title: Natural killer (NK) cell profiles in blood and tumour in women with large and locally advanced breast cancer (LLABC) and their contribution to a pathological complete response (PCR) in the tumour following neoadjuvant chemotherapy (NAC): differential restoration of blood profiles by NAC and surgery.
abstract: BACKGROUND
  NK cells contribute to tumour surveillance, inhibition of growth and dissemination by cytotoxicity, secretion of cytokines and interaction with immune cells. Their precise role in human breast cancer is unclear and the effect of therapy poorly studied. The purpose of our study was to characterise NK cells in women with large (≥3 cm) and locally advanced (T3-4, N1-2, M0) breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC) and surgery, and to ascertain their possible contribution to a pathological complete response (pCR).
  
  
  METHODS
  Women with LLABCs (n = 25) and healthy female donors [HFDs (n = 10)] were studied. Pathological responses in the breast were assessed using established criteria. Blood samples were collected pre and post NAC and surgery. Flow cytometry and labelled monoclonal antibodies established absolute numbers (AbNs) and percentages (%) of NK cells, and expressing granzyme B/perforin and NKG2D. In vitro NK cytotoxicity was assessed and NK cells and cytokines (IL-2, INF-γ, TGF-β) documented in tumours using immunohistochemical techniques. Data was analysed by SPSS.
  
  
  RESULTS
  Women with LLABCs had significantly reduced AbNs (160.00 ± 40.00 cells/µl) but not % of NK cells, compared with HFDs (NK: 266.78 ± 55.00 cells/µl; p = 0.020). NAC enhanced the AbN (p = 0.001) and % (p = 0.006) of NK cells in patients with good pathological responses. Granzyme B(+)/perforin(+) cells were significantly reduced (43.41 ± 4.00%), compared with HFDs (60.26 ± 7.00%; p = 0.003). NAC increased the % in good (p = 0.006) and poor (p = 0.005) pathological responders. Pretreatment NK cytotoxicity was significantly reduced in good (37.80 ± 8.05%) and poor (22.80 ± 7.97%) responders (p = 0.001) but remained unchanged following NAC. NK-NKG2D(+) cells were unaltered and unaffected by NAC; NKG2D expression was increased in patients with a pCR (p = 0.001). Surgery following NAC was not beneficial, except in those with a pCR. Tumour-infiltrating NK cells were infrequent but increased peritumourally (p = 0.005) showing a significant correlation (p = 0.004) between CD56(+) cells and grade of response. Tumour cytokines had no effect.
  
  
  CONCLUSIONS
  Women with LLABCs have inhibited blood innate immunity, variably reversed by NAC (especially with tumour pCRs), which returned to pretreatment levels following surgery. These and in situ tumour findings suggest a role for NK cells in NAC-induced breast pCR.
authors.affiliation: Division of Surgery, Faculty of Medicine and Health Sciences, University of Nottingham, E Floor West Block, Queens Medical Centre, Derby Road, Nottingham, NG7 2UH, UK. Chandan.Verma@nottingham.ac.uk.
authors.forename: Chandan
authors.initials: C
authors.lastname: Verma
authors.affiliation: Division of Surgery, Faculty of Medicine and Health Sciences, University of Nottingham, E Floor West Block, Queens Medical Centre, Derby Road, Nottingham, NG7 2UH, UK. msxvk2@nottingham.ac.uk.
authors.forename: Viriya
authors.initials: V
authors.lastname: Kaewkangsadan
authors.affiliation: Lincoln Breast Unit, Research and Development Department, Lincoln County Hospital, Greetwell Road, Lincoln, LN2 5QY, UK. Jenny.Eremin@ulh.nhs.uk.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Eremin
authors.affiliation: Department of Pathology, PathLinks, Lincoln County Hospital, Greetwell Road, Lincoln, LN2 5QY, UK. Jed.Cowley@ulh.nhs.uk.
authors.forename: Gerard P
authors.initials: GP
authors.lastname: Cowley
authors.affiliation: Academic Department of Pathology, Faculty of Medicine and Health Sciences, University of Nottingham, A Floor West Block, Queens Medical Centre, Derby Road, Nottingham, NG7 2UH, UK. Mohammad.Ilyas@nottingham.ac.uk.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ilyas
authors.affiliation: Lincoln Breast Unit, Research and Development Department, Lincoln County Hospital, Greetwell Road, Lincoln, LN2 5QY, UK. Mohamed.El-Sheemy@ulh.nhs.uk.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: El-Sheemy
authors.affiliation: Division of Surgery, Faculty of Medicine and Health Sciences, University of Nottingham, E Floor West Block, Queens Medical Centre, Derby Road, Nottingham, NG7 2UH, UK. Oleg.Eremin@ulh.nhs.uk.|Lincoln Breast Unit, Research and Development Department, Lincoln County Hospital, Greetwell Road, Lincoln, LN2 5QY, UK. Oleg.Eremin@ulh.nhs.uk.
authors.forename: Oleg
authors.initials: O
authors.lastname: Eremin
mesh_terms: D001706:Biopsy; D001940:Breast / Q000473:pathology; D001943:Breast Neoplasms / Q000097:blood* / Q000188:drug therapy / Q000276:immunology* / Q000601:surgery; D019002:CD56 Antigen / Q000378:metabolism; D002278:Carcinoma in Situ / Q000097:blood / Q000188:drug therapy / Q000276:immunology / Q000601:surgery; D016923:Cell Death; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D007958:Leukocyte Count; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology; D008875:Middle Aged; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D020360:Neoadjuvant Therapy; D009367:Neoplasm Staging; D054353:Perforin / Q000378:metabolism; D016896:Treatment Outcome
chemical_list: D019002:CD56 Antigen; D016207:Cytokines; C527085:KLRK1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K; D054353:Perforin; D053804:Granzymes