Oncology/endocrinePrognostic Significance of VEGF Expression Evaluated by Quantitative Immunohistochemical Analysis in Colorectal Cancer
Introduction
Colorectal cancer remains a major cause of cancer mortality in the Western world both in men and women. It is the second most common malignancy (13.2%) and the second most common cause of cancer death in Europe [1]. Even among patients who undergo potentially curative resection alone, 40% to 50% of them ultimately relapse and die of metastatic disease [2].
Although TNM (tumor-nodes-metastasis) classification is useful for staging colorectal cancer patients and selecting them for specific treatment, it is not sufficient, as many patients at the same stage may have various outcomes, indicating that the conventional staging procedures may be unable to precisely predict cancer prognosis.
Therefore, there is a great need to identify molecular markers of more aggressive colorectal tumor phenotypes to appropriately select patients for adjuvant systemic or targeted therapies. In this regard, many studies have focused on molecular markers playing an important role in tumor angiogenesis.
Several positive regulators of tumor angiogenesis have been identified. Among these, vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor, is the most potent angiogenic factor stimulating endothelial cell proliferation, survival, and vascular maturation [3].
VEGF-A gene is located on chromosome 6p21.3 and undergoes alternative splicing yielding isoforms of 121, 165, 189, and 206 amino acids [4]. The role of VEGF expression as a prognostic factor in colorectal cancer is controversial.
In the present study, the expression of VEGF protein in patients with colorectal cancer was investigated by immunohistochemistry using tissue microarray (TMA) technology [5]. Computerized image analysis was carried out to quantify the intensity of VEGF immunostaining. The findings correlated with clinicopathological characteristics and survival status of the patients.
Section snippets
Patients
One hundred fifty patients who underwent elective potentially radical surgical resection of a histologically proven primary colorectal cancer at the General Hospital of Karditsa between January 1, 1991 and December 31, 2000 were identified from the hospital archives. None of the patients had received chemotherapy or radiotherapy before surgery.
Thirty-three patients were excluded from the analysis for the following reasons: 13 patients died within 4 wk after surgery; one patient was lost to
TMA Construction
Formalin-fixed, paraffin-embedded tissue blocks were used for tissue microarray construction. The paraffin blocks were used to prepare hematoxylin and eosin stained slides. The slides were reviewed by a senior pathologist (A.K.) to identify and mark out representative areas of viable tumor tissue. Taking tumor heterogeneity into account, triplicate tissue cores with a diameter of 1 mm were punched from selected tumor areas of each donor tissue block and brought into a recipient paraffin block
VEGF Expression
VEGF immunoreactivity was observed in the cytoplasm of tumor cells of all colorectal cancer tissues examined, with the majority (>85%) of tumor cells staining positively in all of the cases. Adjacent stromal cells (fibroblasts), wherever present, were noted to stain positively for VEGF. Despite this expression of VEGF by a similar proportion of cells in each case, the intensity of staining was noted to vary between the tumors (Fig. 1). The intensity of VEGF staining as graded by image analysis
Discussion
In addition to known conventional prognostic factors such as the depth of tumor invasion, the number of involved lymph nodes, and the involvement of resection margins, a plethora of molecular markers have been suggested as potential prognostic factors in patients with colorectal cancer, including tumor suppressor genes, cell adhesion molecules, urokinase-type plasminogen activator, cyclins, cyclin-dependent kinase inhibitors, mismatch repair genes, DNA-synthesis related genes, apoptosis
Acknowledgments
The authors thank P. Tzoumakari and G. Vilaras for their excellent technical assistance in the construction of tissue microarrays and immunohistochemistry.
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