title: Improved Natural Killer cell activity and retained anti-tumor CD8(+) T cell responses contribute to the induction of a pathological complete response in HER2-positive breast cancer patients undergoing neoadjuvant chemotherapy.
abstract: BACKGROUND
  Locally advanced HER2-overexpressing breast cancer (BC) patients achieve a high rate of pathological complete responses (pCR) after neoadjuvant chemotherapy (NC). The apparently unaltered immune proficiency of these patients together with the immune-modulating activities of NC drugs suggest a potential contribution of host immunity in mediating clinical responses. We thus performed an extensive immunomonitoring in locally advanced BC patients undergoing NC to identify immunological correlates of pCR induction.
  
  
  METHODS
  The immune profile of 40 HER2-positive and 38 HER2-negative BC patients was characterized at diagnosis and throughout NC (Paclitaxel and Trastuzumab, or Docetaxel and Epirubicin, respectively). The percentages of circulating immune cell subsets including T and B lymphocytes, Natural Killer (NK) cells, regulatory T cells, T helper 17 lymphocytes, were quantified by multiparametric flow cytometry. NK cells functional activity was evaluated through the analysis of NF-kB nuclear translocation by Multispectral flow cytometry, and with the in vitro monitoring of Trastuzumab-mediated antibody-dependent cell cytotoxicity (ADCC). CD8(+) T cell responses against six different tumor-associated antigens (TAA) were characterized by IFN-γ ELISPOT and IFN-γ/IL-2 DualSpot assays.
  
  
  RESULTS
  After NC, HER2-positive patients showed a significant increase in the number of NK cells and regulatory T cells irrespective of the pathological response, whereas patients undergoing a pCR disclosed higher percentages of T helper 17 cells. Notably, a significant increase in the number of activated NK cells was observed only in HER2-positive patients achieving a pCR. Characterization of anti-tumor T cell responses highlighted sustained levels of CD8(+) T cells specific for survivin and mammaglobin-A throughout NC in patients undergoing a pCR in both arms. Moreover, HER2-positive patients achieving a pCR were characterized by a multi-epitopic and polyfunctional anti-tumor T cell response, markedly reduced in case of partial response.
  
  
  CONCLUSIONS
  These results indicate that maintenance of functional T cell responses against selected antigens and improvement of NK cell proficiency during NC are probably critical requirements for pCR induction, especially in HER2-positive BC patients. Trail registration:
  
  
  BACKGROUND
  NCT02307227, registered on ClinicalTrials.gov ( http://www.clinicaltrials.gov , November 26, 2014).
authors.affiliation: Cancer Bio-Immunotherapy Unit, Department of Translational Research, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. emuraro@cro.it.
authors.forename: E
authors.initials: E
authors.lastname: Muraro
authors.affiliation: Cancer Bio-Immunotherapy Unit, Department of Translational Research, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. ecomaro@cro.it.
authors.forename: E
authors.initials: E
authors.lastname: Comaro
authors.affiliation: Unit of Epidemiology and Biostatistics, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. talaminir@gmail.com.
authors.forename: R
authors.initials: R
authors.lastname: Talamini
authors.affiliation: Scientific Direction, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. eturchet@cro.it.
authors.forename: E
authors.initials: E
authors.lastname: Turchet
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. gmiolo@cro.it.
authors.forename: G
authors.initials: G
authors.lastname: Miolo
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. sscalone@cro.it.
authors.forename: S
authors.initials: S
authors.lastname: Scalone
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. lomilitello@gmail.com.
authors.forename: L
authors.initials: L
authors.lastname: Militello
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. dlombardi@cro.it.
authors.forename: D
authors.initials: D
authors.lastname: Lombardi
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. sspazzapan@cro.it.
authors.forename: S
authors.initials: S
authors.lastname: Spazzapan
authors.affiliation: Department of Pathology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. tperin@cro.it.
authors.forename: T
authors.initials: T
authors.lastname: Perin
authors.affiliation: Division of Breast Surgical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. smassarut@cro.it.
authors.forename: S
authors.initials: S
authors.lastname: Massarut
authors.affiliation: Department of Medical Oncology, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. dianacrivellari@gmail.com.
authors.forename: D
authors.initials: D
authors.lastname: Crivellari
authors.affiliation: Cancer Bio-Immunotherapy Unit, Department of Translational Research, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. rdolcetti@cro.it.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Dolcetti
authors.affiliation: Cancer Bio-Immunotherapy Unit, Department of Translational Research, CRO Aviano, IRCCS, National Cancer Institute, Via F. Gallini 2, 33081, Aviano, PN, Italy. dmartorelli@cro.it.
authors.forename: D
authors.initials: D
authors.lastname: Martorelli
mesh_terms: D056704:Adaptive Immunity / Q000187:drug effects; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D005260:Female; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D016130:Immunophenotyping; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology*; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D020360:Neoadjuvant Therapy; D017239:Paclitaxel / Q000494:pharmacology / Q000627:therapeutic use; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D012074:Remission Induction; D000068878:Trastuzumab / Q000494:pharmacology / Q000627:therapeutic use; D016896:Treatment Outcome
chemical_list: D016328:NF-kappa B; D017239:Paclitaxel; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab