title: The significance of bivariate cytokeratin and DNA flow cytometry in paraffin-embedded specimens of non-small cell lung cancer.
abstract: BACKGROUND
  The presence of non-tumor cells inside cancer tissue is one of the causes of errors in cell cycle analysis by DNA flow cytometry. The recent establishment of bivariate cytokeratin and DNA flow cytometry has made feasible the accurate assessment of tumor proliferative activity.
  
  
  METHODS
  Bivariate flow cytometry and immunohistochemistry examinations of paraffin-embedded specimens were performed in 92 patients with non-small cell lung cancer (NSCLC). Determination of the S-phase fraction by flow cytometry, with cytokeratin gating (CK-gated SPF) and without gating (ungated SPF), and the expression of proliferating cell nuclear antigen by immunohistochemistry (PCNA labeling index), were used to assess cancer cell proliferation.
  
  
  RESULTS
  Two tumors had DNA histograms with a coefficient of variation of more than 8.0% and were excluded from the flow cytometric analysis. In DNA diploid tumors (n = 25), the ungated SPFs (8.7 +/- 3.6%) showed a lower distribution than the CK-gated SPFs (14.3 +/- 4.7%) (P < 0.0001). In DNA aneuploid tumors (n = 65), there was no difference in distribution between the ungated SPFs (15.0 +/- 8.3%) and the CK-gated SPFs (15.1 +/- 7.1%) (P = 0.94). The CK-gated SPF and the PCNA labeling index of an individual tumor had a good correlation (P < 0.0001), and this agreed with the result showing that DNA diploid and aneuploid tumors had equal proliferative activity (P = 0.64 and P = 0.63, respectively).
  
  
  CONCLUSIONS
  The technique using CK-gating markedly improved the SPF measurement in DNA diploid tumors. This assessment showed no difference in proliferative activity between DNA diploid and aneuploid tumors in NSCLC. Bivariate cytokeratin and DNA flow cytometry is an accurate and objective method for cancer-specific analysis, and will surely be informative in clinical oncology.
authors.affiliation: First Department of Surgery, Kinki University School of Medicine, 377-2 Ohnohigashi, Osaka-sayama, Osaka 589-8511, Japan. otsuka@surg1.med.kindai.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Otsuka
authors.forename: S
authors.initials: S
authors.lastname: Funai
authors.forename: H
authors.initials: H
authors.lastname: Tsuda
authors.forename: T
authors.initials: T
authors.lastname: Azumi
authors.forename: S
authors.initials: S
authors.lastname: Hara
authors.forename: K
authors.initials: K
authors.lastname: Okuno
authors.forename: M
authors.initials: M
authors.lastname: Yasutomi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000473:pathology*; D002453:Cell Cycle; D002455:Cell Division; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007633:Keratins / Q000032:analysis*; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D018809:Proliferating Cell Nuclear Antigen; D007633:Keratins