Endoglin (CD105) expression in angiogenesis of colon cancer: analysis using tissue microarrays and comparison with other endothelial markers

Some markers of angiogenic endothelial cells are emerging as targets of cancer therapy. The present study compares the expression of CD105 with that of other endothelial markers in all tissue layers during the development of colon cancer. We immunohistochemically analyzed the expression of the colon adenoma–carcinoma sequence by endothelial cells using a panel of eight endothelial markers. We examined sections from endoscopic mucosal resection and surgical resection of tubular adenoma (n=31), carcinoma in adenoma (n=11), and adenocarcinoma (n=34). Cylindrical cores were punched out from donor paraffin blocks of normal mucosa adjacent to tumors, from tumor lesions of mucosa, submucosa, muscularis propria, subserosa, and serosa, and from lymph node metastases. CD31 (PECAM-1) was universally expressed in the blood vessels of adenoma–carcinoma lesions as well as in normal mucosal vessels (80–95%), with no significant differences. In contrast, cancer-associated blood vessels (up to 80%) and cancer cells themselves expressed high levels of CD105. In normal mucosa, CD105 was weakly expressed in endothelial cells of capillaries (≦21%), and significant differences in its expression in endothelial cells between the normal mucosa and adenoma, carcinoma in adenoma, and adenocarcinoma were found. Flt-1, Flk-1, transforming growth factor-β1, transforming growth factor-β receptor II, and CD44 were strongly expressed in the cancer cells but were not expressed in the blood vessels. Vascular endothelial growth factor was expressed at <30% in the blood vessels of adenoma, carcinoma in adenoma, and carcinoma. Moreover, this study provided evidence that CD105 was expressed exclusively in endothelial blood vessels by double immunostaining of CD105 and D2-40. The present study shows that de novo blood vessels of colon cancer specifically express CD105. These findings provide the basis for novel antiangiogenic cancer therapies.     

Endoglin (CD105) expression and angiogenesis status in small cell lung cancer

It is well established that angiogenesis is crucial for tumor development and progression. Among the angiogenesis immunomarkers defined to date, endoglin (CD105) has been shown to be a useful marker of angiogenesis. To investigate the degree of angiogenesis status in small cell lung cancer (SCLC) tissue, we assessed 35 cases of SCLC at autopsy using immunohistochemical staining of CD31 and CD105. The intratumoral area, peritumoral area, and background pulmonary alveoli were then observed under low magnification, and the microvessel density (MVD) for each area was determined. The MVD-CD31 was the highest in the background alveoli, followed by the intratumoral and peritumoral areas. The MVD-CD105 was highest in the intratumoral area, followed by the peritumoral area and the background lung. The ratio of CD105/CD31 revealed that almost 78% of the intratumoral area, 63% of the peritumoral area, and 4.6% of the background lung alveoli were newly formed and expressed CD105. This result indicated that SCLC is predominantly supported by newly formed vessels that are generated by CD105-mediated angiogenesis. These findings suggest that anti-angiogenic therapy, especially CD105-targeting, may prove an effective form of SCLC treatment.     

Significance of CD 105 expression for tumour angiogenesis and prognosis in endometrial carcinomas

Angiogenesis is a key process in tumour growth and metastasis, and Factor-VIII microvascular density has been found to influence prognosis among endometrial carcinoma patients. The CD105/endoglin antibody has been reported to preferentially bind to activated endothelial cells in tissues participating in angiogenesis, and we therefore wanted to compare the prognostic significance of CD105/endoglin to that of Factor-VIII. In a population-based endometrial carcinoma study with long (median 11.5 years) and complete patient follow-up, mean intratumour microvascular density (MVD) assessed using CD105/endoglin was investigated and compared with previous data for MVD assessed using Factor-VIII. MVD by CD105/endoglin was significantly correlated with MVD by Factor-VIII (p=0.001). However, tumours within the two groups defined by the upper and lower quartiles for CD105/endoglin-MVD were both significantly more often metastatic (FIGO-stage III/IV; p=0.03), with high tumour cell proliferation by Ki67 (p=0.007) and with reduced survival (p=0.036) as compared with the intermediate groups. In Cox regression analysis, CD105/endoglin-MVD showed independent prognostic influence when analysed together with patient age, FIGO stage, histologic subtype, histologic grade and Factor-VIII-MVD, while the latter lost its prognostic impact when CD105/endoglin was included. In the subgroup with high MVD, there was a tendency towards improved response to radiation therapy. In conclusion, CD105/endoglin-MVD is significantly associated with FIGO stage, tumour proliferation and prognosis in endometrial carcinoma, indicating that this is a better angiogenic marker in these tumours.     

Sequence analysis and high-throughput immunhistochemical profiling of KIT (CD 117) expression in uveal melanoma using tissue microarrays

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunhistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patients.     

Organ-specific endoglin (CD105) expression in the angiogenesis of human cancers

Some markers of angiogenic endothelial cells are emerging as targets for cancer therapy. The present study compared the expression of CD105 with that of other endothelial markers in cancers from various organs. Surgically resected cancer tissues from 188 patients comprising brain (n = 17), lung (n = 38), breast (n = 30), stomach (n = 30), colon (n = 31), liver (n = 32), and kidney (n = 10) cancers were immunohistochemically analyzed on tissue microarrays using a panel of eight endothelial markers. CD31 was expressed in vascular endothelial cells in cancer lesions as well as in non-cancerous areas (30-100%) in all core tissue samples. CD105 expression was intense and restricted to capillary endothelial cells in cancer lesions (>73%). In contrast, positive expression of CD105 was seen in <20% of non-cancerous areas in the same organs. However, no significant difference in CD105 expression in vascular endothelial cells between cancer lesions and non-cancerous areas from liver and renal cancer samples was found. Vascular endothelial growth factor (VEGF), Flt1, and Flk1 were also expressed, but only sporadically and in few samples (<30%), and transforming growth factor (TGF)-beta1 and TGF-betaRII were negative in vascular endothelial cells but generally positive in cancer cells. CD44 was strongly expressed in sinusoidal endothelial cells of the liver (90-100%). These results show that CD105 is expressed specifically in the tumor angiogenesis of brain, lung, breast, stomach, and colon cancers.     

子宫内膜良、恶性增生组织的CD105(Endoglin)的表达及其意义

目的研究正常、良性及恶性增生子宫内膜组织中新生血管标志物CD105(Endoglin)的表达,与传统总内皮标志物CD34的比较,并探讨组织微血管密度(microvesseldensity,MVD)的临床病理意义.方法采用S-P免疫组织化学染色法检测43例子宫内膜腺癌、15例子内膜不典型增生过长、15例复杂型增生、15例简单型增生及20例正常子宫内膜(10例分泌期内膜和10例增生期内膜)的CD105和CD34的表达情况.结果CD105和CD34标记的组织中的微血管密度(MVD)在子宫内膜从正常子宫内膜到简单型增生、复杂型增生到不典型增生的发展过程中表达逐渐增加,各组间差异有意义(P＜0.05).CD105标记的MVD在不典型增生与内膜癌间则没有明显区别,而CD34标记的MVD在这两组间有显著性差异(P＜0.05).在子宫内膜癌中只有CD105标记的MVD与子宫内膜癌的FIGO分期、病理分级、肌层浸润深度和淋巴结转移相关,CD34标记的MVD则不相关.结论新生血管标志物CD105比通常用的总内皮标记物CD34更好,是一种更有特异性的肿瘤血管内皮标志,它标记的肿瘤内微血管计数(MVD)对评估子宫内膜恶性增生程度有重要意义.     

原发性肝细胞肝癌的家族聚集性研究——附8个家系34例患者的临床分析和家系调查

目的总结原发性肝细胞肝癌（primary hepatocellular carcinomas, PHC）的家族聚集现象和家族性PHC的临床特点。方法回顾性分析了34例家族性PHC的临床表现，并进行了家系调查。结果34例PHC患者分布在8个家系中，平均4．25例（3．9例）；患者发病年龄平均41．3岁（32-62岁），肝炎病毒感染率94．1％（32／34），肝炎或肝硬化率61.8％（21／34）．AFP阳性率82．4％（28／34），就诊时TNM分期Ⅱ期2例，Ⅲ期23例，Ⅳ期9例；生存时间1-25月，平均7．3月。结论通过家族史明确高危人群、有重点地定期检查，提高治疗效果，为目前切实可行的PHC防治策略和思路。     

Endoglin (CD105) and vascular endothelial growth factor as prognostic markers in esophageal adenocarcinoma

Endoglin (CD105), a member of transforming growth factor beta1 receptor complex, has been shown to be a more useful marker to identify tumor angiogenesis than panendothelial markers such as CD31. We investigated endoglin and vascular endothelial growth factor (VEGF) expression as possible prognostic markers in esophageal adenocarcinoma. Surgical specimens from 75 patients with esophageal adenocarcinoma treated with esophagectomy were immunostained for endoglin, CD31, and VEGF. We also included 10 cases of Barrett's esophagus with high-grade dysplasia and 10 cases with Barrett's esophagus low-grade dysplasia. Positively stained microvessels (MVs) were counted in hot spots at magnification of x400. Results were expressed as the highest number of MV identified. For VEGF, intensity of staining was scored on 3-tiered scale. Endoglin demonstrated significantly more vessels than the CD31 (mean, 28.9 +/- 13.2 versus 19.0 +/- 9.4, P < .001). Both endoglin and CD31 MV counts showed significant correlation with stage of the disease (r = 0.59, P < .001; r = 0.52, P < .001, respectively) and patient survival (log rank P < .01). Only endoglin MV count was significantly correlated with the presence of angiolymphatic invasion (r = 0.34, P < .05) and lymph node (LN) metastases (r = 0.48, P < .001). Univariate analysis showed that endoglin MV count is an independent prognostic factor. Endoglin showed a significant increase in MV count in Barrett's esophagus with high-grade dysplasia when compared with Barrett's esophagus low-grade dysplasia (P < .01), whereas CD31 did not show any significant difference. VEGF was expressed in 48 (64%) of 75 cases of adenocarcinoma and was significantly correlated with angiolymphatic invasion, LN metastases, and survival. In conclusion, endoglin is a specific and sensitive marker for tumor angiogenesis. Endoglin staining also showed prognostic significance with positive correlation with the presence of angiolymphatic invasion, LN metastases, tumor stage, and survival.     

Endoglin (CD105) and vascular endothelial growth factor as prognostic markers in prostatic adenocarcinoma

We studied endoglin and vascular endothelial growth factor (VEGF) expression as prognostic markers in prostatic adenocarcinoma in 50 radical prostatectomy specimens. Cases were further categorized by Gleason score as follows: 8 to 10, 9 cases; 7(4 + 3), 9 cases; 7 (3 + 4), 14 cases; 6, 13 cases; and 4 or 5, 5 cases. All cases were immunostained for endoglin, CD31, and VEGF. Positively stained microvessels were counted in densely vascular foci in a x 400 field. VEGF staining intensity was scored on a 2-tiered scale. Results were correlated with survival and other parameters. Endoglin demonstrated significantly more microvessels than did CD31 (mean +/- SD, 37 +/- 15 vs 22 +/- 17; P < .001). VEGF expression was low in 21 cases (42%) and high in 29 (58%). Endoglin correlated positively with Gleason score, lymph node metastases, tumor stage, and preoperative prostate-specific antigen level (P < .05) but not with CD31. VEGF correlated significantly with angiolymphatic invasion and Gleason score (P < .05). A high endoglin microvessel count and VEGF expression correlated with shorter survival. Endoglin is a more specific and sensitive marker for tumor angiogenesis than CD31 and may serve as a prognostic marker for prostatic adenocarcinoma.     

Endoglin (CD105) as a prognostic factor in head and neck squamous cell carcinoma

Endoglin (CD105) is a proliferation-associated protein abundantly expressed in angiogenic endothelial cells. Recent studies revealed that CD105 is intensively expressed in tumor vasculature, whereas intratumoral microvessel density (MVD) determined with the use of antibodies to CD105 has been found to be an important prognostic indicator for the outcome in a number of malignancies. In the current study, we investigated endoglin expression and evaluated MVD in 108 patients with head and neck squamous cell carcinoma. Endoglin was intensively expressed in intratumoral blood vessels, whilst lymphatics were rarely positive for CD105. High microvessel density was associated with a more aggressive tumor phenotype, including advanced clinical stage (p=0.008) and the presence of lymph node metastasis at the time of diagnosis (p=0.02). When microvessel counts were assessed for their prognostic values (high vs low MVD), there was a statistically significant difference in the overall survival among patients with tumors of the oral cavity and larynx (p<0.001) and in the disease-free survival among patients with tumors of the lower lip (p=0.01). The prognostic impact of microvessel density was not dependent on clinical stage or lymph node status. The results of the current study suggest that CD105 is a promising target for tumor imaging and prognosis.     

Protein expression profiles in adenocarcinomas and squamous cell carcinomas of the lung generated using tissue microarrays

With the appearance of defect-targeted therapies, the definition of tumour protein expression profiles has gained increasing importance. Two lung carcinoma tissue microarrays, one including 75 primary adenocarcinomas (ACs) and the other comprising 67 primary squamous cell carcinomas (SQCCs), were generated in the present study. On both arrays, each tumour was represented by an average of five cores. In addition, one punch of normal lung parenchyma adjacent to each tumour was included in the array. Immunohistochemical expression of 86 proteins was evaluated and the results were analysed by non-parametric tests, hierarchical clustering, and principal component analysis. In both tumour entities, parenchyma and tumours were clearly separated by hierarchical clustering. By the same statistical approach, it was possible to distinguish ACs from SQCCs with 98% accuracy and to distinguish parenchyma adjacent to ACs from that adjacent to SQCCs with 96% accuracy. It was also possible to separate ACs into three groups that significantly differed in survival. Cathepsin E and hsp105 were identified as previously unknown predictors of survival in lung AC. In summary, this study has shown that protein profiles are feasible tools for anticipating biological behaviour.     

Endoglin (CD105) and vascular endothelial growth factor as prognostic markers in colorectal cancer.

Endoglin (CD105) has been shown to be a more useful marker to identify proliferating endothelium involved in tumor angiogenesis than panendothelial markers such as CD31. We investigated endoglin and vascular endothelial growth factor expression as possible prognostic markers in colorectal cancer. Surgical specimens from 150 patients with resected colorectal carcinomas were immunostained for endoglin, CD31 and vascular endothelial growth factor. Colorectal carcinoma cases consisted of 50 cases without lymph node metastases, 50 cases with only lymph node metastases and 50 cases with liver metastases (38 cases also had positive lymph nodes). Positively stained microvessels were counted in densely vascular foci (hot spots) at x 400 fields in each specimen. For vascular endothelial growth factor, intensity of staining was scored on a three-tiered scale. Results were correlated with other prognostic parameters. Endoglin demonstrated significantly more proliferating neoplastic microvessels than CD31 (31+/-10 vs 19+/-8/0.15 mm2 field, P<0.001). Low vascular endothelial growth factor expression within tumor cells was seen in 49 (33%) and high expression in 101 cases (67%). There was a positive correlation of endoglin, CD31 counts and vascular endothelial growth factor overexpression with the presence of angiolymphatic invasion and lymph node metastases (P<0.05). Only endoglin counts correlated significantly with liver metastases and positive vascular pedicle lymph nodes (P<0.05), while vascular endothelial growth factor showed significant correlation with the depth of invasion (P<0.01). Endoglin, by staining higher numbers of the proliferating vessels in colon carcinoma, is a more specific and sensitive marker for tumor angiogenesis than the commonly used panendothelial markers. Endoglin staining also showed prognostic significance with positive correlation with angiolymphatic invasion and metastases to lymph nodes and liver.     

Expression of multidrug resistance-associated protein 2 (MRP2) in normal human tissues and carcinomas using tissue microarrays

AIMS: Using tissue microarrays, this study analysed the expression of the multidrug resistance protein, MRP2, by immunohistochemistry with two different MRP2 antibodies. This is the first study to address the expression of MRP2 in various common human neoplasms. METHODS AND RESULTS: Immunohistochemistry was performed on zinc formalin-fixed tissue to evaluate normal tissues and carcinomas using two antibodies against MRP2 (EAG5, a polyclonal antibody, and M2-lll-6, a monoclonal antibody). Immunostaining was localized in neoplastic cells mainly on the cell membrane with M2-lll-6 and cell membrane and cytoplasm with EAG5. In normal tissues MRP2 was expressed in liver, gastrointestinal tract, and kidney tubular epithelial cells with both antibodies. MRP2 was seen in nine of 22 renal cell carcinomas, eight of 13 gastric carcinomas, 25 of 49 breast carcinomas, 14 of 32 lung carcinomas, 39 of 50 colon carcinomas, and 16 of 17 ovarian carcinomas. There was < 10% variability between the two antibodies. MRP2 expression was highest in moderate to poorly differentiated tumours from colon, lung, gastric, and ovarian carcinomas and in grade 2 and 3 breast and renal carcinomas. CONCLUSION: The expression of MRP2 in many solid human tumours indicates that inherent drug resistance may play an important role as a biomarker for predictive chemotherapy treatment.     

Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays

Using global gene expression analyses, multiple novel tumor markers overexpressed in infiltrating ductal adenocarcinomas of the pancreas have recently been identified. However, the expression of these markers in morphologically similar adenocarcinomas of the biliary tree has not been investigated. The purpose of the present study was 3-fold. First, we used 8 markers that have been shown to be overexpressed in whole tissue sections of pancreatic adenocarcinomas to validate tissue microarrays (TMAs) created from a series of pancreatic adenocarcinomas (n=68). The labeling patterns of 6 epithelial markers (fascin, mucin 4, 14-3-3sigma, prostate stem cell antigen, topoisomerase IIalpha, and cdc2/p34) were concordant with previously published studies on whole tissue sections, yet required far fewer slides and reagents. Mesothelin, an epithelial marker, and heat shock protein 47, a marker of peritumoral desmoplasia, showed lower levels of expression in the TMAs when compared with whole tissue sections. Second, we examined the previously unknown expression of the same 8 novel tumor proteins in cancers of the biliary tree by using TMAs created from a series of intrahepatic cholangiocarcinomas, gallbladder adenocarcinomas, and adenocarcinomas of the distal common bile duct (n=38). Each of the 8 markers was overexpressed in the biliary cancers, ranging from 14% demonstrating at least focal labeling with prostate stem cell antigen to 100% labeling with cdc2/p34. Most of the markers showed lower frequencies of expression in the biliary tract carcinomas in comparison to the pancreatic adenocarcinomas. In addition, expression patterns varied with location in the biliary system (intrahepatic versus gallbladder versus distal common bile duct). These differences were statistically significant (P<0.05) for mesothelin, mucin 4, and heat shock protein 47. Finally, the expression of selected markers in neoplastic progression of gallbladder cancer was examined. Two markers, fascin and mesothelin, showed up-regulation of expression with transition from carcinoma in situ to invasive adenocarcinoma, implicating a role for these markers in neoplastic progression. The results of this study indicate that TMA technology provides valid and cost-effective means to screen large numbers of novel tumor markers, even in tumors such as pancreatic and biliary adenocarcinomas that characteristically have abundant desmoplastic stroma. In addition, novel tumor markers of pancreatic adenocarcinomas show similar, yet not identical, expression patterns in biliary carcinomas. Therefore, these markers are potentially useful in developing diagnostic tests and treatment paradigms for tumors involving the biliary system.     

血管内皮生长因子在原发性肝癌组织中的表达及临床意义

目的:探讨血管内皮生长因子(VEGF)在原发性肝癌组织中的表达及其与肿癌的浸润转移的关系。方法:采用SP免疫组化法,检测60例原发性肝癌标本、30例癌旁组织和30例正常组织中VEGF的表达。结果:肝癌组的VEGF阳性表达率为68.3%,显著高于癌旁组(40.0%)和正常对照组(33.3%),有统计学差异(P<0.05);包膜不完整组织中VEGF阳性表达为92.9%,显著高于包膜完整组织(P<0.05);远处转移组织中VEGF阳性表达率为96.6%,显著高于无转移组织(P<0.05);而VEGF阳性表达在不同的TNM分期之间、肿瘤分化程度之间以及不同的病灶大小之间差异无统计学意义(P>0.05)。结论:VEGF在原发性肝癌组织中呈高表达,VEGF表达与肿瘤包膜有无和是否发生远处转移有关,VEGF在肝癌血管生成、生长及转移中起重要作用。     

VEGF in 105 pheochromocytomas: enhanced expression correlates with malignant outcome

Pheochromocytomas are rare sympathoadrenal tumors that are highly vascular. Their malignancy is extremely difficult to estimate on the basis of histopathological features. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors involved in both tumor growth and metastasis. In our search for new prognostic markers, we investigated the expression of VEGF in normal adrenal gland, in 105 primary pheochromocytomas, and in 6 metastases by using immunohistochemistry and Northern blot analysis. We also calculated the microvessel density of these tumors by staining the endothelial cells with monoclonal CD34 antibody. VEGF messenger ribonucleic acid was found in all pheochromocytomas studied. Immunohistochemically, VEGF was not found in normal adrenal medullary cells. Interestingly, all malignant pheochromocytomas (n=8), regardless of their primary location, had strong or moderate VEGF immunoreactivity, while most benign adrenal pheochromocytomas (26 of 37, 70.3%) were either negative or only weakly positive. The staining was heterogenous in extraadrenal pheochromocytomas as well as in a group of tumors that had histologically suspicious features but had not metastasized, here called borderline tumors (n=29). The microvessel density varied greatly in all of the tumor groups, and no statistical difference was found between these groups. Here we report moderate to strong VEGF expression in malignant pheochromocytomas, and negative or weak expression in benign adrenal pheochromocytomas. Normal medullary cells are immunohistochemically negative. Thus, low VEGF expression in pheochromocytomas favors a benign diagnosis.     

原发性肝细胞癌中5种细胞周期蛋白的表达及其临床意义

目的探讨原发性肝细胞癌中5种细胞周期蛋白(cyclin)的表达及其与肝癌细胞的增殖状态、侵袭转移和乙型肝炎病毒(HBV)感染的相关性。方法应用Instrumedics公司生产的组织芯片制作仪,将273例原发性肝癌组织、144例癌旁肝组织和10例尸检非肝病死亡肝组织制成组织芯片,取样针直径2．0am,采用免疫组织化学方法分别检测了cyclin A、cyclin B、cyclin D1、cyclin D3及cyclin E在肝癌、癌旁肝及尸检肝组织的表达率,并分析了肝癌、癌旁肝组织中HBV感染与这5种cyclin表达间的相关性。结果共获得3个肝癌组织芯片蜡块,分别含136、143和148个位点。在273例肝癌组织标本中5种cyclin的阳性表达率分别为cyclin A 52．7％、cyclin B 45．4％、cyclin D1 35．9％、cyclin D3 44．3％和cyclin E 23．1％;在144例癌旁肝组织中分别为8．3％、5．6％、4．9％、6．3％和1．4％;10例尸检肝组织除1例cyclin D1阳性外,其余均为阴性。5种cyclin在肝癌组织中的阳性表达率均显著高于癌旁肝组织(P＜0．01);组织学分级为Ⅱ、Ⅲ级的肝癌组织5种cyclin的表达高于Ⅰ级(P＜0．05);除cyclinA外,伴有门静脉癌栓组的阳性率高于无癌栓组(P＜0．01);HBV感染与cyclin的表达无明显相关性(P＞0．05)。结论各个cyclin在肝癌细胞中呈不同程度的高表达,使癌细胞周期缩短,处于细胞增殖活跃状态,并有利于肝癌细胞的侵袭转移;未发现与HBV感染有关。