title: Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.
abstract: Tumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF).
  
  
  
  We used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen's kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas.
  
  
  
  We found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66-69 % of the variance was attributable to differences between FOVs in the same section and 30-33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20.
  
  
  
  T and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.
authors.affiliation: Department of Pathology, Yale School of Medicine, Yale University, 310 Cedar St., PO Box 208023, New Haven, CT, 06520-8023, USA.
authors.forename: Nikita L
authors.initials: NL
authors.lastname: Mani
authors.affiliation: Department of Pathology, Yale School of Medicine, Yale University, 310 Cedar St., PO Box 208023, New Haven, CT, 06520-8023, USA.|Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Schalper
authors.affiliation: Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Christos
authors.initials: C
authors.lastname: Hatzis
authors.affiliation: Department of Pathology, Moffitt Cancer Center, Tampa, FL, USA.
authors.forename: Ozlen
authors.initials: O
authors.lastname: Saglam
authors.affiliation: Department of Pathology, Yale School of Medicine, Yale University, 310 Cedar St., PO Box 208023, New Haven, CT, 06520-8023, USA.
authors.forename: Fattaneh
authors.initials: F
authors.lastname: Tavassoli
authors.affiliation: Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Meghan
authors.initials: M
authors.lastname: Butler
authors.affiliation: Department of Surgery, Yale School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Anees B
authors.initials: AB
authors.lastname: Chagpar
authors.affiliation: Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Lajos
authors.initials: L
authors.lastname: Pusztai
authors.affiliation: Department of Pathology, Yale School of Medicine, Yale University, 310 Cedar St., PO Box 208023, New Haven, CT, 06520-8023, USA. david.rimm@yale.edu.|Section of Medical Oncology, Department of Internal Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA. david.rimm@yale.edu.
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
mesh_terms: D000328:Adult; D000368:Aged; D015415:Biomarkers; D001943:Breast Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D016130:Immunophenotyping; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D010641:Phenotype; D015203:Reproducibility of Results; D047368:Tumor Burden
chemical_list: D015415:Biomarkers; D016207:Cytokines
keywords: B cells; Breast cancer; Immunofluorescence; Stroma; T cells; Tumor microenvironment