Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance

BACKGROUND: Human macrophage metalloelastase (HME), also referred as matrix metalloproteinase 12, has been shown to convert plasminogen into angiostatin, an essential inhibitor of tumor angiogenesis. The current study was designed to investigate the association of HME mRNA expression with disease progression of in patients with colorectal carcinoma. METHODS: The expression of HME mRNA was quantified by Northern blot analysis in tumorous (T) and nontumorous (N) tissues obtained from 54 patients with primary colorectal carcinoma, and the ratio of these two tissue types was defined as the HME T/N ratio. The prognostic significance of this ratio after surgery, along with its correlation with disease progression and metastatic potential, was evaluated. The tissues also were subjected to in situ hybridization analysis for HME. The microvessel count after immunohistochemical staining of factor VIII was used to assess angiogenesis. RESULTS: The HME T/N ratio showed an inverse correlation with the depth of the intestinal wall invasion, the lymphatic invasion, and the vascular invasion. The patients with overexpression of HME mRNA (HME T/N ratio > 1.191) significantly demonstrated better survival compared with those patients who did not show overexpression of HME mRNA. In situ hybridization identified the colorectal carcinoma cells as mainly responsible for the signal shown in Northern blot analysis. A considerable but not statistically significant lower microvessel density (MVD) was observed in the patients with HME overexpression. CONCLUSIONS: These findings demonstrate that the HME gene plays an important role in the inhibition of tumor progression in patients with colorectal carcinoma and that its overexpression is correlated closely with a better prognosis.     

High level of Nm23-H1 gene expression is associated with local colorectal cancer progression not with metastases.

This study aimed to determine the expression of Nm23-H1 in colorectal cancer and liver metastases and to correlate Nm23-H1 expression with clinicopathological variables. Specimens from 59 primary colorectal cancers and five liver metastases were studied using Northern blot hybridisation. The mean +/- s.e. of tumour/normal (T/N) ratio of Nm23-H1 RNA expression was 4.3 +/- 0.4 (P < 0.001) and 5.1 +/- 0.90 (P < 0.01) for colorectal cancer and liver metastases respectively. No significant relationship was observed between the level of Nm23-H1 RNA and the patient''s age, sex, tumour location, differentiation, presence of lymph node involvement or distant metastases. Nm23-H1 RNA level was 2.6 +/- 0.5 for tumour size less than 3.0 cm and 4.6 +/- 0.5 for those > or = 3.0 cm (P = 0.05). There appeared to be a trend between increasing relative Nm23-H1 RNA and bowel wall invasion, irrespective of metastatic status (T1 = 1.9 +/- 0.3, T2 = 4.1 +/- 0.6, T3 = 4.1 +/- 0.5 and T4 = 6.4 +/- 1.6). This difference was statistically significant when T1 was compared against > or = T2 lesions (P = 0.01). Western blot analysis reveals two Nm23H-1 bands (17.0 kDa and 18.5 kDa). In 16 colorectal patients, the T/N fold-increase in protein expression was 2.66 +/- 0.46 (P < 0.001) and 2.40 +/- 0.32 (P < 0.001) for the 17.0 and 18.5 kDa band respectively. Both Nm23-H1 RNA and protein levels in primary colorectal cancers do not appear to correlate with synchronous regional or distant metastases. Since Nm23-H1 RNA expression is associated with increasing tumour size and tumour local invasion, Nm23-H1 RNA expression may be associated with local disease progression.     

Clinical significance of plasma vascular endothelial growth factor in gastrointestinal cancer

Circulating vascular endothelial growth factor (VEGF) was measured in gastric and colorectal cancer patients using an enzyme-linked immunosorbent assay (ELISA). Firstly, serum and plasma samples were collected from 20 normal controls to compare the values of VEGF and to determine which specimen type was most suitable for detecting circulating VEGF. Seventeen of 20 normal controls had plasma VEGF levels under the limit of detection (15 pg/ml) and the levels of the remaining three controls were 21, 22 and 38 pg/ml. In contrast, all serum samples indicated high levels of VEGF (mean 238 pg/ml), ranging from 44 to 450 pg/ml. In a time-course test of two normal controls serum VEGF values increased markedly between 30 and 60 min and remained high, whilst plasma VEGF values were low up to 480 min. Thus, plasma samples are more suitable for the measurement of circulating VEGF. Next, plasma VEGF levels were examined in 44 patients with gastric cancer (8 early, 7 advanced without remote metastasis and 29 metastatic), 13 with colorectal adenoma (2 with focal cancer) and 26 with colorectal carcinoma (8 advanced without metastasis and 18 metastatic) before treatment. An extremely high plasma concentration of VEGF was seen in some cancer patients with metastasis. To discriminate these patients, a cut-off level was determined, considering both the distribution of the sample concentration and the upper limit of 95% confidence area of VEGF in the cancer patients without metastasis. The cut-off value was 108 pg/ml and most cancer patients without metastases had VEGF levels below the cut-off value. In 11 of 29 metastatic gastric cancer patients (38%) and 9 of 18 metastatic colorectal cancer patients (50%), plasma VEGF levels were higher than the cut-off value. Survival was also analysed in the patients with metastasis. It was significantly longer in the patients with low VEGF levels (below the cut-off) than in those with high VEGF levels (logrank test, P = 0.042). 34 patients with metastasis (19 gastric cancer and 15 colorectal cancer) were treated with systemic chemotherapy, and their pretreatment levels of plasma VEGF and conventional tumour markers (CEA and CA19-9) were evaluated in relation to response. The response to chemotherapy was significantly higher in patients with low VEGF levels (< or = 108 pg/ml) than in those with high VEGF levels (P = 0.047). Such a difference was not observed with CEA/CA19-9. In conclusion, plasma VEGF is a useful marker for tumour metastasis and patient survival, and a possible predictive factor for the response of patients with gastrointestinal cancer to chemotherapy.     

c-MET expression level in primary colon cancer: a predictor of tumor invasion and lymph node metastases.

PURPOSE: Both c-MET and vascular endothelial growth factor (VEGF)-C expression are important factors in primary carcinoma progression. We hypothesized that overexpression of c-MET and/or VEGF-C mRNA in primary colorectal cancer (CRC) can predict tumor invasion and regional metastasis. EXPERIMENTAL DESIGN: The level of c-MET and VEGF-C mRNA expression was assessed using a quantitative RT-RealTime PCR assay on early stage primary CRC tumors (n = 36). RESULTS: The c-MET mRNA copy number ranged from 1.18 x 10(2) to 1.11 x 10(6) copies (median 5.17 x 10(4)) per 250 ng of RNA from CRC specimens. c-MET mRNA copies in CRC specimens was significantly higher than that from normal colon mucosal epithelium (P = 0.0001). c-MET mRNA copies significantly correlated with the depth of invasion: T(1) versus T(2), P = 0.007; T(1) versus T(3)/T(4), P = 0.0001; T(1) versus T(2) versus T(3)/T(4), P = 0.0005; and T(1)/T(2) versus T(3)/T(4), P = 0.011. c-MET copy number in primary CRC of N(1)/N(2) staged patients was significantly higher than N(0) cases (P < 0.03). Expression levels of c-MET mRNA were verified with immunohistochemistry analysis of c-MET protein expression in CRC specimens and normal mucosal epithelium. The VEGF-C mRNA copies of primary CRC assessed ranged from 0 to 1.65 x 10(5) copies (median 580). Although VEGF-C mRNA copies in CRC primary tumors were significantly higher than normal colon mucosal epithelium (P = 0.0008), it did not correlate with any major clinicopathological parameters of CRC. CONCLUSIONS: This study indicates c-MET mRNA overexpression in primary CRC may be an important prognostic marker for early stage invasion and regional disease metastasis.     

Role of the VEGF ligand to receptor ratio in the progression of mismatch repair-proficient colorectal cancer

Background  

The VEGF family of ligands and receptors are intimately involved in tumor angiogenesis, lymphangiogenesis and metastasis. The evaluation of VEGF ligand/receptor ratios may provide a more profound understanding of the involvement of these proteins in colorectal tumour progression. The aim of this study was to elucidate the role of the VEGF ligand/receptor ratios on tumour progression and metastasis in patients with mismatch repair-proficient colorectal cancer.     

Clinicopathological and prognostic significance of EGFR, VEGF, and HER2 expression in cholangiocarcinoma

Epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and human epidermal growth factor receptor 2 (HER2) have been considered as potential therapeutic targets in cholangiocarcinoma, but no studies have yet clarified the clinicopathological or prognostic significance of these molecules. Immunohistochemical expression of these molecules was assessed retrospectively in 236 cases of cholangiocarcinoma, as well as associations between the expression of these molecules and clinicopathological factors or clinical outcome. The proportions of positive cases for EGFR, VEGF, and HER2 overexpression were 27.4, 53.8, and 0.9% in intrahepatic cholangiocarcinoma (IHCC), and 19.2, 59.2, and 8.5% in extrahepatic cholangiocarcinoma (EHCC), respectively. Clinicopathologically, EGFR overexpression was associated with macroscopic type (P=0.0120), lymph node metastasis (P=0.0006), tumour stage (P=0.0424), lymphatic vessel invasion (P=0.0371), and perineural invasion (P=0.0459) in EHCC, and VEGF overexpression with intrahepatic metastasis (P=0.0224) in IHCC. Multivariate analysis showed that EGFR expression was a significant prognostic factor (hazard ratio (HR), 2.67; 95% confidence interval (CI), 1.52-4.69; P=0.0006) and also a risk factor for tumour recurrence (HR, 1.89; 95% CI, 1.05-3.39, P=0.0335) in IHCC. These results suggest that EGFR expression is associated with tumour progression and VEGF expression may be involved in haematogenic metastasis in cholangiocarcinoma.     

Dual function of ERRα in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone.     

Hepatic disseminated tumor cells in colorectal cancer UICC stage 4 patients: prognostic implications

Approximately 30-50% of all colorectal cancer patients with a resectable primary tumor will subsequently develop metastatic disease due to tumor cell dissemination. In the case of limited solid hepatic metastasis, resection of the primary tumor and the respective hepatic metastasis can be curative. Our aim was to evaluate the incidence of hepatic DTC in patients with solid liver metastasis and to describe their prognostic impact. Therefore, we applied a sensitive PCR-RFLP assay detecting one K-ras mutant among one million wild-type cells. Tumor tissue and liver biopsies from 32 colorectal cancer patients staged UICC 4 undergoing palliative surgery could be obtained intra-operatively and were thereupon screened for the presence of hepatic DTC. The primary tumor of 16 patients (50%) harbored a K-ras mutation and survival of K-ras positive patients was reduced as compared to K-ras negatives (P=0.039). In 8 of the patients (50%) with a K-ras mutated primary, no hepatic DTC were detected, whereas the liver of the remaining 8 patients (50%), showed a coincidence of solid liver metastasis and hepatic DTC. Median survival of patients with solid liver metastasis and hepatic DTC was decreased as compared with patients without any hepatic DTC burden (median survival 165 vs. 240 days; log-rank P=0.951). A subgroup analysis revealed that survival was significantly decreased in the case of bilobal DTC affection as compared with monolobal hepatic DTC affection (median survival 68 vs. 355 days; log-rank P=0.002). We conclude that the detection of hepatic DTC is a powerful prognostic factor. Furthermore, bilobal hepatic DTC involvement might be a contraindication for the resection of solid liver metastasis.     

Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in sera of cancer patients.

The concentration of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as ''elevated''. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic factors. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factors compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas.     

Molecular determinants of cetuximab efficacy.

PURPOSE: To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS: There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION: This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.     

Targeted therapy with bevacizumab (Avastin) for metastatic colorectal cancer

During the last decade, the development of new drugs known as targeted therapies was the result of a better understanding of the processes involved in the transformation of normal cells into cancer. The term targeted therapy refers to drugs that selectively target specific molecular pathways involved in tumorigenesis or tumour progression. Angiogenesis is important for tumour growth and metastasis, and is an important target for new biological agents. Bevacizumab is a humanised recombinant antibody that prevents vascular endothelial growth factor (VEGF) receptor binding, and inhibits angiogenesis and tumour growth. On February 26, 2004, the Food and Drug Administration approved bevacizumab as first-line treatment for patients with metastatic colorectal cancer (CRC). The integration of targeted therapies in the treatment of colon cancer has resulted in significant improvements in efficacy outcomes. The efficacy of bevacizumab in the treatment of metastatic CRC is presented in this review article.     

DLL4和VEGF蛋白在大肠癌中的表达及其临床意义

目的:检测大肠癌组织中DLL4和VEGF的表达,探讨其与临床病理参数的关系、临床意义及两者的相关性。方法:采用免疫组化Envision法检测60例结直肠癌及21例癌旁正常组织中DLL4、VEGF的表达情况。结果:DLL4和VEGF在大肠癌组织中表达的阳性率分别为75.0%、61.67%,明显高于癌旁正常组织(P<0.05)。DLL4及VEGF蛋白在大肠癌中的表达与肿瘤浸润深度、淋巴结转移、远处转移及TNM分期密切相关(P<0.05);二者在大肠癌组织中的表达与患者性别、年龄、肿瘤部位、肿瘤大小及组织学分化程度无关(P>0.05)。DLL4阳性表达病例中的VEGF表达率明显高于DLL4阴性表达的病例,DLL4和VEGF蛋白表达水平呈明显正相关(r=0.257,P<0.05)。结论:DLL4和VEGF在大肠癌和正常大肠组织中的表达差异有统计学意义,DLL4在大肠癌组织中的表达上调与VEGF有关,它们可能共同调控肿瘤新生血管发生,参与大肠癌的侵袭和转移,从而影响患者的预后。     

Urokinase receptor and vascular endothelial growth factor are synergistically associated with the liver metastasis of colorectal cancer.

Considering recent findings that the urokinase plasminogen activation (PA) system is involved in invasion and vascular endothelial growth factor (VEGF) is involved in angiogenesis of colorectal cancer, we evaluated these factors in the liver metastasis of primary colorectal cancer. Cancer tissues from 71 colorectal cancer patients were assayed quantitatively for antigen levels of urokinase type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2), and were also assayed immunohistochemically for expression of VEGF protein. Among the PA system factors, both the levels of uPAR and PAI-1 were significantly higher in larger tumors than in smaller ones, and were also significantly higher in tumors that invaded subserosa, serosa or adjacent organs than in mucosal, submucosal tumors or in tumors that invaded the muscle layer. The uPAR levels were significantly higher in tumors with liver metastasis than in those without. VEGF overexpression was significantly more frequent in tumors with lymph node involvement or liver metastasis than in those without. Among the PA system factors, the uPAR levels were significantly higher in tumors with VEGF overexpression and a multivariate analysis revealed that high uPA level and VEGF overexpression were independent risk factors for liver metastasis. The combination of high uPAR level and overexpression of VEGF was associated with the worst prognosis in patients with colorectal cancer. These results suggest that uPAR and VEGF might contribute synergistically to the liver metastasis of colorectal cancer.     

Urokinase Receptor and Vascular Endothelial Growth Factor Are Synergistically Associated with the Liver Metastasis of Colorectal Cancer

Considering recent findings that the urokinase plasniinogen activation (PA) system is involved in invasion and vascular endothelial growth factor (VEGF) is involved in angiogenesis of colorectal cancer, we evaluated these factors in the liver metastasis of primary colorectal cancer. Cancer tissues from 71 colorectal cancer patients were assayed quantitatively for antigen levels of urokinase type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2), and were also assayed immunohistochemically for expression of VEGF protein. Among the PA system factors, both the levels of uPAR and PAI-1 were significantly higher in larger tumors than in smaller ones, and were also significantly higher in tumors that invaded subserosa, serosa or adjacent organs than in mucosal, submucosal tumors or in tumors that invaded the muscle layer. The uPAR levels were significantly higher in tumors with liver metastasis than in those without. VEGF overexpression was significantly more frequent in tumors with lymph node involvement or liver metastasis than in those without. Among the PA system factors, the uPAR levels were significantly higher in tumors with VEGF overexpression and a multivariate analysis revealed that high uPA level and VEGF overexpression were independent risk factors for liver metastasis. The combination of high uPAR level and overexpression of VEGF was associated with the worst prognosis in patients with colorectal cancer. These results suggest that uPAR and VEGF might contribute Synergistically to the liver metastasis of colorectal cancer.     

大肠肿瘤血管内皮生长因子的表达及其作用机制探讨

目的 :通过检测血管内皮生因子 (VEGF)的表达来明确它与大肠肿瘤临床病理因素的关系。方法 :应用免疫组织化学方法 (SABC法 ) ,采用VEGF多克隆抗体测定 55例病理证实的大肠癌标本及 17例大肠腺瘤标本中VEGF的表达。结果 :大肠癌VEGF的阳性例数为 36,表达率 ( 65% )高于大肠腺瘤 ,差别显著 (P <0 .0 5) ;发生淋巴结转移大肠癌VEGF的表达率高于未发生转移的大肠癌 ,差别显著 (P<0 .0 5) ,未发生淋巴结转移大肠癌VEGF的表达率高于大肠腺瘤 ,差别显著 (P <0 .0 5) ,结论 :VEGF的过度表达揭示大肠肿瘤恶变及其转移能力的增加