title: Multiplexed quantitative analysis of CD3, CD8, and CD20 predicts response to neoadjuvant chemotherapy in breast cancer.
abstract: OBJECTIVE
  Although tumor-infiltrating lymphocytes (TIL) have been associated with response to neoadjuvant therapy, measurement typically is subjective, semiquantitative, and unable to differentiate among subpopulations. Here, we describe a quantitative objective method for analyzing lymphocyte subpopulations and assessing their predictive value.
  
  
  METHODS
  We developed a quantitative immunofluorescence assay to measure stromal expression of CD3, CD8, and CD20 on one slide. We validated this assay by comparison with flow cytometry on tonsil specimens and assessed predictive value in breast cancer on a neoadjuvant cohort (n = 95). Then, each marker was tested for prediction of pathologic complete response (pCR) compared with pathologist estimation of the percentage of lymphocyte infiltrate.
  
  
  RESULTS
  The lymphocyte percentage and CD3, CD8, and CD20 proportions were similar between flow cytometry and quantitative immunofluorescence on tonsil specimens. Pathologist TIL count predicted pCR [P = 0.043; OR, 4.77; 95% confidence interval (CI), 1.05-21.6] despite fair interobserver reproducibility (κ = 0.393). Stromal AQUA (automated quantitative analysis) scores for CD3 (P = 0.023; OR, 2.51; 95% CI, 1.13-5.57), CD8 (P = 0.029; OR, 2.00; 95% CI, 1.08-3.72), and CD20 (P = 0.005; OR, 1.80; 95% CI, 1.19-2.72) predicted pCR in univariate analysis. CD20 AQUA score predicted pCR (P = 0.019; OR, 5.37; 95% CI, 1.32-21.8) independently of age, size, nuclear grade, nodal status, ER, PR, HER2, and Ki-67, whereas CD3, CD8, and pathologist estimation did not.
  
  
  CONCLUSIONS
  We have developed and validated an objective, quantitative assay measuring TILs in breast cancer. Although this work provides analytic validity, future larger studies will be required to prove clinical utility.
authors.affiliation: Department of Pathology, Yale University Medical School, New Haven, Connecticut.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Brown
authors.affiliation: Department of Pathology, Yale University Medical School, New Haven, Connecticut.
authors.forename: Hallie
authors.initials: H
authors.lastname: Wimberly
authors.affiliation: Department of Surgery, Yale University Medical School, New Haven, Connecticut.
authors.forename: Donald R
authors.initials: DR
authors.lastname: Lannin
authors.affiliation: Department of Pathology, Yale University Medical School, New Haven, Connecticut.
authors.forename: Christian
authors.initials: C
authors.lastname: Nixon
authors.affiliation: Department of Pathology, Yale University Medical School, New Haven, Connecticut. david.rimm@yale.edu.
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
authors.affiliation: Department of Pathology, Yale University Medical School, New Haven, Connecticut.
authors.forename: Veerle
authors.initials: V
authors.lastname: Bossuyt
mesh_terms: D000328:Adult; D000368:Aged; D018951:Antigens, CD20 / Q000378:metabolism*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D017252:CD3 Complex / Q000378:metabolism*; D016827:CD8 Antigens / Q000378:metabolism*; D017024:Chemotherapy, Adjuvant; D005260:Female; D006801:Humans; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology / Q000378:metabolism*; D008875:Middle Aged; D020360:Neoadjuvant Therapy; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011379:Prognosis; D015203:Reproducibility of Results; D016896:Treatment Outcome; D047368:Tumor Burden
chemical_list: D018951:Antigens, CD20; D014408:Biomarkers, Tumor; D017252:CD3 Complex; D016827:CD8 Antigens