Determination of microvessel density by quantitative real-time PCR in esophageal cancer: correlation with histologic methods, angiogenic growth factor expression, and lymph node metastasis.
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.
Loges
Sonja
S
Department of Internal Medicine II, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
Clausen
Henning
H
Reichelt
Uta
U
Bubenheim
Michael
M
Erbersdobler
Andreas
A
Schurr
Paulus
P
Yekebas
Emre
E
Schuch
Gunter
G
Izbicki
Jakob
J
Pantel
Klaus
K
Bokemeyer
Carsten
C
Fiedler
Walter
W
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
D015703:Antigens, CD; D051929:CD146 Antigen; D015820:Cadherins; D017931:DNA Primers; D000099152:Cadherin 5