title: The predominance of M2-polarized macrophages in the stroma of low-hypoxic bladder tumors is associated with BCG immunotherapy failure.
abstract: OBJECTIVE
  Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard treatment for superficial bladder tumors with intermediate/high risk of recurrence or progression. However, approximately 30% of patients fail to respond to the treatment. Effective BCG therapy needs precise activation of the type 1 helper cells immune pathway. Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype and may directly interfere with the BCG-induced antitumor immune response. Thus, we aim to clarify the influence of TAMs, in particular of the M2 phenotype in stroma and tumor areas, in BCG treatment outcome.
  
  
  METHODS
  The study included 99 patients with bladder cancer treated with BCG. Tumors resected before treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. CD68(+) and CD163(+) macrophages were evaluated within the stroma and tumor areas, and high density of infiltrating cells spots were selected for counting. Hypoxia, an event known to modulate macrophage phenotype, was also assessed through hypoxia induced factor (HIF)-1α expression.
  
  
  RESULTS
  Patients in whom BCG failed had high stroma-predominant CD163(+) macrophage counts (high stroma but low tumor CD163(+) macrophages counts) when compared with the ones with a successful treatment (71% vs. 47%, P = 0.017). Furthermore, patients presenting this phenotype showed decreased recurrence-free survival (log rank, P = 0.008) and a clear 2-fold increased risk of BCG treatment failure was observed in univariate analysis (hazard ratio = 2.343; 95% CI: 1.197-4.587; P = 0.013). Even when adjusted for potential confounders, such as age and therapeutic scheme, multivariate analysis revealed 2.6-fold increased risk of recurrence (hazard ratio = 2.627; 95% CI: 1.340-5.150; P = 0.005). High stroma-predominant CD163(+) macrophage counts were also associated with low expression of HIF-1α in tumor areas, whereas high counts of CD163(+) in the tumor presented high expression of HIF-1α in tumor nests.
  
  
  CONCLUSIONS
  TAMs evaluation using CD163 is a good indicator of BCG treatment failure. Moreover, elevated infiltration of CD163(+) macrophages, predominantly in stroma areas but not in the tumor, may be a useful indicator of BCG treatment outcome, possibly owing to its immunosuppressive phenotype.
authors.affiliation: Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal; ICBAS, Abel Salazar Biomedical Sciences Institute, University of Porto, Porto, Portugal; Núcleo de Investigação em Farmácia-Centro de Investigação em Saúde e Ambiente (CISA), School of Allied Health Sciences-Polytechnic Institute of Oporto, Porto, Portugal; Research Department, LPCC-Portuguese League Against Cancer (NRNorte), Portugal. Electronic address: luis14lima@gmail.com.
authors.forename: Luís
authors.initials: L
authors.lastname: Lima
authors.affiliation: Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Oliveira
authors.affiliation: Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal; Department of Pathology, Portuguese Institute of Oncology, Porto, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Tavares
authors.affiliation: Department of Pathology, Hospital Pedro Hispano, Matosinhos, Portugal.
authors.forename: Teresina
authors.initials: T
authors.lastname: Amaro
authors.affiliation: Department of Urology, Portuguese Institute of Oncology, Porto, Portugal.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cruz
authors.affiliation: INEB-Institute of Biomedical Engineering, Porto University, Portugal; Department of Pathology e Oncology, Faculty of Medicine, Porto University, Portugal; Department of Biology, Faculty of Sciences, Porto University, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Oliveira
authors.affiliation: Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal; QOPNA, Mass Spectrometry Center of the University of Aveiro, Campus de Santiago, Aveiro, Portugal.
authors.forename: José A
authors.initials: JA
authors.lastname: Ferreira
authors.affiliation: Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal; Health Faculty of University Fernando Pessoa, Porto, Portugal; Department of Surgical Oncology, Portuguese Institute of Oncology, Porto, Portugal.
authors.forename: Lúcio
authors.initials: L
authors.lastname: Santos
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001500:BCG Vaccine / Q000009:adverse effects*; D016022:Case-Control Studies; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D000860:Hypoxia / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007167:Immunotherapy / Q000009:adverse effects*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D060787:Neoplasm Grading; D009364:Neoplasm Recurrence, Local / Q000401:mortality / Q000473:pathology* / Q000628:therapy; D009367:Neoplasm Staging; D011446:Prospective Studies; D011956:Receptors, Cell Surface / Q000378:metabolism; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D015996:Survival Rate; D017211:Treatment Failure; D059016:Tumor Microenvironment / Q000187:drug effects / Q000276:immunology; D001749:Urinary Bladder Neoplasms / Q000401:mortality / Q000473:pathology* / Q000628:therapy; D000098902:CD163 Antigen; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D001500:BCG Vaccine; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011956:Receptors, Cell Surface; D000098902:CD163 Antigen; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C497442:HIF1A protein, human
keywords: BCG immunotherapy; Bladder cancer; CD163; CD68; Tumor-associated macrophages