Sialyl Lewis X expression and lymphatic microvessel density in primary tumors of node-negative colorectal cancer patients predict disease recurrence

Abstract

Up to 30% of curatively resected colorectal cancer patients with tumor-negative lymph nodes, show disease recurrence. We assessed whether these high-risk patients can be identified by examining primary tumors for the following blood and lymphatic vasculature markers: A) sialyl Lewis X (sLeX), vascular endothelial growth factor (VEGF)-C and VEGF-D expression; B) blood and lymphatic microvessel density (BMVD/LMVD); and C) the presence of blood and lymphatic vessel invasion. Thirty-six cases (disease recurrence within 5 years) and 72 controls (no disease recurrence for at least 5 years) were selected in a case-control design. Tumor sections were stained by antibodies CSLEX1 (sLeX), anti-VEGF-C, anti-VEGF-D, anti-CD31 (BMVD) or D2-40 (LMVD) to determine the parameters as mentioned above. A multivariate analysis showed sLeX expression and high LMVD (odds ratio 5.1, 95% confidence interval 1.3-20.0 and odds ratio 3.1, 95% confidence interval 1.0-10.0, respectively) to be independent factors predicting disease recurrence. Expression of sLeX correlated with liver metastases (P = 0.015). A high LMVD was related to regional intra-abdominal or intrapelvic metastases in lymph nodes and distant metastases other than in the liver and lungs such as peritoneum, bones, brain and adrenal glands (P = 0.004). A high BMVD in the invasive front correlated with lung metastases (P = 0.018). We show that high-risk node-negative colorectal cancer patients can be identified by primary tumor assessment for sLeX expression and LMVD. Our results are consistent with the notion that both lymphatic and hematogenous metastasis play a role in colorectal cancer.

Fig. 1

CSLEX1, VEGF-C and VEGF-D expression in colorectal tumors. A tumor negatively stained (a) and positively stained (b) for CSLEX1; this tumor showed both membrane and cytoplasmic brown staining. A tumor with negative (c) and positive cytoplasmic brown staining (d) for VEGF-C; similar faint cytoplasmic brown staining as shown in negatively stained tumors for VEGF-C was seen in phosphate buffered saline controles for the VEGF-C staining; this background staining could be clearly differentiated from positive cytoplasmic brown staining for VEGF-C. A tumor with negative (e) and positive cytoplasmic brown staining (f) for VEGF-D; a brown stromal background staining was seen

Fig. 2

Visualization of lymphatic and blood vessels in colorectal tumors. Lymphatic vessels were stained brown when using immunohistochemistry by the antibodies D2–40 (a). These lymphatic vessels showed no staining by immunohistochemistry when using antibodies CD31 (b). Blood vessels were not recognized by D2–40 (c) but were brown stained by CD31 (d)

Fig. 3

Tumor cell invasion in lymphatic and blood vessels in colorectal tumors. Lymphatic vessel invasion seen on a hematoxylin and eosin stained section (a) and immunohistochemically stained section by the antibodies D2–40 (b); lymphatic vessels were brown stained by D2–40. Blood vessel invasion seen on a hematoxylin and eosin stained section (c) and immunohistochemically stained section by the antibodies CD31 (d); blood vessels were brown stained by CD31