title: Detection of occult breast cancer micrometastases in axillary lymph nodes using a multimarker reverse transcriptase-polymerase chain reaction panel.
abstract: BACKGROUND
  Axillary lymph node status in breast cancer patients remains the single most important predictor of outcomes. Current methods of histopathologic analysis may be inadequate because 30% of node-negative patients recur. The purpose of this study was to test the hypothesis that a multigene reverse transcriptase-polymerase chain reaction (RT-PCR) panel provides a more sensitive method to detect axillary lymph node metastases than routine pathologic examination.
  
  
  METHODS
  Sixty-one consecutive breast cancer patients were evaluated, with nine normal control patients. Nodes > 1 cm were bisected for histopathologic and RT-PCR analysis. Nodal tissue was homogenized, and total RNA was converted into cDNA with reverse transcriptase. Reverse transcriptase-polymerase chain reaction analysis was performed with primers specific for keratin-19, c-myc, prolactin inducible protein (PIP), and beta-actin using ethidium bromide gel electrophoresis. Reverse transcriptase-polymerase chain reaction positive/ pathology negative axillary lymph nodes were reevaluated using step sectioning and immunohistochemical staining.
  
  
  RESULTS
  Thirty-seven patients had pathologically negative axillary lymph nodes, of which 15 (40%) were positive by RT-PCR analysis. Two RT-PCR negative results (one probably from tissue processing error and the other secondary to sampling error) among the 24 histologically positive specimens were detected (8%). The number of patients in each pathologic stage was 26 patients in stage I; 18, stage IIA; 7, stage IIB; 7, stage IIIA; 3, stage IIIB; and 0 patients in stage IV. By RT-PCR staging, 8 of 26 patients went from stage I to IIA (30%), and 7 of 18 from stage IIA to IIB (39%). Of the RT-PCR positive individuals who were stage I by pathologic analysis, 100% were found to be c-myc positive, 0% keratin-19 positive, and 0% PIP positive; for stage IIIB patients these markers were 50%, 100%, and 100% respectively. Additionally, an increasing number of positive markers per specimen appeared to correlate with larger primary tumor size (p < 0.01) and decreased predicted 5-year survival (r = 0.950, p < 0.002).
  
  
  CONCLUSIONS
  Multimarker RT-PCR analysis appears to be a readily available and highly sensitive method for the detection of axillary lymph node micrometastases. Longterm followup of RT-PCR positive patients will be required to determine its clinical relevance. If validated as a predictor of disease recurrence, this method would provide a powerful complement to routine histopathologic analysis of axillary lymph nodes.
authors.affiliation: Department of Surgery, Hollings Cancer Center and Medical University of South Carolina, Charleston 29425, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Lockett
authors.forename: P L
authors.initials: PL
authors.lastname: Baron
authors.forename: P H
authors.initials: PH
authors.lastname: O'Brien
authors.forename: B M
authors.initials: BM
authors.lastname: Elliott
authors.forename: J G
authors.initials: JG
authors.lastname: Robison
authors.forename: N
authors.initials: N
authors.lastname: Maitre
authors.forename: J S
authors.initials: JS
authors.lastname: Metcalf
authors.forename: D J
authors.initials: DJ
authors.lastname: Cole
mesh_terms: D001365:Axilla; D001943:Breast Neoplasms / Q000473:pathology*; D005260:Female; D006801:Humans; D008198:Lymph Nodes / Q000473:pathology; D008207:Lymphatic Metastasis / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D012194:RNA-Directed DNA Polymerase; D012720:Severity of Illness Index
chemical_list: D012194:RNA-Directed DNA Polymerase