not available for evaluation. Details on biopsies are provided in Supplementary Table S3. a PD-1 and PD-L1 staining in brown is shown in samples from a representative patient. Magnification is × 600 in each image and bars designate 40 μm. b The expression scores for PD-1 and PD-L1 for tumor biopsy samples from the 11 patients assayed. Score definitions are provided in the “Methods” section. Values represent the maximum measured PD-1 or PD-L1 values in cases where a second measurement was made (all values shown in Supplementary Figure S3). Gray shaded patients had blood samples acquired within 1 month of tumor biopsies and were utilized for blood to tumor comparisons in Fig. 5. c Positive correlation between PD-L1 score (y-axis) and PD-1 score (x-axis) from the 11 patients evaluated using maximum values from panel b. Open squares designate overlapping datapoints from two patients. P and R values were computed from a Spearman test. The line is a least-squares fit to the data that is provided for visual purposes
Fig. 5
Fig. 5
Correlations between PD-L1 expression in tumor samples by IHC and various immune parameters in peripheral blood by flow cytometry. Flow cytometry data from the eight patients that provided blood samples within 1 month of tumor biopsies (Fig. 4b) were correlated with PD-L1 scores in their tumors. Scores for PD-L1 staining from tumor biopsies in individual patients are shown on the x-axis in each panel with y-axes showing values for significantly correlated flow cytometry parameters from the same patients: a Granzyme B GMFI in CD56dim NK cells, b granzyme B GMFI in CD4+ T cells, c perforin GMFI in CD4+ T cells, d numbers of CD8+ effector memory T cells/μl of peripheral blood, e % of total CD8+ T cells with the naïve phenotype. Datapoints represent the maximum measured PD-L1 values in cases where a second measurement was made, as in Fig. 4. P and R values were computed from a Spearman test. The lines are least-squares fits to the data that are provided for visual purposes
Fig. 6
Fig. 6
Correlations between PD-1 and PD-L1 expression in tumor samples and infiltrating T and B cells by IHC. The eight tumor samples analyzed in Fig. 5 were stained for CD3 to mark infiltrating T cells, CD20 to mark infiltrating B cells, and hematoxylin. The sample from patient 10 had limited available tissue, so this was excluded from the CD3 staining analysis. a CD3 and CD20 staining in brown is shown in samples from a representative patient. Magnification is × 100 in each image and bars designate 200 μm. Percentages of CD3+ or CD20+ TIL were assessed by a pathologist as percentage of positive cells relative to total nucleated cells in tumor and immediately peri-tumoral areas. Percentage scores for staining of TIL (x-axis) were correlated to scores for PD-1 and PD-L1 (y-axis) from the same tumor biopsies in individual patients are shown as: b % CD20+ TIL vs. PD-1, c % CD20+ TIL vs. PD-L1, and d % CD3+ TIL vs. PD-1. Numbers next to square icons designate the number of multiple superimposed data points at that position
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