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High Numbers of Circulating CD57+ NK Cells Associate with Resistance to HER2-Specific Therapeutic Antibodies in HER2+ Primary Breast Cancer

Aura Muntasell, Sònia Servitja, Mariona Cabo, Begoña Bermejo, Sandra Pérez-Buira, Federico Rojo, Marcel Costa-García, Oriol Arpí, Manuela Moraru, Laia Serrano, Ignasi Tusquets, María Teresa Martínez, Gemma Heredia, Andrea Vera, María Martínez-García, Laura Soria, Laura Comerma, Sara Santana-Hernández, Pilar Eroles, Ana Rovira, Carlos Vilches, Ana Lluch, Joan Albanell and Miguel López-Botet
Aura Muntasell
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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  • ORCID record for Aura Muntasell
  • For correspondence: amuntasell@imim.es jalbanell@hospitaldelmar.cat
Sònia Servitja
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
3Department of Medical Oncology, Hospital del Mar-CIBERONC, Barcelona, Spain.
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Mariona Cabo
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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Begoña Bermejo
4Department of Oncology, Hospital Clinico de Valencia-CIBERONC, Valencia, Spain.
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Sandra Pérez-Buira
5Department of Pathology, IIS “Fundacion Jimenez Diaz University Hospital,” Madrid, Spain.
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Federico Rojo
5Department of Pathology, IIS “Fundacion Jimenez Diaz University Hospital,” Madrid, Spain.
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Marcel Costa-García
6Universitat Pompeu Fabra, Barcelona, Spain.
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Oriol Arpí
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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Manuela Moraru
7HLA-Immunogenetics Department, Instituto Hospital Universitario Puerta de Hierro, Majadahonda, Spain.
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Laia Serrano
8Department of Pathology, Hospital del Mar, Barcelona, Spain.
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Ignasi Tusquets
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
3Department of Medical Oncology, Hospital del Mar-CIBERONC, Barcelona, Spain.
6Universitat Pompeu Fabra, Barcelona, Spain.
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María Teresa Martínez
4Department of Oncology, Hospital Clinico de Valencia-CIBERONC, Valencia, Spain.
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  • ORCID record for María Teresa Martínez
Gemma Heredia
6Universitat Pompeu Fabra, Barcelona, Spain.
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Andrea Vera
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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María Martínez-García
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
3Department of Medical Oncology, Hospital del Mar-CIBERONC, Barcelona, Spain.
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Laura Soria
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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Laura Comerma
8Department of Pathology, Hospital del Mar, Barcelona, Spain.
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Sara Santana-Hernández
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
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Pilar Eroles
4Department of Oncology, Hospital Clinico de Valencia-CIBERONC, Valencia, Spain.
9Biomedical Research Institute, INCLIVA, Valencia, Spain.
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Ana Rovira
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
3Department of Medical Oncology, Hospital del Mar-CIBERONC, Barcelona, Spain.
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Carlos Vilches
7HLA-Immunogenetics Department, Instituto Hospital Universitario Puerta de Hierro, Majadahonda, Spain.
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Ana Lluch
4Department of Oncology, Hospital Clinico de Valencia-CIBERONC, Valencia, Spain.
9Biomedical Research Institute, INCLIVA, Valencia, Spain.
10Universitat de Valencia, Valencia, Spain.
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Joan Albanell
2Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
3Department of Medical Oncology, Hospital del Mar-CIBERONC, Barcelona, Spain.
6Universitat Pompeu Fabra, Barcelona, Spain.
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  • For correspondence: amuntasell@imim.es jalbanell@hospitaldelmar.cat
Miguel López-Botet
1Immunity and Infection, IMIM (Institut Hospital del Mar d’Investigacions Mèdiques), Barcelona, Spain.
6Universitat Pompeu Fabra, Barcelona, Spain.
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DOI: 10.1158/2326-6066.CIR-18-0896 Published August 2019
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  • Figure 1.
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    Figure 1.

    Baseline CD57+ NK cells associate with pCR in patients with HER2+ breast cancer treated with neoadjuvant HER2-specific antibody–based treatment. The NK-cell phenotype was analyzed by multiparametric flow cytometry in baseline PBMC samples from patients with HER2+ breast cancer. A, Frequencies and absolute numbers of total, CD57+, NKG2A+, and KIR2D+ (including KIR2DL1+ and KIR2DL2/L3/S2+ NK cells as identified by the combination of DM-1 and CHL mAbs) and LILRB1+ NK cells in patients stratified by pCR to treatment including HER2-specific antibodies. B, Proportions of CD57+ NK cells in patients categorized by their response to treatment according to Miller–Payne criteria. C, pCR rates in patients stratified according to CD57+ NK-cell cutoffs. Patients with < or ≥ 65% and 164 cells/μL are labeled as CD57low and CD57high, respectively. ORs and 95% CIs for pCR are indicated. Kaplan–Meier curves for DFS in patients stratified for pCR to neoadjuvant treatment (D) as well as by baseline proportions of CD57+ NK cells (≥ and < 65%; E), or CD57+ NK-cell numbers (≥ and < 164 NK cells/μL; F).

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    Figure 2.

    pCR achievement, trastuzumab-induced NK-cell activation, and CD57+ NK cells in breast cancer patients with distinct CD16A 158V/F genotype. The CD16A 158V/F genotype was analyzed in DNA samples from patients with primary HER2+ breast cancer (n = 60) by PCR. A, pCR rates in patients categorized according to their CD16A 158F/F or CD16A 158V/V and V/F genotypes. B, NK-cell degranulation analyzed in baseline PBMC samples from patients with HER2+ breast cancer upon coculture with trastuzumab-coated SKBR3 cells by flow cytometry. Percentage of CD107a+ CD56dim NK cells in response to trastuzumab-coated SKBR3 cells in patients with CD16 158 F/F (n = 20) as compared with patients harboring CD16 158 V/F or V/V genotype (n = 21). P values calculated by Mann–Whitney U test. C and D, Relative and absolute numbers of circulating CD57+ NK cells in baseline samples from patients stratified by their CD16 158V/F genotype (*P < 0.05; Mann–Whitney test).

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    Figure 3.

    Reduced presence of CD57+ lymphocytes in tumor-associated as compared with circulating paired samples from patients with breast cancer. Tumor-associated immune cells, isolated by mechanical and enzymatic digestion, and paired PBMC samples, isolated by Ficoll gradient, were analyzed by multiparametric flow cytometry. CD45+ DAPI− cells with lymphocyte morphology were gated for further analysis. A, t-SNE plot showing lymphocyte clusters defined by CD3, CD8, CD4, CD56, CD16, and CD57 expression in circulating and tumor-associated lymphocyte samples. Color scale represents high expression (red) or low expression (blue) of the indicated marker. Numbers indicate the CD57+CD16+ NK-cell cluster (1), the CD57+ CD8+ T-cell cluster (2), and the CD57+CD4+ T-cell cluster (3). B, Density maps in the t-SNE field displaying peripheral blood-derived (top) or tumor-associated (bottom) samples compiled separately. C–F, Proportions of NK cells (CD56+ CD3−), CD16+ NK cells, CD16+ CD57+, and CD16+CD57− NK cells in paired circulating and tumor-associated samples from patients with breast cancer (n = 7). Statistical significance by Mann–Whitney U test.

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    Figure 4.

    Circulating CD57+ NK cells show reduced CXCR3 expression and CD16-induced proliferation. Surface expression of CXCR1, CXCR3, CX3CR1, CCR7, and CD62L was analyzed by multiparametric flow cytometry in CD57+ and CD57− CD56dim NK cells from patients with HER2+ breast cancer. A and B, Dot plots showing CXCR1, CX3CR1, CXCR3, CCR7, CD62L, and CD57 coexpression in circulating NK cells. Data from four representative patients. C, Proportions of CD57+ and CD57− NK cells positive for each molecule. Each dot represents a single determination in individual patients (n = 6). Statistical comparison by Wilcoxon paired test. D–F, CFSE dilution assays by flow cytometry using purified NK cells from healthy volunteers cultured for 6 days with plate bound anti-CD16 agonist at 0.5 or 5 μg/mL in the presence of IL2. D, Dot plots and histograms showing CFSE labeling in CD57+ and CD57− NK cells in the indicated conditions. Data from a representative experiment out of 6 performed are shown. E, Percentage of CD57+ and CD57− NK cells ≥ 4 divisions at day 6 (mean ± SEM; n = 6). F, Numbers of total and CD57+ NK cells recovered at day 6 (mean ± SEM; n = 6).

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    Figure 5.

    Comparable responses of CD57+ and CD57− circulating NK cells from HER2+ breast cancer patients in trastuzumab-induced ADCC in vitro assays. A–C, NK-cell activation as analyzed in baseline PBMC samples from patients with HER2+ breast cancer by flow cytometry upon coculture with trastuzumab-coated SKBR3 cells. A, Frequencies of CD57+ NK cells in PBMC samples from patients categorized as CD57high (≥65% CD57+ NK cells, n = 18) or CD57low (<65% of CD57+ NK cells, n = 16). B and C, Percentage of CD107a+ or TNFα+ CD56dim NK cells in patients with high or low proportions of CD57+ NK cells. P values calculated by Mann–Whitney U test. D–G, PBMCs from patients with HER2+ breast cancer and healthy controls paired by their CD16 genotype and NKG2C profile were cocultured with trastuzumab-coated SKBR3 for 4 hours and degranulation was analyzed by CD107a mobilization assay by flow cytometry (D). Dot plots showing the proportions of CD107a+ NK cells according to CD57 expression in NK cells from patients and controls paired by their CD16 genotype. Data from two representative examples are shown. E–G, Percentage of total as well as CD57+ and CD57− NK cells degranulating against trastuzumab-coated SKBR3 cells in PBMC samples from healthy controls and patients with HER2+ breast cancer. Control and patient samples with the same CD16 genotype were assayed in parallel.

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    Figure 6.

    Circulating CD57+ and tumor-infiltrating NK-cell numbers, respectively, predict resistance and complete response to HER2 therapeutic antibodies. Association between circulating CD57+ NK cells, tumor-infiltrating NK-cell numbers and pCR analyzed in a fraction of patients for whom both parameters were available (n = 40). A, IHC staining of NK cells identified as CD56+ (red) CD3− (green) lymphocytes on diagnostic tumor biopsies. B, Correlation between tumor-infiltrating NK-cell and circulating CD57+ NK-cell numbers. C and D, pCR rate in patients stratified according to ≥3 TI-NK cell/50× HPF or by ≥164 circulating CD57+ NK cell/μL cutoffs; sensitivity and specificity of both biomarkers are indicated.

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  • Table 1.

    Patient characteristics

    Cohort
    No. of patients66
    Mean age58
    Tumor size (n/%)
     T1–T251 (77%)
     T3–T415 (23%)
    Lymph node status (n/%)
     N025 (38%)
     N+41 (61%)
    Histologic type (n/%)
     DIC58 (87%)
     Others8 (10%)
    Histologic grading (n/%)
     G1–G233 (50%)
     G330 (45%)
     NA3 (4%)
    Hormonal status (n/%)
     ER+47 (71%)
     ER−19 (29%)
     PR+30 (45%)
     PR−36 (55%)
    Ki67 Index (n/%)
     <20%8 (12%)
     ≥20%57 (86%)
     NA1 (1%)
    Anti-HER2 (n/%)
     Trastuzumab45 (69%)
     Dual anti-HER220 (31%)
    Chemo regimen (n/%)
     Anthracylines and taxanes43 (73%)
     Taxanes12 (20%)
     Hormonal4 (7%)
    pCR
     pCR27 (41%)
     No pCR37 (56%)
     NA2 (3%)
    Clinical follow-up
     Median (IQR)26 (20–33)
    • NOTE: Dual anti-HER2 includes: trastuzumab + pertuzumab (n = 17), trastuzumab + lapatinib (n = 2), and trastuzumab + neratinib (n = 1).

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    • Supplementary Figures and Tables - Supplementary Figures 1-6 and Tables 1-3
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Cancer Immunology Research: 7 (8)
August 2019
Volume 7, Issue 8
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High Numbers of Circulating CD57+ NK Cells Associate with Resistance to HER2-Specific Therapeutic Antibodies in HER2+ Primary Breast Cancer
Aura Muntasell, Sònia Servitja, Mariona Cabo, Begoña Bermejo, Sandra Pérez-Buira, Federico Rojo, Marcel Costa-García, Oriol Arpí, Manuela Moraru, Laia Serrano, Ignasi Tusquets, María Teresa Martínez, Gemma Heredia, Andrea Vera, María Martínez-García, Laura Soria, Laura Comerma, Sara Santana-Hernández, Pilar Eroles, Ana Rovira, Carlos Vilches, Ana Lluch, Joan Albanell and Miguel López-Botet
Cancer Immunol Res August 1 2019 (7) (8) 1280-1292; DOI: 10.1158/2326-6066.CIR-18-0896

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High Numbers of Circulating CD57+ NK Cells Associate with Resistance to HER2-Specific Therapeutic Antibodies in HER2+ Primary Breast Cancer
Aura Muntasell, Sònia Servitja, Mariona Cabo, Begoña Bermejo, Sandra Pérez-Buira, Federico Rojo, Marcel Costa-García, Oriol Arpí, Manuela Moraru, Laia Serrano, Ignasi Tusquets, María Teresa Martínez, Gemma Heredia, Andrea Vera, María Martínez-García, Laura Soria, Laura Comerma, Sara Santana-Hernández, Pilar Eroles, Ana Rovira, Carlos Vilches, Ana Lluch, Joan Albanell and Miguel López-Botet
Cancer Immunol Res August 1 2019 (7) (8) 1280-1292; DOI: 10.1158/2326-6066.CIR-18-0896
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