Preoperative assessment of gastric cancer vascularity by flash echo imaging

BACKGROUND: Tumor vascularity as indicated by immunohistochemical staining is a significant prognostic factor in gastric and other cancers. Non-invasive preoperative assessment of the vascularity of gastric cancers has not been possible. We aim to determine the reliability of harmonic flash echo imaging (FEI) for assessment of vascularity of gastric cancers by comparison with CD34 staining of resected specimens. METHODS: Twelve patients undergoing surgical resection of advanced gastric cancer were studied. An ultrasound system transmitting ultrasound pulses at 2.3 MHz and receiving them at 4.6 MHz (second harmonic image) was used for harmonic FEI. Approximately 30 s after intravenous injection of ultrasonic contrast medium (SHU 508A, Levovist), second harmonics (4.6 MHz) emitted from microbubbles were obtained to enhance the B-mode images. Using the tumor image showing strongest enhancement in each FEI series, regions of interest were determined to measure mean echo intensity in the tumor. Immunohistochemistry using antibodies against CD34 was carried out in resected specimens. Tumor vascularity was determined by counting stained microvessels. RESULTS: A significant positive correlation was noted between sonographic amplitude determined preoperatively by FEI analysis and number of CD34-stained microvessels in tumor specimens (r = 0.869, P = 0.004). CONCLUSION: Vascularity of gastric cancers now can be evaluated non-invasively by harmonic FEI.     

Evaluation of colon cancer vascularity by flash echo imaging

OBJECTIVE: Tumor vascularity is strongly related to the prognoses of patients with various malignant primary tumors, including colorectal cancer. The aim of this study was to confirm the usefulness of harmonic flash echo imaging (H-FEI) for the preoperative evaluation of tumor vascularity in colorectal cancer by comparison with CD34 staining of resected specimens. MATERIAL AND METHODS: Seventeen patients undergoing surgical resection for advanced colorectal cancer (9 M, 8 F) were enrolled in this study. An ultrasound system transmitting ultrasound pulses at 2.3 MHz and receiving them at 4.6 MHz (second harmonic) was used for H-FEI. After intravenous injection of ultrasonic contrast medium (SHU 508A), intermittent harmonic scanning was carried out for 120 s at intervals of 4 s. On the tumor image of the strongest enhancement in each patient, 5 regions of interest were taken to decide the mean echo intensity of the tumor. Immunohistochemistry using antibodies against CD34 was carried out on resected specimens. The mean microvessel density was decided by counting microvessels on 5 fields (x 400) of CD34-stained specimens. RESULTS: The mean value of H-FEI intensity was 5.44+/-1.10, and the mean microvessel count with CD34 staining was 35.0+/-7.95. A significant positive linear correlation was observed between the intensity of H-FEI determined preoperatively and the microvessel count in CD34-stained specimens (r=0.741, p=0.0006). CONCLUSION: Vascularity of colon cancer can be evaluated non-invasively using H-FEI.     

Gastric Wall Structure Using a 30 MHz Endoscopic Ultrasound Probe,Focusing upon Delineation of the Muscularis Mucosae

Background: The normal gastric wall has been reported to appear to be a five‐layered structure. The structure of the gastric wall using a 30 MHz endoscopic ultrasound probe and especially the identification of the muscularis mucosae (MM), has not been analyzed clearly. Methods: In a basic study, 11 sections of normal gastric wall with 26 horizontally inserted nylon sutures were immersed in water. The sections were scanned and the findings correlated using standard histology. In a clinical study, 15 early gastric cancers were examined by a 30 MHz endoscopic ultrasound probe. Results: In a basic study, layers deeper than the lower part of the submucosa could not be seen using ultrasonography. The first to fourth layers represented the mucosal layer except the MM, the fifth layer (high‐echo layer) represented the boundary echo and a part of the MM, while the sixth layer (low‐echo layer) represented the rest of the MM. The muscularis mucosae was seen clearly in all samples. In a clinical study the layers deeper than the submucosal layer could not be seen and the MM was visible in 87% of cases. The depth of invasion was estimated accurately in 90% of mucosal cancers and in 80% of submucosal cancers. Conclusion: A 30 MHz endoscopic ultrasound probe, which cannot image the entire gastric wall, can visualize the MM and may help to confirm the structure of gastric wall layers and improve the ability to determine the depth of invasion in gastric cancer.     

Correlation between expression of cyclooxygenase-2 and angiogenesis in human gastric adenocarcinoma

AIM: To evaluate the expression of cyclooxygenase (COX2) and the relationship with tumor angiogenesis and advancement in gastric adenocarcinoma.METHODS: Immunohistochemical stain was used for detecting the expression of COX-2 in 45 resected specimens of gastric adenocarcinoma; the monoclonal antibody against CD34 was used for displaying vascular endothelial cells, and microvascular density (MVD) was detected by counting of CD34-positive vascular endothelial cells. Paracancerous tissues were examined as control.RESULTS: Immunohistological staining with COX-2-specific polyclonal antibody showed cytoplasmic staining in the cancer cells, some atypical hyperplasia and intestinal metaplasia,as well as angiogenic vasculature present within the tumors and prexisting vasculature adjacent to cancer lesions. The rate of expression of COX-2 and MVD index in gastric cancers were significantly increased, compared with those in the paracancerous tissues (77.78 vs 33.33 %, 58.13±19.99 vs 24.02±10.28, P＜0.01, P＜0.05, respectively). In 36 gastric carcinoma specimens with lymph node metastasis, the rate of COX-2 expression and MVD were higher than those in the specimens without metostasis (86.11 vs 44.44 %,58.60±18.24 vs 43.54±15.05, P＜0.05, P＜0.05, respectively).The rate of COX-2 expression and MVD in the specimens with invasive serosa were significantly higher than those in the specimens without invasion to serosa (87.88 vs 50.0 %,57.01±18.79 vs42.35±14.65, P＜0.05, P＜0.05). Moreover,MVD in COX-2-positive specimens was higher than that in COX-2-negative specimens (61.29±14.31 vs 45.38±12.42,P＜0.05). COX-2 expression was positively correlated with MVD (r=0.63, P＜0.05).CONCLUSION: COX-2 expression might correlate with the occurance and advancement of gastric carcinoma and is involved in tumor angiogenesis in gastric carcinoma. It is likely that COX-2 by inducing angiogenesis can be one of mechanisms which promotes invasion and metastasis of gastric carcinoma. It may become a new therapeutic target for anti-angiogenesis.     

Tumor angiogenesis increases with nuclear p53 accumulation in gastric carcinoma

BACKGROUND/AIMS: Tumor angiogenesis is associated with disease stage in gastric carcinoma. The tumor suppressor p53 was recently reported to regulate angiogenesis. We investigated the relationship between nuclear p53 accumulation and tumor vascularity in gastric carcinoma. METHODOLOGY: Patients with gastric carcinoma undergoing surgery were randomly enrolled. Nuclear p53 expression assessed by immunohistochemistry on tumor sections was categorized as higher and lower groups. Tumor vascularity was assessed by counting microvessel density on anti-CD34 stained sections. Tumor vascularity between different p53 expressions was compared based on the different clinicopathologic characteristics. RESULTS: Twenty-three patients had gastric carcinoma with higher nuclear p53 expression and 42 with lower nuclear p53 expression. Vascularity in higher p53 group (54.3 +/- 35.3) was significantly higher than that in lower p53 group (32.1 +/- 29.9) (p = 0.009). Further analysis showed that higher nuclear p53 immunoreactivity was associated with significantly higher vascularity in the advanced gastric carcinoma group (p = 0.023), the intestinal type group (p = 0.003), the non-cardiac group (p = 0.019), and the H. pylori-negative group (p = 0.014). CONCLUSIONS: The present study demonstrated that nuclear p53 protein expression positively associates vascularity in gastric carcinoma although their relationship varies in the different clinicopathologic subsets.     

Endoscopic ultrasound-guided fine-needle aspiration biopsy for the diagnosis of gastrointestinal stromal tumors in the stomach

Background For the diagnosis of gastric submucosal tumors (SMTs), endoscopic ultrasound (EUS) alone does not reveal the complete pathology, such as the degree of malignancy, and EUS-guided fine-needle aspiration biopsy (EUS-FNAB) has been reported to be more useful. Recently, most cases initially diagnosed as leiomyosarcomas have received further study with immunohistochemical staining and have been given the new diagnosis of gastrointestinal stromal tumors (GISTs). The degree of malignancy of GISTs differs widely in clinical aspects. In this study, we examined whether EUS-FNAB was useful in diagnosing GISTs and differentiating their degrees of malignancy.Methods From January 1997 to March 2002, 21 cases of gastric GISTs were diagnosed from the immunohistochemical staining of specimens resected at Aichi Cancer Center Hospital. Of these 21 patients, 14 (5 with high-grade malignancy and 9 with low-grade malignancy) underwent EUS-FNAB preoperatively, and were examined further: their EUS-FNAB specimens were submitted for additional immunohistochemical testing.Results The EUS-FNAB specimens from all patients were positive for c-kit and CD34 immunohistochemical testing, coinciding with the staining results of the resected specimens. The MIB-1 labeling indices in specimens of high-grade malignancy were significantly higher than those of low-grade malignancy. If we assumed that a tumor with an MIB-1 labeling index of more than 5% was a high-grade malignancy, the diagnostic accuracy was 85.7%.Conclusions The EUS-FNAB procedure is a useful tool for diagnosing GISTs of the stomach with immunohistochemical staining. When used with MIB-1 staining, the procedure may indicate GIST prognosis and influence decisions regarding therapeutic strategies.     

Eph-B4受体及其配体Ephrin-B2在胃癌组织中的表达及其病理学意义

目的 :探讨Eph B4受体与其配体Ephrin B2在胃癌组织中的表达及其对血管生成和肿瘤生物学行为的影响。方法 :应用免疫组化SABC法检测 93例胃癌和 30例癌旁组织中Enp B4受体与其配体Ephrin B2的表达及间质微血管密度 (MVD)。结果 :胃癌组织中肿瘤细胞、间质新生血管的Eph B4与Ephrin B2高度表达 ,癌组织中Eph B4与Ephrin B2的阳性表达 (分别为 49.46 %和 50 .54 % )明显高于癌旁组织 (分别为 2 0 .0 %和 2 3 .3 % ,P <0 .0 1 )。Enp B4与Ephrin B2阳性表达组的平均MVD值 (分别为 56 .2 1± 1 5 .3和 62 .41± 1 6 .9)明显高于Enp B4与Ephrin B2阴性表达组 (分别为 32 .2 2± 1 2 .9和 34 .1 2± 1 4 .7,P <0 .0 5和P <0 .0 1 )。此外 ,Enp B4与Ephrin B2表达程度还与胃癌的转移和癌浸润程度密切相关。结论 :Enp B4与Ephrin B2可以促进肿瘤间质的血管生成 ,加速肿瘤浸润和转移     

Magnifying pharmacoendoscopy: response of microvessels to epinephrine stimulation in differentiated early gastric cancers

BACKGROUND: Magnifying endoscopy is a promising modality for fine observation of minute surface structures and microvessel architecture in gastric lesions. OBJECTIVE: To observe the response of microvessels to epinephrine stimulation in early gastric cancer tissues and to assess the usefulness of magnifying pharmacoendoscopy for histologic diagnosis. DESIGN: This was a prospective pilot study. SETTING: This study was conducted at an academic hospital. PATIENTS: Twenty-nine patients with differentiated early gastric cancer were enrolled. INTERVENTIONS: Microvessels in both the cancerous lesion and its adjacent non-neoplastic gastric mucosa were observed by magnifying endoscopy before and after focal spray with epinephrine solution (0.05 mg/mL). MAIN OUTCOME MEASUREMENTS AND RESULTS: After epinephrine stimulation, noncancerous gastric mucosa surrounding the cancerous lesion showed a change in color from red to white; no microvessels were evident. On the other hand, all the cancerous lesions examined clearly showed enhancement of tumor microvessels. The rate of detection of tumor microvessels by magnifying pharmacoendoscopy (100%) was significantly higher than that by magnifying endoscopy alone (41.3%). LIMITATIONS: This was small pilot study. CONCLUSIONS: Magnifying pharmacoendoscopy with epinephrine is a powerful tool for assessing tumor vascularity and may contribute to the histologic diagnosis of differentiated early gastric cancers before endoscopic treatment.     

Harmonic imaging for improving diagnosis of liver tumors--preliminary report

Contrast harmonic imaging (CHI) is a new Doppler technology using the non-linear properties of ultrasound contrast agents. AIM: In this pilot study contrast harmonic imaging was evaluated for the detection and characterization of liver lesions. METHODS: Tumor vascularity patterns and diagnostic accuracy in the differential diagnosis of liver lesions were compared in 50 liver lesions using gray-scale sonography, unenhanced and enhanced power Doppler sonography and CHI. In addition, CHI was compared to spiral-CT for the detection of liver metastases in 28 patients. RESULTS: CHI was superior to power Doppler sonography in the visualization of vascularity pattern. Due to the better resolution and image quality of tumor vascularity the diagnostic accuracy of CHI in the differential diagnosis of liver lesions was higher (88% CHI versus 40% unenhanced power Doppler, and 58% contrast-enhanced power Doppler). In the detection of liver metastasis CHI showed a higher sensitivity (96.8%) compared to spiral-CT (74.6%) in 28 patients with 63 metastases. CONCLUSIONS: CHI is a promising new ultrasound technology to further improve the differential diagnosis of liver lesions and the detection of metastases.     

Predicting the need for surgery in Crohn's disease with contrast harmonic ultrasound

OBJECTIVE: Harmonic flash echo imaging (H-FEI), an intermittent scanning method using injection of Levovist, enables the evaluation of microperfusion of the gastrointestinal wall. The aim of this study was prospectively to investigate the relationship between bowel blood flow and the likelihood of the need for surgery in 70 patients with active Crohn's disease (CD), using conventional ultrasonography (US) and H-FEI. MATERIAL AND METHODS: For H-FEI, Levovist injection was followed by scanning of the bowel segment of interest with intermittent harmonic imaging for 2 min (interval, 4 s; transmission pulse at 2.3 MHz; reception at 4.6 MHz). We calculated the mean echo-intensity for the bowel wall showing strongest opacity in the FEI series for each patient. Maximum bowel-wall thickness, wall stratification, maximum echo-intensity obtained by H-FEI, CD activity index, and C-reactive protein concentration were compared between patients treated successfully with medication after H-FEI (group M) and those who proved to need surgery (group S). RESULTS: No significant differences in clinical variables were evident between groups M (n=45) and S (n=25). The subsequent need for surgery was significantly indicated by multivariate analysis, showing loss of stratification in the bowel wall (odds ratio (OR)=5.98, 95% confidence interval (CI)=1.4-25.1, p=0.015) and high echo-intensity according to H-FEI (OR=1.02, CI=1.005-1.034, p=0.007). CONCLUSION: In active CD, patients with bowel segments showing a loss of stratification and rich perfusion by H-FEI are likely to need surgical treatment.     

Clinicopathological and prognostic implications of endoglin （CD105） expression in hepatocellular carcinoma and its adjacent non-tumorous liver

AIM: The expression pattern of endoglin (CD105) in hepatocellular carcinoma (HCC) has not been reported so far. We hypothesized that CD105 could differentially highlight a subset of microvessels in HCC, and intratumoral microvessel density (IMVD) by CD105 immunostaining (IMVD-CD105) could provide better prognostic information than IMVD by CD34 immunostaining (IMVD-CD34). METHODS: Paraffin blocks of tumor and adjacent non-tumorous liver tissues from 86 patients who underwent curative resection of HCC were used for this study. Serial sections were stained for CD105 and CD34, respectively, to highlight the microvessels. IMVD was counted according to a standard protocol. RESULTS: In the HCC tissues, CD105 was either negatively or positively stained only in a subset of microvessels. In contrast, CD34 showed positive and more extensive microvessel staining in all cases examined. However, in the adjacent non-tumorous liver sections, CD105 showed a diffuse pattern of microvessel staining in 20 of 86 cases, while CD34 showed negative or only focal staining of the sinusoids around portal area. Correlation with clinicopathological data demonstrated that lower scores of IMVD-CD105 were found in larger sized tumors [mean 41.4/0.74 mm2 (>5 cm tumor) vs 65.9/0.74 mm2 (≤5 cm tumor), P= 0.043] and more aggressive tumors, as indicated by venous infiltration [36.8/0.74 mm2 (present) vs 64.2/0.74 mm2 (absent), P = 0.020], microsatellite nodules [35.1/0.74 mm2 (present) vs 65.9/0.74 mm2 (absent), P= 0.012], and advanced TNM tumor stage [38.8/0.74 mm2 (stage 3 or 4) vs 68.3/0.74 mm2 (stage 1 or 2), P= 0.014]. No prognostic significance was observed when median values were used as cut-off points using either IMVD-CD105 or IMVD-CD34. However, the presence of the diffuse pattern of CD105 expression in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival (median 8.6 vs 21.5 mo, P= 0.026). CONCLUSION: Our data demonstrate that a lower IMVD-CD105 is associated with larger and more aggressive tumors. In this study, IMVD-CD105 did not provide significant prognostic information. However, active angiogenesis as highlighted by diffuse CD105 staining of the microvessels in the adjacent non-tumorous liver tissues is predictive of early recurrence.     

Vascularity of hepatic VX2 tumors of rabbits： Assessment with conventional power Doppler US and contrast enhanced harmonic power Doppler US

AIM: To investigate the characteristics of the vascularity of hepatic metastasis. METHODS: Six New Zealand rabbits, weighing averagely 2.7+/-0.4 kg, were selected and operated to establish hepatic VX2 tumor carrier model. Hepatic VX2 tumors were then imaged with conventional B mode US, second harmonic imaging (SHI), color Doppler flow imaging (CDFI), power Doppler imaging (PDI) and harmonic PDI by a transducer S8 connected to HP-5 500 ultrasound system. A kind of self made echo contrast agent was intravenously injected at a dose of 0.01 mL/kg through ear vein, and then the venous passage was cleaned with sterilized saline. RESULTS: Totally, 6 hypoechoic lesions and 3 hyperechoic lesions were found in the six carrier rabbits with a mean size about 2.1+/-0.4 cm under conventional B mode ultrasound, they were oval or round in shape with a clear outline or a hypoechoic halo at the margin of the lesions. Contrast agent could not change the echogenicity of the lesions under conventional B mode and SHI, however, it could greatly increase the flow sensitivity of the lesions under PDI and harmonic PDI. Nutrient artery of these metastatic lesions might also be well depicted under contrast enhanced PDI and harmonic PDI. CONCLUSION: Our result suggested that contrast enhanced PDI, especially harmonic PDI, was a promised method in the detection of vascularity of hepatic tumor nodules.     

STUDY OF THE TUMOR VESSELS IN DEPRESSED‐TYPE EARLY GASTRIC CANCERS USING NARROW BAND IMAGING MAGNIFYING ENDOSCOPY AND CDNA ARRAY ANALYSIS

Background: Since endoscopic mucosal resection has been applied to differentiated gastric cancers with invasion limited to the mucosal layer, the diagnosis of their differentiation is important. The degree of differentiation varies depending on the size and location of the tumors. Correct diagnosis by biopsy can be difficult because depressed‐type early gastric cancers sometimes contain mixed histology. Methods: Fifteen patients with depressed‐type early gastric cancers were observed by magnifying endoscopy with a narrow band lighting system. The fine mucosal vascular pattern was recorded and compared with the histological differentiation and features of vessels by staining with CD34. In some patients, cDNA array analysis was performed to determine differences among histological types. Results: Tumor vascular patterns were classified into two categories. Grid‐like network patterns not only characterized differentiated type but were also associated with high microvascular density. Short twig‐like patterns typified the undifferentiated type and a low vascular density. Differentiated types highly expressed some angiogenic factors, such as VEGFc and Flt‐4. Conclusions: Tumor vessel pattern of depressed‐type early gastric cancer obtained by narrow band imaging magnifying endoscopy reflects both the histological features and the degree of expression of angiogenic factors.     

PGP9.5 overexpression in esophageal squamous cell carcinoma

BACKGROUND/AIMS: PGP9.5 is a ubiquitin hydrolase widely expressed in neuronal tissue at all stages of neuronal differentiation and has been used as a neuroendocrine marker. Recently, it has been proved that PGP9.5 expression was highly observed in squamous cell carcinoma of lung cancer, suggesting that it might be a tumor marker for squamous cell carcinoma. To better characterize its role in digestive tract cancers, we examined PGP9.5 expression retrospectively in esophageal cancers. METHODOLOGY: We examined PGP9.5 expression retrospectively in 40 resected esophageal cancers (squamous cell carcinoma) and 10 gastric cancers (adenocarcinoma) using immunohistochemistry. RESULTS: Of 40 esophageal cancer specimens, 19 (48%) exhibited positive staining with PGP9.5 in most tumor cells, while no PGP9.5 expression was observed in any of the 10 gastric cancers. CONCLUSIONS: Although the precise mechanism underlying the effect of PGP9.5 on oncogenicity remains to be proven, it was confirmed that it may be a potential marker for esophageal squamous cell carcinoma.     

Imaging diagnosis of portal tumor thrombus secondary to gastric cancer

Tumor thrombus in the portal vein secondary to advanced gastric cancer was diagnosed by ultrasound (US) and computed tomography (CT), and was confirmed at autopsy in a 54-year-old man. The register data for 3176 patients of resected gastric cancer disclosed that there was no patient with portal tumor thrombus secondary to gastric cancer.     

The clinical meaning of mucin phenotype and Epstein-Barr virus infection in gastric cancer

BACKGROUND/AIMS: Differentiated type adenocarcinomas producing gastric type mucin are receiving much attention because of their degree of clinical malignancy. Most Epstein-Barr virus (EBV)-associated gastric cancers are undifferentiated type, and correlate with gastric type mucin. We analyzed the clinical meaning of mucin phenotypes and the detection of EBV in gastric cancers. METHODOLOGY: The objects of study were 120 consecutive gastric cancer lesions, resected endoscopically (EMR group, n=54) or surgically (surgery group, n=66). The mucin phenotypes were determined using immunostaining for human gastric mucin (HGM), MUC2, and CD10. Changes in histological type within the lesions were examined. The presence of EBV was determined using in situ hybridization for EBV-encoded small RNA 1 (EBER-1). RESULTS: The incomplete intestinal phenotype accounted for 83% of the EMR group, and the gastric phenotype for only 13%. None of the EMR group lesions had changes in the degree of differentiation, and there was no EBER-1-positive lesion. In the surgery group, the gastric phenotype accounted for 29%, significantly more than in the EMR group (p=0.0363). The incomplete intestinal phenotype accounted for 64% of surgically resected lesions. Changes in the degree of differentiation were significantly more common in the surgery group (16/66) than in the EMR group (0/54) (p=0.0001), tending to be more common in the gastric phenotype lesions. There were 3 EBER-1-positive lesions in the surgery group, accounting for 5%, and all were HGM positive. CONCLUSIONS: There appears to be little need to determine the mucin phenotype or EBV status of endoscopically resected lesions. In cases of gastric cancer where surgical resection is indicated, however, where the preoperative findings indicate a depth of invasion to SM or greater, and/or an undifferentiated lesion, then mucin phenotyping of biopsy specimens may be useful in predicting the predominant histological type of the tumor.     

Risk factors for lymphatic and venous involvement in endoscopically resected gastric cancer

Background

Lymphatic and venous involvement is a critical factor in the curability assessment of endoscopically resected gastric cancers; however, the risk factors for lymphatic and venous involvement in endoscopically resected gastric cancers remain unknown.

Methods

To identify risk factors for lymphatic and venous involvement in endoscopically resected gastric cancers, we retrospectively reviewed a consecutive series of 1229 endoscopically resected gastric cancers in 1083 patients treated between January 2009 and December 2011.

Results

Lymphatic and venous involvement was detected in 57 (4.6 %) and 32 (2.6 %) lesions, respectively. A multivariate analysis identified a larger tumor size, deeper invasion (submucosal invasion or deeper), and the presence of a papillary or an undifferentiated-type adenocarcinoma component as independent risk factors for lymphatic involvement. As for venous involvement, deeper invasion (≥500 μm submucosal invasion), a macroscopically elevated type, and the presence of an undifferentiated-type adenocarcinoma component were identified as independent risk factors.

Conclusions

The present study identified the independent risk factors for lymphatic and venous involvement in endoscopically resected gastric cancers. The recognition of these risk factors would help in the selection of lesions that may require a particularly careful histological evaluation.     

Three-dimensional reconstruction of tumor microvasculature: simultaneous visualization of multiple components in paraffin-embedded tissue

Three-dimensional (3D) visualization of microscopic structures may provide useful information about the exact 3D configuration, and offers a useful tool to examine the spatial relationship between different components in tissues. A promising field for 3D investigation is the microvascular architecture in normal and pathological tissue, especially because pathological angiogenesis plays a key role in tumor growth and metastasis formation. This paper describes an improved method for 3D reconstruction of microvessels and other microscopic structures in transmitted light microscopy. Serial tissue sections were stained for the endothelial marker CD34 to highlight microvessels and corresponding images were selected and aligned. Alignment of stored images was further improved by automated non-rigid image registration, and automated segmentation of microvessels was performed. Using this technique, 3D reconstructions were produced of the vasculature of the normal brain. Also, to illustrate the complexity of tumor vasculature, 3D reconstructions of two brain tumors were performed: a hemangioblastoma and a glioblastoma multiforme. The possibility of multiple component visualization was shown in a 3D reconstruction of endothelium and pericytes of normal cerebellar cortex and a hemangioblastoma using alternate staining for CD34 and alpha-smooth muscle actin in serial sections, and of a GBM using immunohistochemical double staining. In conclusion, the described 3D reconstruction procedure provides a promising tool for simultaneous visualization of microscopic structures.     

Relationship between the expression of iNOS,VEGF,tumor angiogenesis and gastric cancer

AIM：To in vestigate the relationship between the expression of inducible nitric oxide synthase(iNOS),vascular endothelial growth factor(VEGF),the microvascular density(MVD)and the pathological features and clinical staging of gastric cancer.METHODS:Immunohistochemical staining was used for detecting the expression of iNOS and VEGFin46resected specimens of gastric carcinoma;the monoclonal antibody against CD34 was used for displaying vascular endothelial cells,and MVD was detected by counting of CD34-positive vascular endothelial cells.RESULTS:Of 46resected specimens of gastric carcinoma,the rates of expressions of iNOS and VEGF were 58.70%and76.09%,respectively,and MVDaveraged55.59&#177;19.39,Judged by the standard TNM criteria,the rate of expression of iNOS in stageⅣ（84.46%)was higher than those in stageⅠ,Ⅱ,Ⅲ（Fish exact probabilities test,P=0.019,0.023and 0.033,respectively);the rates of expression of VEGFin stage Ⅲ,Ⅳ（76.0%,92.31%,respectively)were higher than those in stageⅠ,Ⅱ（Fis exact probabilities test,P=0.031,0.017,0.022and0.019).MVDs in stageⅢ,Ⅳ（64.72&#177;14.96,67.09&#177;18.29,respectively)were higher than those in stageⅠ,Ⅱ(t\2.378,4.015,2.503and2.450,P&lt;0.05,P&lt;0.001,P&lt;0.001,P&lt;0.05,respectively),In37gastric carcinoma specimens with lymph node metastasis,MVD(68.69&#177;18.07)and the rates of expression of iNOS and VEGF(70.27%,83.78%,respectively)were higher than those in the specimens with absence of metastasis(t=2.205,X^2=6.3587,X^2=6.2584,P&lt;0.01,P&lt;0.05,P&lt;0.05,respectively),MVD and the expressions of iNOS and EGF were not correlated to the location,size or grade of tumor,nor with the depth of invasion of tumor;MVDs in the positive iNOS and VEGF specimens(59.88&#177;18.02,58.39&#177;17.73,repectively)were higher than those in the negative iNOS and VEGF specimens(X^2=6.3587and 6.1574,P&lt;0.05,P&lt;0.05,respectively);thus the expressions of iNOS and VEGF was correlated to MVD,but the expression of iNOS was not correlated to that of VEGF,In addition.of the 46 surviving patients,the 5-year survival rate of patients with positive iNOS or VEGF tumors was significantly less than that of patients with negative iNOS-or VEGF tumors(X^2=4.3842and 5.4073,P&lt;0.05,P&lt;0.05.respectively).CONCLUSION:The expressions of iNOS and VEGF are colosely related to tumor angiogenesis,and are involved in the advancement and the lymph node metastasis;thusMVD and the expressions of iNOS and EGF may serve indexes for evaluating staging of gastric carcinoma and forecasting its risk of metastasis,which will help establish a comprehensive therapeutical measure of post-operative patients and provide a new approach to tumor therapy.     

Phagocytosis(cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas

AIM:To identify those with a micropapillary pattern,ascertain relative frequency and document clinicopathological characteristics by reviewing gastric carcinomas.METHODS:One hundred and fifty-one patients diagnosed with gastric cancer who underwent gastrectomy were retrospectively studied and the presence of a regional invasive micropapillary component was evaluated by light microscopy.All available hematoxylin-eosin(HE)-stained slides were histologically reviewed and 5 tumors were selected as putative micropapillary carcinoma when cancer cell clusters without a vascular core within empty lymphatic-like space comprised at least5%of the tumor.Tumor tissues from these 5 invasive gastric carcinomas were immunostained using an antimucin 1(MUC1)antibody(clone MA695)to detect the characteristic inside-out pattern and with D2-40antibody to determine the presence of intratumoral lymph vessels.Detection of intraepithelial neutrophil apoptosis was evaluated in consecutive histological tissue sections by three independent methods,namely light microscopy with HE staining,the conventional terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end-labeling(TUNEL)method and immunohistochemistry for activated caspase-3(clone C92-605).RESULTS:Among 151 gastric cancers resected for cure,5(3.3%)were adenocarcinomas with a micropapillary component.Four of the patients died of disease from 6 to 23 mo and one patient was alive with metastases at 9 mo.All patients had advanced-stage cancer(≥p T2)and lymph node metastasis.Positive MUC1 immunostaining on the stroma-facing surface(inside-out pattern)of the carcinomatous cluster cells,together with negative immunostaining for D2-40 in the cells limiting lymphatic-like spaces,confirmed the true micropapillary pattern in these gastric neoplasms.In all five cases,several micropapillae were infiltrated by neutrophils.HE staining,TUNEL assay and immunostaining for caspase-3 demonstrated apoptoticneutrophils within cytoplasmic vacuoles of tumor cells.These data suggest phagocytosis(cannibalism)of apoptotic neutrophils by micropapillary tumor cells.Tumor cell cannibalism is usually found in aggressive tumors with anaplastic morphology.Our data extend these observations to gastric micropapillary carcinoma:a tumor histotype analogously characterized by aggressive behavior and poor prognosis.The results are of interest because they raise the intriguing possibility that neutrophil cannibalism by tumor cells may be one of the mechanisms favoring tumor growth in gastric micropapillary carcinomas.CONCLUSION:This is the first study showing phagocytosis(cannibalism)of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.