title: Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients.
abstract: BACKGROUND
  Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients.
  
  
  METHODS
  We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the "Secondary Adjuvant Taxotere Treatment" (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive.
  
  
  RESULTS
  The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers.
  
  
  CONCLUSIONS
  The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data.
  
  
  BACKGROUND
  ClinicalTrials.gov: NCT00248703.
authors.affiliation: Department of Hematology and Oncology, Stavanger University Hospital, Stavanger, Norway. bjgilje@gmail.com.
authors.forename: Bjørnar
authors.initials: B
authors.lastname: Gilje
authors.forename: Oddmund
authors.initials: O
authors.lastname: Nordgård
authors.forename: Kjersti
authors.initials: K
authors.lastname: Tjensvoll
authors.forename: Elin
authors.initials: E
authors.lastname: Borgen
authors.forename: Marit
authors.initials: M
authors.lastname: Synnestvedt
authors.forename: Rune
authors.initials: R
authors.lastname: Smaaland
authors.forename: Bjørn
authors.initials: B
authors.lastname: Naume
mesh_terms: D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000032:analysis*; D001853:Bone Marrow / Q000473:pathology*; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000188:drug therapy / Q000473:pathology; D000077143:Docetaxel; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D043823:Taxoids / Q000627:therapeutic use; D016896:Treatment Outcome
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D043823:Taxoids; D000077143:Docetaxel