Hiroko Bando

Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications

Since the discovery that cancer development requires the growth of new blood vessels, many investigations have revealed the key molecules in the regulation of new vessel formation. One of the most important of these molecules is vascular endothelial growth factor (VEGF)--an endothelial-cell-specific mitogen and survival factor. VEGF also causes increased vascular permeability and recruits progenitor endothelial cells from the bone marrow. Clinical observations have confirmed that VEGF status is closely associated with the extent of neovascularisation and prognosis in many solid tumours. VEGF status is predictive of resistance to various treatments, including radiotherapy, chemotherapy, and endocrine therapy. Preliminary results also indicate that anti-VEGF treatment suppresses cancer progression without serious toxic effects. Various approaches for the control of cancers involving inhibition of the activity of VEGF are currently being investigated. This review considers the clinical implications of VEGF, particularly its prognostic, predictive, and therapeutic value.

Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer

Angiogenesis, the formation of new blood vessels, is controlled by a balance between positive and negative endothelial regulatory factors. Soluble vascular endothelial growth factor receptor‐1 (sVEGFR1), a naturally occurring soluble form of VEGFR1, is a negative counterpart of the vascular endothelial growth factor (VEGF) signaling pathway, which has been characterized as one of the most important endothelial regulators in human tumor angiogenesis. In our study, we examined the expression of sVEGFR1 in 110 primary breast carcinomas, and assessed its clinical significance. Ninety‐four of 110 tumors showed ≥0.1 ng/mg protein of sVEGFR1 (range:0. 1–6.9 ng/mg protein; median: 1.03 ng/mg protein) as determined by a specific enzyme‐linked immunosorbent assay (ELISA). Immunoblot analysis confirmed the presence of sVEGFR1 in breast tumor tissues. The levels of sVEGFR1 were correlated significantly with the levels of VEGF. There was no significant correlation between the levels of sVEGFR1 and any clinico‐pathological factors including age, menopause, nodal involvement and hormone receptor status. A univariate prognosis analysis showed that the intratumoral VEGF status, as determined by ELISA, was a significant prognostic indicator, but sVEGFR1 status was not. In the combined analysis, however, the ratio of sVEGFR1 and VEGF levels provided more statistically significant prognostic value than VEGF status alone. Tumors in which the sVEGFR1 levels exceeded VEGF levels 10‐fold had a markedly favorable prognosis. Multivariate analysis also demonstrated that the ratio of sVEGFR1 and VEGF was an independent prognostic indicator after nodal status. In conclusion, sVEGFR1, an intrinsic inhibitor of VEGF, frequently co‐expressed with VEGF in primary breast cancer tissues. The intratumoral balance between sVEGFR1 and VEGF levels might be crucial for the progression of breast cancer.

Thymidine phosphorylase (platelet-derived endothelial-cell growth factor) in cancer biology and treatment.

Thymidine phosphorylase (TP) is often induced in the tumour microenvironment by physiological and chemical stress. Its induction protects cells from apoptosis and helps cell survival by stimulating nucleoside metabolism and angiogenesis. Chemotherapy often upregulates TP, which acts in cell rescue; this result indicates that TP is a crucial therapeutic target. Clinical trials for metastatic diseases have shown that TP-targeting chemotherapy with fluorouracil derivatives greatly improves the effectiveness of conventional chemotherapy for not only response but also prognosis. This new idea, the improvement of TP-inducible therapy with TP-targeting therapy, should be further investigated for early disease states, and inhibitors of TP warrant extensive investigation.

Expression of Macrophage Migration Inhibitory Factor in Human Breast Cancer: Association with Nodal Spread

Macrophage migration inhibitory factor (MIF) is known to exert pleiotropic functions including inhibition of macrophage migration, anchoring, and counteraction of the anti‐inflammatory and immunosuppressive activity of glucocorticoids. Ninety‐three primary breast cancer tissues and 64 sera of primary breast cancer patients were analyzed for the expression of MIF. The clinico‐pathological significance of MIF expression was evaluated. It was found that MIF was frequently over‐expressed in primary breast cancer tissues. RT‐PCR and western blotting analysis confirmed that wild‐type MIF is expressed, and immunohistochemical analysis showed that MIF expression was localized at tumor cells as well as stromal cells, including tumor‐associated macrophages. Intra‐tumoral MIF protein concentrations detected by enzyme‐linked immunosorbent assay (ELISA) varied with a median value of 1821 ng/mg protein (range: 8–8126 ng/mg protein), and correlated inversely with nodal involvement (P=0.039). No significant correlation was observed with other clinico‐pathological factors including tumor size, menopausal status and hormone receptors. The circulating level of MIF protein ranged up to 105.7 ng/ml (median: 17.3 ng/ml), and it was also found to correlate inversely with the number of involved nodes (P=0.02). A comparative study with other soluble inflammatory mediators showed that intratumoral levels of MIF were significantly associated with those of interleukin‐1β, suggesting that interactions between tumor cells and tumor‐associated macrophages play an important role in the up‐regulation of MIF. The multifunctional inflammatory/immune mediator MIF was frequently expressed in primary breast cancer, and its expression level was inversely associated with nodal spread. Thus, MIF seems to play a role in tumor‐stroma interactions of primary breast cancers, particularly those with a phenotype of node‐negative or minimal nodal spread.

Expression of multidrug resistance-related transporters in human breast carcinoma

The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance‐related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin‐based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi‐quantitative reverse‐transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin‐based chemotherapy. P‐glycoprotein (P‐gp)/MDRl, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.

Overexpression of soluble vascular endothelial growth factor receptor 1 in colorectal cancer: Association with progression and prognosis.

We examined the expression of sVEGFR1 in colorectal cancer tissue and corresponding normal colorectal mucosa to assess the clinical significance of sVEGFR1 in colorectal cancer. We also assessed the relationship between sVEGFR1 levels and various clinicopathologic factors and prognoses. sVEGFR1 and VEGF levels were measured in fresh‐frozen tumor extracts from 84 primary colorectal cancer tissues and 27 corresponding normal mucosa using ELISA. Mean of sVEGFR1 levels were 3.17 ng/mg protein. sVEGFR1 levels increased significantly in cancer tissue compared with normal mucosa. Although VEGF levels increased in cancer tissues, the ratio of sVEGFR1/VEGF in cancer tissue was significantly lower than that in normal tissue. No significant correlation between sVEGFR1 or VEGF levels and any clinicopathologic parameter was found. Overexpression of sVEGFR1 was significantly associated with a favorable prognosis. Based on sVEGFR1 levels in colorectal cancer without distant metastases, patients with higher sVEGFR1 levels (≥1.5 ng/mg protein) demonstrated significant longer recurrence‐free survival than patients with lower sVEGFR1 levels (<1.5 ng/mg protein) (P = 0.0017). Multivariate analysis showed that the sVEGFR1 levels in cancer tissue were an independent prognostic indicator of disease progression. sVEGFR1 expression was significantly elevated in colorectal cancer tissue compared with normal mucosa and the intratumoral balance between sVEGFR1 and VEGF was significantly different between tumor tissue and normal controls. Furthermore, sVEGFR1 levels showed a significant prognostic value. Further studies concerning the biologic and therapeutic significance of sVEGFR1 in colorectal cancer are warranted. (Cancer Sci 2007; 98: 405–410)

Immunodetection and quantification of vascular endothelial growth factor receptor‐3 in human malignant tumor tissues

Vascular endothelial growth factor recpetor‐3 (VEGFR‐3) and its ligands, vascular endothelial growth factor‐C (VEGF‐C) and –D (VEGF‐D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme‐linked immunosorbent assay (ELISA) for the quantification of VEGFR‐3 in different human cell and tissue lysates. A combination of the goat polyclonal anti‐VEGFR‐3 antibody and the mouse monoclonal anti‐human VEGFR‐3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2–25 ng/ml. The assay was specific for VEGFR‐3, with no cross‐reactivity to VEGFR‐1 or VEGFR‐2. Complex formation with VEGF‐C and VEGF‐D had no effect on the sensitivity of the assay. The VEGFR‐3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14‐fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR‐3 were in the range of 0.6–16.7 ng/mg protein. Importantly, the level of VEGFR‐3 protein detected in the ELISA correlated significantly with the number of VEGFR‐3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR‐3‐positive vessel density. The protein levels of VEGFR‐3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF‐C (p<0.001), or biological active, free VEGF‐A (p<0.0001), but not with VEGFR‐1 or total VEGF‐A. This assay provides a useful tool for the investigations of the expression levels of VEGFR‐3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis‐related disease.

Copper‐transporting P‐Type Adenosine Triphosphatase (ATP7B) Is Expressed in Human Breast Carcinoma

This is the first report to show that a copper‐transporting P‐type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT‐PCR and immunohistochemistry. ATP7B gene/protein could be detected in 22.0% (9/41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B‐positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well‐/moderately differentiated carcinoma (P=0.012). Furthermore, we found no association between the ATP7B gene/protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.

Expression of uridine and thymidine phosphorylase genes in human breast carcinoma

Uridine phosphorylase (UPase) and an angiogenic enzyme, thymidine phosphorylase (dThdPase) are involved in degradation of the pyrimidine nucleosides through phosphorolysis. The expression levels of UPase and dThdPase are higher in human solid tumors including breast carcinomas than in normal tissues. To clarify the correlation between the expression levels of UPase and dThdPase genes and the clinicopathological factors, mRNA levels of these enzymes were examined by RT‐PCR in 43 breast carcinomas. UPase gene expression was not correlated with dThdPase gene expression (regression coefficient R = 0.032). Although the expression level of the dThdPase gene was correlated with angiogenesis, detected by immunostaining endothelial cells (R = 0.66), that of UPase gene was not (R = 0.044). These results suggest that UPase does not have a strong angiogenic activity. The UPase gene expression levels in tumors of patients who relapsed were significantly higher than in those from patients who did not (p = 0.039). Although the expression levels of neither UPase or dThdPase were associated with age, pT, pN, pM, estrogen or progesterone receptor positivity, the patients with the higher levels of UPase gene expression had worse survival (p = 0.0038) than those with lower levels. In contrast, the expression of dThdPase gene was not related to relapse or survival of these patients with breast carcinoma. Our findings suggest that the expression level of UPase gene may be an independent prognostic marker in human breast carcinoma.

Evaluation of circulating tumor cells in patients with breast cancer: multi- institutional clinical trial in Japan

BackgroundWith the development of the CellSearch System, it has become possible to measure circulating tumor cell (CTC) levels with high reproducibility, and the CTC test is currently being used clinically for patients with metastatic breast cancer in the United States. It is imperative that the clinical significance of the CTC test also be examined in Japan.MethodsUsing the CellSearch System, CTC levels were evaluated in 57 healthy individuals and patients with benign breast disease; 30 patients with primary breast cancer (stages 1–3); and 38 patients with metastatic breast cancer. First, the relationship between CTC levels and the presence of metastasis was examined using a cutoff score of 2 CTCs per 7.5 ml whole blood. Then, the patients with metastatic breast cancer were divided into two groups, using a cutoff score of 5 CTCs per 7.5 ml blood, and progression-free survival (PFS) and overall survival (OS) were compared in the two groups.ResultsWhen the clinical cutoff score was set at 2 CTCs per 7.5 ml blood, 0% of the healthy individuals and patients with benign breast disease (0/57), 3.3% of the patients with primary breast cancer (1/30), and 50% of the patients with metastatic breast cancer (19/38) were identified as as having 2 CTCs per 7.5 ml blood. Additionally, with a cutoff score of 5 CTCs, 11 patients were reported to have 5 or more CTCs and both PFS (P = 0.0036) and OS (P = 0.04) were worse for this patient population than for the population with fewer than 5 CTCs.ConclusionAs concluded in a similar clinical trial in the United States, for patients with breast cancer, measuring CTC levels can be both an accurate indicator of metastases and an important measure of patient prognosis.