title: Determination of threshold values for determining the size of the fraction of steroid hormone receptor-positive tumor cells in paraffin-embedded breast carcinomas.
abstract: BACKGROUND
  For 4 years we used a multiparameter DNA flow cytometric (MP-FCM) technique to assess steroid hormone receptor expression in the diagnostic workup of routinely processed formalin-fixed, paraffin-embedded breast carcinomas as an alternative to immunohistochemistry (IHC) for the quantification of hormone receptor-positive cells. In all cases a positive fraction of hormone receptor-expressing epithelial cells was detected. This observation raised the question of what the cutoff value might be to distinguish receptor-negative from receptor-positive tumors.
  
  
  METHODS
  In our search for a possible threshold value of positivity for estrogen receptor (ER) and progesterone receptor (PR) in MP-FCM, we developed four steps. First, we compared IHC results in our own laboratory with the results obtained by MP-FCM on a small series of breast tumors (n = 42). Second, after collecting our first 843 tumors, we made a comparison with the literature of the distribution of receptor positivity according to age classes. Third, using the most likely threshold that resulted from this comparison, we compared a subset of 340 node-negative tumors for their combined ER/PR profiles with the data from a similar group of node-negative tumor cases from the National Cancer Institute's Surveillance, Epidemiology and End-Result (SEER) study. Fourth, with the results of these comparisons, we prospectively collected IHC data and MP-FCM results of the same tumor samples for a period of 1 year. In this way, we collected data for an additional 180 tumors.
  
  
  RESULTS
  The first step in this process resulted in an previous publication where 20% of steroid hormone receptor-positive cells seemed to be an acceptable cutoff point for positivity. However, the second step provided the best correlation at approximately 35% of ER reactive cells in the cytokeratin-positive cell population. With this cutoff, the distribution of combined ER/PR profiles in our patient population of node-negative breast cancers also showed a distribution similar to the data from the SEER study. The fourth step, using the 35% threshold value, resulted in a good correlation (r = 0.85, P < 0.0001) for ER and PR between IHC and MP-FCM in the 180 tumors investigated.
  
  
  CONCLUSIONS
  By comparing in-house data with those from large external data collections in the literature, a threshold percentage can be defined that distinguishes steroid hormone receptor-negative from hormone receptor-positive tumors. As a result, information about DNA content and cell cycle distribution can be obtained. This observational study provides additional support to our opinion that MP-FCM is an alternative for IHC determination of ER and PR positivity. It is more objective and quantification can be done more appropriately. The additional value of this approach is that we generate continuous variables of ER/PR content instead of categorical classes, which can be used at different threshold levels for evaluation of clinical relevance.
authors.affiliation: Department of Clinical Chemistry and Hematology, Atrium Medical Center Heerlen, Heerlen, The Netherlands. m.leers@atriummc.nl
authors.forename: M P G
authors.initials: MP
authors.lastname: Leers
authors.forename: J G M
authors.initials: JG
authors.lastname: Hoop
authors.forename: M
authors.initials: M
authors.lastname: van Beers
authors.forename: N
authors.initials: N
authors.lastname: van Rodijnen
authors.forename: M
authors.initials: M
authors.lastname: Pannebakker
authors.forename: M
authors.initials: M
authors.lastname: Nap
mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis; D008198:Lymph Nodes / Q000473:pathology; D008875:Middle Aged; D009367:Neoplasm Staging; D016612:Paraffin Embedding; D011003:Ploidies; D011237:Predictive Value of Tests; D011960:Receptors, Estrogen / Q000032:analysis; D011980:Receptors, Progesterone / Q000032:analysis; D011987:Receptors, Steroid / Q000032:analysis*; D012016:Reference Values; D018426:SEER Program
chemical_list: D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D011987:Receptors, Steroid; D007633:Keratins