Pre-existing effector T-cell levels and augmented myeloid cell composition denote response to CDK4/6 inhibitor palbociclib and pembrolizumab in hormone receptor-positive metastatic breast cancer

Colt Egelston, Weihua Guo, Susan Yost, Jin Sun Lee, David Rose, Christian Avalos, Jian Ye, Paul Frankel, Daniel Schmolze, James Waisman, Peter Lee, Yuan Yuan, Colt Egelston, Weihua Guo, Susan Yost, Jin Sun Lee, David Rose, Christian Avalos, Jian Ye, Paul Frankel, Daniel Schmolze, James Waisman, Peter Lee, Yuan Yuan

Abstract

Background: Single-agent pembrolizumab treatment of hormone receptor-positive metastatic breast cancer (MBC) has demonstrated modest clinical responses. Little is known about potential biomarkers or mechanisms of response to immune checkpoint inhibitors (ICIs) in patients with HR+ MBC. The present study presents novel immune correlates of clinical responses to combined treatment with CDK4/6i and ICI.

Methods: A combined analysis of two independent phase I clinical trials treating patients with HR+ MBC was performed. Patients treated with the combination of the CDK4/6i palbociclib+the ICI pembrolizumab+the aromatase inhibitor (AI) letrozole (palbo+pembro+AI) were compared with patients treated with pembrolizumab+AI (pembro+AI). Peripheral blood mononuclear cells collected at pretreatment, 3 weeks (cycle 2 day 1) and 9 weeks (cycle 4 day 1) were characterized by high-parameter flow cytometry to assess baseline immune subset composition and longitudinal changes in response to therapy.

Results: In the peripheral blood, higher pretreatment frequencies of effector memory CD45RA+ CD8+ T cells and effector memory CD4+ T cells were observed in responders to palbo+pembro+AI. In contrast, this was not observed in pembro+AI-treated patients. We further characterized T-cell subsets of effector-like killer cell lectin-like receptor subfamily G member 1 (KLRG1+) ICOS+ CD4+ T cells and KLRG1+ CD45RA+ CD8+ T cells as baseline biomarkers of response. In comparison, pretreatment levels of tumor-infiltrating lymphocyte, tumor mutation burden, tumor programmed death-ligand 1 expression, and overall immune composition did not associate with clinical responses. Over the course of treatment, significant shifts in myeloid cell composition and phenotype were observed in palbo+pembro+AI-treated patients, but not in those treated with pembro+AI. We identified increased fractions of type 1 conventional dendritic cells (cDC1s) within circulating dendritic cells and decreased classical monocytes (cMO) within circulating monocytes only in patients treated with palbociclib. We also demonstrated that in palbociclib-treated patients, cDC1 and cMO displayed increased CD83 and human leukocyte antigen-DR isotype (HLA-DR) expression, respectively, suggesting increased maturation and antigen presentation capacity.

Conclusions: Pre-existing circulating effector CD8+ and CD4+ T cells and dynamic modulation of circulating myeloid cell composition denote response to combined pembrolizumab and palbociclib therapy for patients with HR+ MBC.

Trial registration number: NCT02778685 and NCI02648477.

Keywords: CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; breast neoplasms; immunotherapy.

Conflict of interest statement

Competing interests: YY has contracted clinical trials and research projects sponsored by Merck, Eisai, Novartis, Puma, Genentech, and Pfizer. She is a consultant for Puma and is on the Speakers Bureau for Eisai. There are no patents or products in development or marketed products associated with this research to declare. There are no restrictions on sharing of data and/or materials.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1
Figure 1
Baseline tumor and peripheral blood characterization of HR+ MBC responders and non-responders to palbo+pembro+AI. Baseline tumor biopsies were assessed for TILs (A), PD-L1 expression (B, reported as fraction of each cohort with PD-L1+ scored tumors), and tumor mutation burden (C, reported as m/MB). Baseline peripheral blood samples were examined by flow cytometry for lymphocyte composition frequencies, including CD4+ T cells, CD8+ T cells, CD3+ CD56+ NKTs, CD19+ B cells, or CD3− CD56+ NKs (D). The composition of NKs was further assessed for CD56bright, CD16bright, and CD56dim CD16dim NK subsets (E). Monocyte subsets were assessed as a fraction of a myeloid cell gate for frequencies of CD14+ CD16− cMOs, CD14+CD16+ iMOs, and CD14− CD16+ ncMOs (F). Dendritic cell subsets were similarly assessed as a fraction of a myeloid cell gate for frequencies of CD141+ dendritic cells (cDC1), CD1c+ dendritic cells (cDC2), and CD123+ pDCs (G). NR patients are depicted in blue; responder (R) patients are depicted in red. (D) NR, n=7; R, n=9. (E–G) NR, n=7; R, n=10. AI, aromatase inhibitor; cDC, conventional dendritic cell; cDC1, type 1 conventional dendritic cell; cDC2, type 2 conventional dendritic cell; cMO, classical monocyte; HR, hormone receptor; iMO, intermediate monocyte; m/MB, mutations/megabase; MBC, metastatic breast cancer; ncMO, non-classical monocyte; NK, natural killer; NKT, natural killer T cell; NR, non-responder; palbo, palbociclib; pDC, plasmacytoid dendritic cell; PD-L1, programmed death-ligand 1; pembro, pembrolizumab; R, responder; TIL, tumor-infiltrating lymphocyte; TMB, tumor mutation burden.
Figure 2
Figure 2
Distinct pre-existing effector T-cell subsets in responders to palbo+pembro+ AI. CD8+ T cells (A) and CD4+ T cells (B) were further assessed by flow cytometry for naïve (CCR7+ and CD45RA+), CM (CCR7+ and CD45RA−), EM (CCR7− and CD45RA−), and EmrA (CCR7− and CD45RA+) cell subsets. Dimensionality reduction by the FlowSOM algorithm was performed to identify T-cell metaclusters in an unbiased manner as shown in t-distributed stochastic neighbor embedding (tSNE) projections of R and NR patients (C). A heatmap of identified clusters displays expression of various surface proteins used for each identified cell cluster. Percentages of identified non-naïve CD8+ T-cell clusters as a fraction of total CD8+ T cells are shown in NR and R patients (D). Percentages of identified non-naïve CD4+ T-cell clusters as a fraction of total CD4+ T cells are shown in NR and R patients (E). Percentages of non-classical CD4+ CD8+ DP cells and CD8dim cells as a fraction of total lymphocytes (F). *P<0.05, **P<0.01; unpaired, two-tailed t-tests. NR patients are depicted in blue (n=7) and R patients are depicted in red (n=9). AI, aromatase inhibitor; CM, central memory; DP, double-positive; EM, effector memory; EMRA, effector memory CD45RA+; NR, non-responder; palbo, palbociclib; pembro, pembrolizumab; R, responder.
Figure 3
Figure 3
Peripheral immune composition changes in Rs to palbo+pembro+ AI compared with NRs to pembro+AI. Baseline immune subset composition in responders to palbo+pembro+AI treatment (red) was compared with non-responders to pembro+AI treatment (green). Major lymphocyte subsets (A), CD8+ T-cell memory subsets (B), CD4+ T-cell memory subsets (C), NK subsets (D), monocyte subsets (E), and dendritic cell subsets (F) were assessed by flow cytometry. Changes in immune composition from baseline to C2D1 were also assessed by flow cytometry for all major non-T-cell immune subsets (G) and T-cell clusters previously identified (H) by calculating fold change from baseline. Log2 transformed fold changes are depicted and populations are sorted from least significant difference (top) to most significant difference (bottom). C2D1 palbo+pembro+AI (gold); C2D1 pembro+AI (green). *P<0.05, **P<0.01; unpaired, two-tailed t-tests. (A–C) R: palbo+pembro+AI, n=9; NR: pembro+AI, n=10. (DcF) R: palbo+pembro+AI, n=10; NR: pembro AI, n=10. (G–H) palbo+pembro+AI, n=14; pembro+AI, n=10. AI, aromatase inhibitor; C2D1, cycle 2 day 1; cDC1, type 1 conventional dendritic cell; cDC2, type 2 conventional dendritic cell; CM, central memory; cMO, classical monocyte; EM, effector memory; EMRA, effector memory CD45RA+; iMO, intermediate monocyte; ncMO, non-classical monocyte; NK, natural killer cell; NKT, natural killer T cell; NR, non-responder; palbo, palbociclib; pDC, plasmacytoid dendritic cell; pembro, pembrolizumab; R, responder.
Figure 4
Figure 4
Dynamic peripheral myeloid cell changes in patients treated with palbo+pembro+AI compared with pembro+AI Changes in cell population percentages over the course of therapy were compared between palbo+pembro+AI-treated and pembro+AI-treated patients. Changes in frequencies of cDC1 (A), cDC2 (B), pDC (C), cMO (D), iMO (E), and ncMO (F) were evaluated as fractions of myeloid cell gates. (A–F) Data are normalized by subtraction of cell population percentages identified at baseline in the same patient. cDC1 frequencies among total circulating dendritic cells (cDC1+cDC2) are shown over the course of treatment (G). cMO frequencies among total circulating monocytes (cMO+iMO+ncMO) are shown over the course of treatment (H). Baseline palbo+pembro+AI depicted by gray open circle (n=17); C2D1 palbo pembro+AI depicted in gold (n=14); C4D1 palbo+pembro+AI depicted in purple (n=13), C2D1 pembro+AI depicted in green (n=10). *P

Figure 5

Palbociclib yields increased frequencies of…

Figure 5

Palbociclib yields increased frequencies of circulating mature cDC1 and cMOs. Circulating dendritic cell…

Figure 5
Palbociclib yields increased frequencies of circulating mature cDC1 and cMOs. Circulating dendritic cell and monocyte subsets were assessed by flow cytometry for changes in maturation status over the course of therapy. Changes in CD83 expression compared with baseline pretreatment were examined in cDC1 (A) and cDC2 (B) cells. Monocyte subsets were examined for changes in frequencies of human leukocyte antigen-DR isotype (HLA-DR)high cells compared with baseline (C). Changes in HLA-DR expression as measured by change in MFI relative to baseline MFI on the same cell subsets was assessed in cMO (D), iMO (E), and ncMO (F). Representative histograms are shown, with the dotted line displaying matched baseline cell subset marker expression. Baseline palbo+pembro+AI depicted by gray open circle (n=17); C2D1 palbo+pembro+AI depicted in gold (n=14); C4D1 palbo+pembro+AI depicted in purple (n=13), C2D1pembro+AI depicted in green (n=10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Dunnett’s multiple comparisons test. AI, aromatase inhibitor; C2D1; cycle 2 day 1; C4D1, cycle 4 day 1; cDC1, type 1 conventional dendritic cell; cDC2, type 2 conventional dendritic cell; cMO, classical monocyte; iMO, intermediate monocyte; MFI, median fluorescent intensity; ncMO, non-classical monocyte; palbo, palbociclib; pDC, plasmacytoid dendritic cell; pembro, pembrolizumab.
Figure 5
Figure 5
Palbociclib yields increased frequencies of circulating mature cDC1 and cMOs. Circulating dendritic cell and monocyte subsets were assessed by flow cytometry for changes in maturation status over the course of therapy. Changes in CD83 expression compared with baseline pretreatment were examined in cDC1 (A) and cDC2 (B) cells. Monocyte subsets were examined for changes in frequencies of human leukocyte antigen-DR isotype (HLA-DR)high cells compared with baseline (C). Changes in HLA-DR expression as measured by change in MFI relative to baseline MFI on the same cell subsets was assessed in cMO (D), iMO (E), and ncMO (F). Representative histograms are shown, with the dotted line displaying matched baseline cell subset marker expression. Baseline palbo+pembro+AI depicted by gray open circle (n=17); C2D1 palbo+pembro+AI depicted in gold (n=14); C4D1 palbo+pembro+AI depicted in purple (n=13), C2D1pembro+AI depicted in green (n=10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Dunnett’s multiple comparisons test. AI, aromatase inhibitor; C2D1; cycle 2 day 1; C4D1, cycle 4 day 1; cDC1, type 1 conventional dendritic cell; cDC2, type 2 conventional dendritic cell; cMO, classical monocyte; iMO, intermediate monocyte; MFI, median fluorescent intensity; ncMO, non-classical monocyte; palbo, palbociclib; pDC, plasmacytoid dendritic cell; pembro, pembrolizumab.

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