Metastasis to the regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer and an important prognostic factor in many types of cancer. Recent evidence suggests that VEGF-C promotes lymphangiogenesis, and that tumor lymphangiogenesis in turn promotes lymphatic metastasis. We have studied the role of LVD in breast cancer, and examined whether LVD is associated with lymph node metastasis, VEGF-C expression, or prognosis. In addition, we examined whether VEGF-C mRNA transcript levels were associated with lymph node metastasis and LVD. We began by investigating the lymphatics in primary human breast carcinoma with long-term follow-up (113 cases of invasive ductal and other breast cancers) by quantitative immunohistochemical staining for podoplanin. We then analyzed the relationship between LVD and lymph node status as well as VEGF-C immunoreactivity and other established clinicopathological parameters. The relationship between LVD and prognosis was also studied. VEGF-C mRNA transcript levels were examined by quantitative real-time RT-PCR, in 55 invasive ductal breast carcinomas. This was followed by an analysis of the relationship between VEGF-C mRNA transcript levels and lymph node metastasis as well as LVD. Mean LVD of 'hot spots' was 10.2 +/- 7.4/each case. LVD was significantly correlated with lymph node metastasis (p < 0.0001), VEGF-C immunoreactivity (p = 0.0084), and podoplanin positive lymphatic invasion (p < 0.0001). Survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that high LVD was associated with both worse disease free survival (p = 0.0033) and overall survival (p = 0.0391). VEGF-C mRNA transcript levels were also correlated with lymph node metastasis (p = 0.0074) and LVD (p = 0.0409). Increased LVD was correlated with lymph node metastasis and VEGF-C expression. High LVD may be a significant unfavorable prognostic factor for long-term survival in breast cancer.

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Expression of cell-cycle modulators at the G 1 -S boundary, retinoblastoma gene product (pRb), p21, p16, p27, p53, cyclin D 1 , and cyclin E was investigated with 104 hepatocellular carcinomas (HCC), as well as 90 of their adjacent noncancerous lesions and 9 normal liver control specimens. The labeling indices (LI) of pRb, p21, p16, and p27 were higher in HCC lesions than in the adjacent noncancerous lesions and normal controls. Especially, p27 LI in noncancerous lesions was significantly higher than that in normal livers (P ‫؍‬ .011). Aberrant p53 expression and cyclin D 1 and E overexpression were observed exclusively in HCC lesions. pRb was positive in 85.6% of the HCC cases and was not related to any clinicopathological parameters. The p21 LI was generally low (average, 5.5 ؎ 9.8). Although a negative regulator, p21 LI was higher in cases with intrahepatic metastasis (P ‫؍‬ .0359). The p16 LI was significantly decreased (P ‫؍‬ .0121) in cases with advanced stage. p27 LI was significantly decreased in cases with portal invasion (P ‫؍‬ .0409), poor differentiation (P F .0001), larger size (P ‫؍‬ .0421), and intrahepatic metastasis (P ‫؍‬ .0878, borderline significance). On the other hand, aberrant p53 expression showed positive relationships with poor differentiation (P ‫؍‬ .0004) and Ki-67 LI (P ‫؍‬ .0047). Cyclin D 1 overexpression was found in 32.6% of the cases and occurred more frequently in those with high Ki-67 LI (P ‫؍‬ .0032), pRb expression (P ‫؍‬ .0202), poor differentiation (P ‫؍‬ .0612, borderline significance), and intrahepatic metastasis (P ‫؍‬ .0675, borderline significance). Cyclin E was overexpressed in 35.5% and had positive relationships with Ki-67 LI (P ‫؍‬ .0269) and stage (P ‫؍‬ .0125). In univariate analysis, cases with p27 LI F 50 (P ‫؍‬ .0004), cyclin D 1 overexpression (P ‫؍‬ .0041), and cyclin E overexpression (P ‫؍‬ .0572, borderline significance) showed poorer outcomes for disease-free survival (DFS). In multivariate analysis, p27 expression could be recognized as an independent prognostic marker for DFS. These findings suggest that in HCC: 1) p27 is active against HCC progression in early phases and, possibly, hepatocarcinogenesis as a negative regulator and can be a novel prognostic marker for DFS; and 2) cyclin D 1 predominantly works for cell-cycle progression at the G 1 -S boundary. (HEPATOLOGY 1999;30:90-99.)

Purpose: Metastasis to regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer. Recent evidence suggests that tumor production of vascular endothelial growth factor-C (VEGF-C) promotes lymphagiogenesis, which in turn promotes lymphatic metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers. Experimental Design: Nitrite/nitrate levels and VEGF-C production were assessed in MDA-MB-231 breast cancer cells after induction and/or inhibition of NO synthesis. Formation of nitrotyrosine, a biomarker for peroxynitrate formation from NO in vivo, was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels and lymph node status, VEGF-C immunoreactivity, and other established clinicopathologic variables, as well as prognosis, was analyzed. Results: Production of nitrite/nitrate and VEGF-C in MDA-MB-231 cells was increased by treatment with the NO donor DETA NONOate. The NO synthase inhibitor N G -nitro-L-arginine methyl ester eliminated this increase. High-grade nitrotyrosine staining was observed in 57.5% (65 of 113) of the invasive breast carcinomas. Nitrotyrosine levels were significantly correlated with VEGF-C immunoreactivity and lymph node metastasis. Survival curves determined by the Kaplan-Meier method showed that high nitrotyrosine levels were associated with reduced disease-free and overall survival. In multivariate analysis, high nitrotyrosine levels emerged as a significant independent predictor for overall survival. Conclusions: Our data showed a role for NO in stimulating VEGF-C expression in vitro. Formation of its biomarker nitrotyrosine was also correlated with VEGF-C expression and lymph node metastasis. Furthermore, high nitrotyrosine levels may serve as a significant prognostic factor for long-term survival in breast cancer.

These results implied that differentiated thyroid carcinomas with superficially limited invasion could be treated successfully by nonresectional management of the trachea and that those with deep invasion should be treated by resection of the invaded trachea.