title: Determination of microvessel density by quantitative real-time PCR in esophageal cancer: correlation with histologic methods, angiogenic growth factor expression, and lymph node metastasis.
abstract: OBJECTIVE
  Angiogenesis and lymphangiogenesis are important steps in tumor growth and dissemination and are of prognostic importance in solid tumors. The determination of microvessel density (MVD) by immunohistology is subject to considerable variability between different laboratories and observers. We compared MVD determination by immunohistology and quantitative real-time PCR and correlated the results with clinical variables.
  
  
  METHODS
  The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2, and lymphatic endothelial markers VEGFR-3, Prox, and LYVE was assessed by quantitative PCR (qPCR) in primary surgical samples. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was quantified by PCR and correlated with MVD and clinical variables.
  
  
  RESULTS
  The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2 correlated with each other in 54 samples of primary esophageal cancer (P < 0.0001 for all comparisons). MVD determined immunohistologically by CD31 staining in a subgroup of 35 patients correlated significantly with the qPCR method. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was significantly associated with MVD (P < 0.0001 for all comparisons). Analysis of the expression of lymphendothelial markers VEGFR-3, Prox, and LYVE revealed concordant results, indicating that quantification of lymphendothelial cells is possible by qPCR. The presence of lymph node metastasis on surgical specimens was significantly correlated with MVD (P < 0.003), VEGFR-2 (P < 0.048), and VEGF-C (P < 0.042) expression.
  
  
  CONCLUSIONS
  These results indicate that quantification of MVD by qPCR in surgical samples of esophageal carcinoma yields similar results with immunohistology. Interestingly, the extent of angiogenesis and lymphangiogenesis was not related in individual tumor samples. Lymph node metastases could be predicted by MVD and VEGF-C expression.
authors.affiliation: Department of Internal Medicine II, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Loges
authors.forename: Henning
authors.initials: H
authors.lastname: Clausen
authors.forename: Uta
authors.initials: U
authors.lastname: Reichelt
authors.forename: Michael
authors.initials: M
authors.lastname: Bubenheim
authors.forename: Andreas
authors.initials: A
authors.lastname: Erbersdobler
authors.forename: Paulus
authors.initials: P
authors.lastname: Schurr
authors.forename: Emre
authors.initials: E
authors.lastname: Yekebas
authors.forename: Gunter
authors.initials: G
authors.lastname: Schuch
authors.forename: Jakob
authors.initials: J
authors.lastname: Izbicki
authors.forename: Klaus
authors.initials: K
authors.lastname: Pantel
authors.forename: Carsten
authors.initials: C
authors.lastname: Bokemeyer
authors.forename: Walter
authors.initials: W
authors.lastname: Fiedler
mesh_terms: D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000096:biosynthesis; D051929:CD146 Antigen / Q000096:biosynthesis; D015820:Cadherins / Q000096:biosynthesis; D002277:Carcinoma / Q000175:diagnosis / Q000378:metabolism* / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D004938:Esophageal Neoplasms / Q000175:diagnosis / Q000378:metabolism* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008207:Lymphatic Metastasis; D008297:Male; D008833:Microcirculation; D008875:Middle Aged; D009389:Neovascularization, Pathologic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D051929:CD146 Antigen; D015820:Cadherins; D017931:DNA Primers; D000099152:Cadherin 5