This study analyzed canine haemangioma and haemangiosarcoma samples immunohistochemically to better characterize the biology and identify potential therapeutic targets. It found greater expression of CD117, VEGFR-3 and CD44 in haemangiosarcoma compared to haemangioma, suggesting these proteins may be suitable targets. It also found marked mast cell infiltration in haemangioma, indicating a possible role for mast cells in benign vascular neoplasia in dogs.
Objective: Tongue squamous cell carcinoma (TSCC) is a prominent type of oral cancer. Despite the numerous research studies on SCC and microRNAs (miRs), the relation between TSCC and miR-135b-5p is poorly discussed. This experiment aims to find out the possible effect of miR-135b-5p on TSCC with the network of its downstream genes.
Study Design: TSCC tissues and adjacent normal tissues were harvested. Then, expression of miR-135b-5p and AT-rich interactive domain‑containing protein 1A gene (ARID1A) and the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) pathway was analyzed. After the transfection of miR-135b-5p inhibitor and its negative control into TSCC cells, functional assays were employed to measure cell proliferation, apoptosis, and cycle. Next, the target relation between miR-135b-5p and ARID1A was confirmed. In addition, the fact that miR-135b-5p promoted TSCC development via mediating ARID1A was demonstrated by functional rescue experiment.
Results: miR-135b-5p was upregulated in TSCC tissues and cells, while ARID1A was suppressed (p< 0.05). Silenced miR-135b-5p discouraged TSCC cell proliferation, improved apoptosis, induced cell cycle arrest, and increased ARID1A expression while inactivating the PI3K/AKT axis (p<0.05). Furthermore, knockdown of ARID1A reversed the impacts on TSCC cell proliferation and apoptosis exerted by silencing miR-135b-5p.
Conclusion: This research supported that silenced miR-135b-5p impeded TSCC proliferation and apoptosis by promoting ARID1A and inactivating the PI3K/AKT axis, which may provide some indications for TSCC alleviation.
Keywords: apoptosis; ARID1A; ARID1A protein, human; carcinoma, squamous cell; cell line, tumor; cell proliferation; drug resistance, neoplasm; microRNA-135b-5p; microRNAs; PI3K/AKT pathway; neoplasm metastasis; neoplastic stem cells; proliferation; protein binding; tongue; tongue squamous cell carcinoma
Objective: To probe into the influence of miR-21 on the proliferation as well as apoptosis of oral squamous cell carcinoma (OSCC) and its causative role.
Study Design: We adopted microarray for detecting the differentially expressed genes in OSCC tumor tis-sues and paracancerous tissues. We assessed the link of miR-21 expression with tumor size, lymph node metastasis, and tumor differentiation. We employed CCK-8 and EdU assay for detecting the impact of miR-21 inhibitor and miR-21 mimic on Cal-27 cell proliferation, as well as TUNEL and AnnexinV-FITC/PI double staining for detecting miR-21 expression on cell apoptosis. We forecasted the possible target of miR-21 via TargetScan, as well as detected the interaction of miR-21 with PTEN via luciferase reporter experiment. The function of miR-21 expression in PTEN signaling pathway was monitored via western blot. We constructed PTEN overexpression plasmid and conducted rescue experiment to evaluate overexpressed PTEN on miR-21–induced proliferation.
Results: Microarray and RT-qPCR indicated that miR-21 expression increased demonstrably in OSCC. Subsequently, statistical analysis showed that miR-21 expression was plainly correlated with tumor size, lymph node metastasis, tumor differentiation, and smoking history. CCK-8 and EdU method exhibited that miR-21 mimics manifestly promoted Cal-27 cell proliferation, while miR-21 inhibitor blatantly inhibited Cal-27 cell proliferation. TUNEL and V-FITC/PI double staining assay showed that miR-21 inhibitor conspicuously promoted Cal-27 cell apoptosis. CCK-8 and EdU assay exhibited that overexpressed PTEN abolished the pro-proliferation influence of miR-21 mimic. TUNEL and V-FITC/PI experiments pointed out that knocking down PTEN abrogated the pro-apoptosis impact of miR-21 inhibitor.
Conclusion: miR-21 contributes to OSCC cell proliferation via targeting PTEN and inhibits its apoptosis.
Keywords: Akt/PKB signaling pathway; apoptosis; biomarkers, tumor; carcinoma, squamous cell; cell line, tumor; cell proliferation; microRNAs; miR-21; miRNA-21; mouth neoplasms; oral cancer; oral squamous cell carcinoma; proliferation; real time PCR
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Objective: To analyze the sonographic features of different histopathological subtypes of borderline ovarian tumors (BOTs) confirmed by pathology, and to study the ultrasound performances of various types in borderline ovarian tumors.
Study Design: Retrospective analysis was performed on the pathological results and ultrasound projection findings of 129 patients diagnosed as BOTs by ultrasound department of our hospital from January 2012 to November 2019. All patients were confirmed by surgical pathology and scanned consecutively by the investigators using transabdominal or transvaginal ultrasound examination.
Results: Serous borderline tumors (SBOTs) were observed, and the prevalence rate (53%) was significantly higher than that of other subtypes, and the probability of bilateral lesions was higher (40%). The sonogram often showed ultrasound features of papillary neoplasm in the lesion and good internal echo (p<0.05). Mucinous borderline ovarian tumors (MBOTs) were mostly unilateral lesions (86%). The prevalence was second only to SBOTs. Histomorphological examinations were divided into gastrointestinal-type and endocervical-type. Among them, the gastrointestinal type of MBOTs were mostly unilateral, and their incidence was higher than that of endocervical-type of MBOTs. Compared with other pathological subtypes, the gastrointestinal type is more likely to show the sonographic characteristics of huge space occupying in the pelvic and abdominal cavity (mean diameter >10 cm), polycystic, multiple septums, and poor internal echo (p<0.05). The ultrasonographic features of the endocervical-type of MBOTs were similar to those of SBOTs. Compared with gastrointestinal type, the sonographic images showed smaller lesion diameter, less septal or cyst, and more papillary excrescences in the tumor (p<0.05). The borderline clear cell tumor is the intermediate transition between the clear cell adenofibroma and the clear cell carcinoma. The clinical manifestations are diverse and lack specificity. The histology of sonography was mainly solid, and the multiple microcapsules were honeycomb-like. It can also be shown as cystic. Among the 169 patients with BOTs, 20 cases of SBOTs, 17 cases of MBOTs, and 10 cases of other rare subtypes were complicated with other diseases or multiple subtypes. This study did not find significant ultrasonic characteristics were used for distinguish them from other subtypes.
Conclusion: BOTs is a common disease in women during the reproductive period. It is characterized by the development of malignant tumors. Its clinical and pathological subtypes are complex and diverse. It leads many doctors to use the terms “large pelvic mass” and “solid ovarian mass” for diagnosis because of their lack of experience and understanding.
Keywords: adenocarcinoma, mucinous; adenocarcinoma, serous; borderline ovarian tumors; diagnostic imaging; ovarian neoplasms; papillary neoplasms; prognosis; transvaginal ultrasound, ultrasonography
Objective: Ischemia-reperfusion (I/R) leads to reactive oxygen species formation and cell death in kidney tissue with injury and organ transplantation. Simvastatin (SIM) is an antioxidant, anti-inflammatory, and anticoagulant agent. Alterations in I/R-induced acute kidney injury model with SIM treatment were analyzed.
Study Design: Wistar rats (n=28) were grouped into Sham, Ischemia, I/R, and I/R+SIM treated. Left rat kidney renal vessels were clamped for 60 minutes for ischemia, and the I/R group had 6 hours of reperfusion. 10 mg/kg SIM was given orally for 28 days. MDA, GSH, and MPO were analyzed. Kidney tissues were paraffin embedded, and primary antibodies TNF-α and caspase-3 were applied for immunohistochemistry.
Results: In the I/R group, intense inflammatory cell infiltration around the vessels and necrosis in the glomerular structures were observed. In the treated group, proximal and distal tubular cells were found to be close to normal. Immunoexpression of caspase-3 in the ischemia group was positive in degenerative glomeruli. In the treated group, TNF-α expression was negative in the glomerular structures. MDA and MPO levels were significantly increased in ischemia and I/R.
Conclusion: We suggest that SIM treatment improved kidney tissue structure and function in a model of I/R injury.
Keywords: caspase-3; immunohistochemistry; ischemia/reperfusion; kidney; MPO; simvastatin
Objective: The association between telomerase reverse transcriptase (TERT) promoter mutation and outcome of melanoma is unclear and controversial. We aim to conduct a meta-analysis and investigate whether the TERT promoter mutation is a prognostic factor of melanoma.
Study Design: Appropriate studies were searched in 3 databases: PubMed, Web of Science, and Embase. Pooled hazard ratios (HRs) were counted through random effects model.
Results: Heterogeneity was moderate in overall survival (OS) (I2=43.7%, p=0.059) and low in disease-free survival (DFS) (I2=0.0%, p=0.587). Sensitivity analysis indicated that the removal of any of the study did not affect the final results. Evidence for publication bias was not found (Begg’s test, p=0.281; Egger’s test, p=0.078). The pooled OS HRs from combined effects analysis was determined (HR 1.07; 95% CI 0.83–1.39, p=0.585), together with the pooled HRs of DFS (HR 1.65; 95% CI 1.02–2.66, p=0.042). TERT promoter mutation predicted a good outcome in meta-static melanoma patients (HR 0.66; 95% CI 0.46–0.96, p=0.042). The pooled HRs of combined mutation in TERT promoter and BRAF (HR 6.27; 95% CI 2.7–14.58, p=0.000) predicted a bad outcome in melanoma patients.
Conclusion: TERT promoter mutation significantly predicted poor DFS outcome but, on the contrary, predicted a good outcome in metastatic melanoma patients. The combined TERT promoter and BRAF mutation was a significant independent factor of OS in melanoma patients.
Keywords: melanoma; meta-analysis; mutation; prognosis; promoter regions, genetic; skin neoplasms; telomerase; TERT promoter mutation; TERT protein, human
Objective: The prognostic indictors of age-related poor outcomes in patients with acute myeloid leukemia (AML) are still controversial. The aim of this work was to provide comprehensive insights into the effect of different hemocytes and to investigate the association between age and clinical features in adult patients with AML.
Study Design: A retrospective study was performed to determine the role of age in the therapeutic outcomes of AML. A total of 166 newly diagnosed adult patients’ data from January 2015 to November 2019 in Zhongshan Hospital of Xiamen University were collected and analyzed.
Results: Older patients presented a poorer prognosis (p=0.001) with shorter overall survival, which is served as age-related outcomes. Binary logistic regression demonstrated that cytogenetic risk (OR=4.508, 95% CI 2.733–7.435), leukocyte (OR=7.410, 95% CI 1.139–5.910), and bone marrow blast cells (OR=3.261, 95% CI 1.075–5.615) were independent indictors for age-related prognosis. In addition, Kaplan-Meier curve also revealed that the above factors were associated with overall survival (all p values <0.001).
Conclusion: Cytogenetic risk, leukocyte, and bone marrow blast cells are dominant factors which account for the age-related poor outcomes and shorter overall survival in AML.
Keywords: acute myeloid leukemia, adult, cytogenetic risk, hemocyte, leukemia, overall survival
Objective: Tongue squamous cell carcinoma (TSCC) is a prominent type of oral cancer. Despite the numerous research studies on SCC and microRNAs (miRs), the relation between TSCC and miR-135b-5p is poorly discussed. This experiment aims to find out the possible effect of miR-135b-5p on TSCC with the network of its downstream genes.
Study Design: TSCC tissues and adjacent normal tissues were harvested. Then, expression of miR-135b-5p and AT-rich interactive domain‑containing protein 1A gene (ARID1A) and the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) pathway was analyzed. After the transfection of miR-135b-5p inhibitor and its negative control into TSCC cells, functional assays were employed to measure cell proliferation, apoptosis, and cycle. Next, the target relation between miR-135b-5p and ARID1A was confirmed. In addition, the fact that miR-135b-5p promoted TSCC development via mediating ARID1A was demonstrated by functional rescue experiment.
Results: miR-135b-5p was upregulated in TSCC tissues and cells, while ARID1A was suppressed (p< 0.05). Silenced miR-135b-5p discouraged TSCC cell proliferation, improved apoptosis, induced cell cycle arrest, and increased ARID1A expression while inactivating the PI3K/AKT axis (p<0.05). Furthermore, knockdown of ARID1A reversed the impacts on TSCC cell proliferation and apoptosis exerted by silencing miR-135b-5p.
Conclusion: This research supported that silenced miR-135b-5p impeded TSCC proliferation and apoptosis by promoting ARID1A and inactivating the PI3K/AKT axis, which may provide some indications for TSCC alleviation.
Keywords: apoptosis; ARID1A; ARID1A protein, human; carcinoma, squamous cell; cell line, tumor; cell proliferation; drug resistance, neoplasm; microRNA-135b-5p; microRNAs; PI3K/AKT pathway; neoplasm metastasis; neoplastic stem cells; proliferation; protein binding; tongue; tongue squamous cell carcinoma
Objective: To probe into the influence of miR-21 on the proliferation as well as apoptosis of oral squamous cell carcinoma (OSCC) and its causative role.
Study Design: We adopted microarray for detecting the differentially expressed genes in OSCC tumor tis-sues and paracancerous tissues. We assessed the link of miR-21 expression with tumor size, lymph node metastasis, and tumor differentiation. We employed CCK-8 and EdU assay for detecting the impact of miR-21 inhibitor and miR-21 mimic on Cal-27 cell proliferation, as well as TUNEL and AnnexinV-FITC/PI double staining for detecting miR-21 expression on cell apoptosis. We forecasted the possible target of miR-21 via TargetScan, as well as detected the interaction of miR-21 with PTEN via luciferase reporter experiment. The function of miR-21 expression in PTEN signaling pathway was monitored via western blot. We constructed PTEN overexpression plasmid and conducted rescue experiment to evaluate overexpressed PTEN on miR-21–induced proliferation.
Results: Microarray and RT-qPCR indicated that miR-21 expression increased demonstrably in OSCC. Subsequently, statistical analysis showed that miR-21 expression was plainly correlated with tumor size, lymph node metastasis, tumor differentiation, and smoking history. CCK-8 and EdU method exhibited that miR-21 mimics manifestly promoted Cal-27 cell proliferation, while miR-21 inhibitor blatantly inhibited Cal-27 cell proliferation. TUNEL and V-FITC/PI double staining assay showed that miR-21 inhibitor conspicuously promoted Cal-27 cell apoptosis. CCK-8 and EdU assay exhibited that overexpressed PTEN abolished the pro-proliferation influence of miR-21 mimic. TUNEL and V-FITC/PI experiments pointed out that knocking down PTEN abrogated the pro-apoptosis impact of miR-21 inhibitor.
Conclusion: miR-21 contributes to OSCC cell proliferation via targeting PTEN and inhibits its apoptosis.
Keywords: Akt/PKB signaling pathway; apoptosis; biomarkers, tumor; carcinoma, squamous cell; cell line, tumor; cell proliferation; microRNAs; miR-21; miRNA-21; mouth neoplasms; oral cancer; oral squamous cell carcinoma; proliferation; real time PCR
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Objective: To analyze the sonographic features of different histopathological subtypes of borderline ovarian tumors (BOTs) confirmed by pathology, and to study the ultrasound performances of various types in borderline ovarian tumors.
Study Design: Retrospective analysis was performed on the pathological results and ultrasound projection findings of 129 patients diagnosed as BOTs by ultrasound department of our hospital from January 2012 to November 2019. All patients were confirmed by surgical pathology and scanned consecutively by the investigators using transabdominal or transvaginal ultrasound examination.
Results: Serous borderline tumors (SBOTs) were observed, and the prevalence rate (53%) was significantly higher than that of other subtypes, and the probability of bilateral lesions was higher (40%). The sonogram often showed ultrasound features of papillary neoplasm in the lesion and good internal echo (p<0.05). Mucinous borderline ovarian tumors (MBOTs) were mostly unilateral lesions (86%). The prevalence was second only to SBOTs. Histomorphological examinations were divided into gastrointestinal-type and endocervical-type. Among them, the gastrointestinal type of MBOTs were mostly unilateral, and their incidence was higher than that of endocervical-type of MBOTs. Compared with other pathological subtypes, the gastrointestinal type is more likely to show the sonographic characteristics of huge space occupying in the pelvic and abdominal cavity (mean diameter >10 cm), polycystic, multiple septums, and poor internal echo (p<0.05). The ultrasonographic features of the endocervical-type of MBOTs were similar to those of SBOTs. Compared with gastrointestinal type, the sonographic images showed smaller lesion diameter, less septal or cyst, and more papillary excrescences in the tumor (p<0.05). The borderline clear cell tumor is the intermediate transition between the clear cell adenofibroma and the clear cell carcinoma. The clinical manifestations are diverse and lack specificity. The histology of sonography was mainly solid, and the multiple microcapsules were honeycomb-like. It can also be shown as cystic. Among the 169 patients with BOTs, 20 cases of SBOTs, 17 cases of MBOTs, and 10 cases of other rare subtypes were complicated with other diseases or multiple subtypes. This study did not find significant ultrasonic characteristics were used for distinguish them from other subtypes.
Conclusion: BOTs is a common disease in women during the reproductive period. It is characterized by the development of malignant tumors. Its clinical and pathological subtypes are complex and diverse. It leads many doctors to use the terms “large pelvic mass” and “solid ovarian mass” for diagnosis because of their lack of experience and understanding.
Keywords: adenocarcinoma, mucinous; adenocarcinoma, serous; borderline ovarian tumors; diagnostic imaging; ovarian neoplasms; papillary neoplasms; prognosis; transvaginal ultrasound, ultrasonography
Objective: Ischemia-reperfusion (I/R) leads to reactive oxygen species formation and cell death in kidney tissue with injury and organ transplantation. Simvastatin (SIM) is an antioxidant, anti-inflammatory, and anticoagulant agent. Alterations in I/R-induced acute kidney injury model with SIM treatment were analyzed.
Study Design: Wistar rats (n=28) were grouped into Sham, Ischemia, I/R, and I/R+SIM treated. Left rat kidney renal vessels were clamped for 60 minutes for ischemia, and the I/R group had 6 hours of reperfusion. 10 mg/kg SIM was given orally for 28 days. MDA, GSH, and MPO were analyzed. Kidney tissues were paraffin embedded, and primary antibodies TNF-α and caspase-3 were applied for immunohistochemistry.
Results: In the I/R group, intense inflammatory cell infiltration around the vessels and necrosis in the glomerular structures were observed. In the treated group, proximal and distal tubular cells were found to be close to normal. Immunoexpression of caspase-3 in the ischemia group was positive in degenerative glomeruli. In the treated group, TNF-α expression was negative in the glomerular structures. MDA and MPO levels were significantly increased in ischemia and I/R.
Conclusion: We suggest that SIM treatment improved kidney tissue structure and function in a model of I/R injury.
Keywords: caspase-3; immunohistochemistry; ischemia/reperfusion; kidney; MPO; simvastatin
Objective: The association between telomerase reverse transcriptase (TERT) promoter mutation and outcome of melanoma is unclear and controversial. We aim to conduct a meta-analysis and investigate whether the TERT promoter mutation is a prognostic factor of melanoma.
Study Design: Appropriate studies were searched in 3 databases: PubMed, Web of Science, and Embase. Pooled hazard ratios (HRs) were counted through random effects model.
Results: Heterogeneity was moderate in overall survival (OS) (I2=43.7%, p=0.059) and low in disease-free survival (DFS) (I2=0.0%, p=0.587). Sensitivity analysis indicated that the removal of any of the study did not affect the final results. Evidence for publication bias was not found (Begg’s test, p=0.281; Egger’s test, p=0.078). The pooled OS HRs from combined effects analysis was determined (HR 1.07; 95% CI 0.83–1.39, p=0.585), together with the pooled HRs of DFS (HR 1.65; 95% CI 1.02–2.66, p=0.042). TERT promoter mutation predicted a good outcome in meta-static melanoma patients (HR 0.66; 95% CI 0.46–0.96, p=0.042). The pooled HRs of combined mutation in TERT promoter and BRAF (HR 6.27; 95% CI 2.7–14.58, p=0.000) predicted a bad outcome in melanoma patients.
Conclusion: TERT promoter mutation significantly predicted poor DFS outcome but, on the contrary, predicted a good outcome in metastatic melanoma patients. The combined TERT promoter and BRAF mutation was a significant independent factor of OS in melanoma patients.
Keywords: melanoma; meta-analysis; mutation; prognosis; promoter regions, genetic; skin neoplasms; telomerase; TERT promoter mutation; TERT protein, human
Objective: The prognostic indictors of age-related poor outcomes in patients with acute myeloid leukemia (AML) are still controversial. The aim of this work was to provide comprehensive insights into the effect of different hemocytes and to investigate the association between age and clinical features in adult patients with AML.
Study Design: A retrospective study was performed to determine the role of age in the therapeutic outcomes of AML. A total of 166 newly diagnosed adult patients’ data from January 2015 to November 2019 in Zhongshan Hospital of Xiamen University were collected and analyzed.
Results: Older patients presented a poorer prognosis (p=0.001) with shorter overall survival, which is served as age-related outcomes. Binary logistic regression demonstrated that cytogenetic risk (OR=4.508, 95% CI 2.733–7.435), leukocyte (OR=7.410, 95% CI 1.139–5.910), and bone marrow blast cells (OR=3.261, 95% CI 1.075–5.615) were independent indictors for age-related prognosis. In addition, Kaplan-Meier curve also revealed that the above factors were associated with overall survival (all p values <0.001).
Conclusion: Cytogenetic risk, leukocyte, and bone marrow blast cells are dominant factors which account for the age-related poor outcomes and shorter overall survival in AML.
Keywords: acute myeloid leukemia, adult, cytogenetic risk, hemocyte, leukemia, overall survival
Content Cytotoxicity Studies of Colorectal Carcinoma Cells Using Printed Impe...journalBEEI
Monitoring the effectiveness of drugs on cancer cells is crucial for chemotherapeutics studies. In-vitro cell-based biosensors can be used as an alternative for characteristic studies of cells’ response to drugs. Cell-based sensors provide real-time measurements and require smaller sample volumes compared to conventional T-flask measurement methods. This paper presents a biosensor that detects in real-time, impedance variations of human colon cancer, HCT-116 cells when treated with anti-cancer agent, 5-Fluorouracil (5-FU). Two different extracellular matrix (ECM); polyaniline and gelatin were tested and evaluated in terms of attachment quality. Polyaniline was found to provide the best attachment for HCT-116 cells and was used for cytotoxicity studies. Cytokinetic behavior indicated that 5-FU inhibited HCT-116 cells at IC50 of 6.8 µg/mL. Trypan blue exclusion method for testing cell viability was used to validate the impedance measurements, where the cancer cell concentrations were reduced to ~35% when treated with 2.5 µg/mL, and 50% when treated with 6.8 µg/mL. The results generated by the microfabricated impedance biosensor are comparable to the Trypan blue method since both gave similar cell growth trend. It can be concluded that the impedance biosensor has potential to be used as an alternative method in drug testing applications.
Molecular Detection and Therapeutic Management of Feline MycoplasmosisIOSRJAVS
Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum (formerly known as Haemobartonellafelis) are the causes of hemotropic mycoplasmosis in cats. The parasites attach to the surface of the red blood cell, and have the potential to cause severe alterations of the cell’s shape, resulting in anaemia. A three-year-old tom cat was presented in University Veterinary Hospital with symptoms of lethargy, reduced appetite and fever for past 3 days. Clinical examination revealed increased temperature(103º – 105º F), blanched mucous membranes and lymphadenopathy. Upon peripheral blood smear examination small coccoid organisms could be noticed in the periphery of the RBCs. Hematobiochemical examination revealed anaemia, thrombocytopaenia and decreased haematocrit values. The blood samples were subjected to DNA extraction and followed by Polymerase Chain Reaction which confirmed mycoplasmosis due to Mycoplasma haemofelis. The animal was treated with intravenous administration of oxytetracycline@ 10mg/kg BW for five days along with prednisolone and vitamin supplements. Uneventful clinical recovery was noticed 7 days post therapy.
POLYMORPHISMS OF GLUTATHIONE S-TRANSFERASE M1 AND T1: GENETIC RISK FACTOR FOR...VR Foundation
Introduction: vitiligo is the result of the interaction of genetic, biochemical, environmental and immunological factors. Oxidative stress has a significant pathogenic role in vitiligo, and is normally contrasted by various non-enzymatic and enzymatic antioxidant systems, including glutathione S-transferases (GSTs). The “null” allele (which causes the lack of production of the corresponding protein) of GST isoforms M1 and T1 may be found, with inter-ethnically variable frequency, in a relevant part of the general population, and is associated to various oxidative-stress related diseases.
Aims & Scope: to define the prevalence of GSTM1 and GSTT1 null polymorphisms in vitiligo patients and healthy controls from Sicily and Calabria, and the correlation of such genotypes with the disease.
Material and Methods: polymerase chain reaction was performed on buccal swabs of 58 non-segmental vitiligo patients (28 males, 30 females) and 150 healthy controls (71 males, 79 females) to define their GSTM1 and GSTT1 genotype (null/active).
Results: the GSTM1/GSTT1 double-null genotype is significantly more frequent in vitiligo patients than in controls (p=0.041), while this is not true for either single-null genotype. Neither the GSTM1 genotype nor the GSTT1 genotype are correlated to the disease.
Comments: the two other papers on GSTM1/GSTT1 genotype and vitiligo agree with our data for which concerns the association of the double-null genotype with a significantly increased risk for the disease, while they differ about disease correlation with either GST null genotype (GSTM1 in one paper, GSTT1 in another, none of the above in our casuistic). Discrepancies are probably due to a different “genetic setup” of the populations studied, which determines a different relative importance of GSTM1 and GSTT1 in the global balance of the antioxidant system. Thus, possession of the null allele for one GST isoform can variably increase the risk for vitiligo, depending on the relative importance of that isoform, while the reduction of the antioxidant potential due to the simultaneous lack of both GST isoforms (double null genotype) is significant in any configuration of the antioxidant system and always constitutes a risk factor for the disease.
Oxidative stress and antioxidant response could be linked to the dysregulated response observed in vitiligo. As suggested in a recent paper, cultured keratinocytes derived from vitiligo patients have higher levels of reactive oxygen species (ROS) and a characteristic dysregulation of the cytokine, chemokine and growth factor pattern.
Future perspectives include :
- GST genotyping of other populations, ethnically different from those currently described in literature, because of the high variability of the frequency of GST - Disclaimer-
This PPT is loaded as student material "as is", from the VRF Vitiligo Master Class Barcelona November 2011; VRF does not endorse or otherwise approve it.
Paludisme grave : pourquoi doit-on développer des modèles in vitro sur le terrain ? - Conférence de la 8e édition du Cours international « Atelier Paludisme » - RAZAKANDRAINIBE Romy - Madagascar - romy@pasteur.mg
Content Cytotoxicity Studies of Colorectal Carcinoma Cells Using Printed Impe...journalBEEI
Monitoring the effectiveness of drugs on cancer cells is crucial for chemotherapeutics studies. In-vitro cell-based biosensors can be used as an alternative for characteristic studies of cells’ response to drugs. Cell-based sensors provide real-time measurements and require smaller sample volumes compared to conventional T-flask measurement methods. This paper presents a biosensor that detects in real-time, impedance variations of human colon cancer, HCT-116 cells when treated with anti-cancer agent, 5-Fluorouracil (5-FU). Two different extracellular matrix (ECM); polyaniline and gelatin were tested and evaluated in terms of attachment quality. Polyaniline was found to provide the best attachment for HCT-116 cells and was used for cytotoxicity studies. Cytokinetic behavior indicated that 5-FU inhibited HCT-116 cells at IC50 of 6.8 µg/mL. Trypan blue exclusion method for testing cell viability was used to validate the impedance measurements, where the cancer cell concentrations were reduced to ~35% when treated with 2.5 µg/mL, and 50% when treated with 6.8 µg/mL. The results generated by the microfabricated impedance biosensor are comparable to the Trypan blue method since both gave similar cell growth trend. It can be concluded that the impedance biosensor has potential to be used as an alternative method in drug testing applications.
Molecular Detection and Therapeutic Management of Feline MycoplasmosisIOSRJAVS
Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum (formerly known as Haemobartonellafelis) are the causes of hemotropic mycoplasmosis in cats. The parasites attach to the surface of the red blood cell, and have the potential to cause severe alterations of the cell’s shape, resulting in anaemia. A three-year-old tom cat was presented in University Veterinary Hospital with symptoms of lethargy, reduced appetite and fever for past 3 days. Clinical examination revealed increased temperature(103º – 105º F), blanched mucous membranes and lymphadenopathy. Upon peripheral blood smear examination small coccoid organisms could be noticed in the periphery of the RBCs. Hematobiochemical examination revealed anaemia, thrombocytopaenia and decreased haematocrit values. The blood samples were subjected to DNA extraction and followed by Polymerase Chain Reaction which confirmed mycoplasmosis due to Mycoplasma haemofelis. The animal was treated with intravenous administration of oxytetracycline@ 10mg/kg BW for five days along with prednisolone and vitamin supplements. Uneventful clinical recovery was noticed 7 days post therapy.
POLYMORPHISMS OF GLUTATHIONE S-TRANSFERASE M1 AND T1: GENETIC RISK FACTOR FOR...VR Foundation
Introduction: vitiligo is the result of the interaction of genetic, biochemical, environmental and immunological factors. Oxidative stress has a significant pathogenic role in vitiligo, and is normally contrasted by various non-enzymatic and enzymatic antioxidant systems, including glutathione S-transferases (GSTs). The “null” allele (which causes the lack of production of the corresponding protein) of GST isoforms M1 and T1 may be found, with inter-ethnically variable frequency, in a relevant part of the general population, and is associated to various oxidative-stress related diseases.
Aims & Scope: to define the prevalence of GSTM1 and GSTT1 null polymorphisms in vitiligo patients and healthy controls from Sicily and Calabria, and the correlation of such genotypes with the disease.
Material and Methods: polymerase chain reaction was performed on buccal swabs of 58 non-segmental vitiligo patients (28 males, 30 females) and 150 healthy controls (71 males, 79 females) to define their GSTM1 and GSTT1 genotype (null/active).
Results: the GSTM1/GSTT1 double-null genotype is significantly more frequent in vitiligo patients than in controls (p=0.041), while this is not true for either single-null genotype. Neither the GSTM1 genotype nor the GSTT1 genotype are correlated to the disease.
Comments: the two other papers on GSTM1/GSTT1 genotype and vitiligo agree with our data for which concerns the association of the double-null genotype with a significantly increased risk for the disease, while they differ about disease correlation with either GST null genotype (GSTM1 in one paper, GSTT1 in another, none of the above in our casuistic). Discrepancies are probably due to a different “genetic setup” of the populations studied, which determines a different relative importance of GSTM1 and GSTT1 in the global balance of the antioxidant system. Thus, possession of the null allele for one GST isoform can variably increase the risk for vitiligo, depending on the relative importance of that isoform, while the reduction of the antioxidant potential due to the simultaneous lack of both GST isoforms (double null genotype) is significant in any configuration of the antioxidant system and always constitutes a risk factor for the disease.
Oxidative stress and antioxidant response could be linked to the dysregulated response observed in vitiligo. As suggested in a recent paper, cultured keratinocytes derived from vitiligo patients have higher levels of reactive oxygen species (ROS) and a characteristic dysregulation of the cytokine, chemokine and growth factor pattern.
Future perspectives include :
- GST genotyping of other populations, ethnically different from those currently described in literature, because of the high variability of the frequency of GST - Disclaimer-
This PPT is loaded as student material "as is", from the VRF Vitiligo Master Class Barcelona November 2011; VRF does not endorse or otherwise approve it.
Paludisme grave : pourquoi doit-on développer des modèles in vitro sur le terrain ? - Conférence de la 8e édition du Cours international « Atelier Paludisme » - RAZAKANDRAINIBE Romy - Madagascar - romy@pasteur.mg
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An immunohistochemical analysis of Canine Haemangioma and Haemangiosarcoma
1. An Immunohistochemical Analysis of Canine
Haemangioma and Haemangiosarcoma
S. Sabattini and G. Bettini
Department of Veterinary Public Health and Animal Pathology, Faculty of Veterinary Medicine, Alma Mater Studiorum,
University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy
Summary
The aim of the present study was to investigate immunohistochemically aspects of the biology of canine endo-
thelial neoplasia. Forty samples of canine cutaneous and visceral haemangiosarcoma (HSA), 29 samples of
cutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelled
with antisera specific for vimentin, smooth muscle actin, von Willebrand factor (vWF), CD117 (KIT), vascu-
lar endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGFC) and
CD44. Further antisera were employed to determine the level of cellular proliferation (MIB-1 index) and to-
luidine blue staining was used to detect populations of tumour-infiltrating mast cells (MCs). There was greater
expression of CD117, VEGFR-3 and CD44 in HSA than in HA, suggesting that these proteins might be suit-
able targets for the future development of novel therapeutic approaches to canine HSA. Marked infiltration of
MC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neo-
plasia in the dog.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: dog; haemangioma; haemangiosarcoma; immunohistochemistry; mast cells
Introduction
Haemangioma (HA) and haemangiosarcoma (HSA)
are benign and malignant neoplasms of vascular en-
dothelial cells (EC) that are common in the dog.
HA generally arises in the skin, with a particular pre-
dilection for dogs with short hair coats and lightly pig-
mented skin (Hargis et al., 1992). HSA most often
arises in the spleen, right atrium or skin (Pearson
and Head, 1976). Canine HSA is locally infiltrative
and readily metastasizes, particularly to the lung
and liver (Oksanen, 1978; Brown et al., 1985). Most
affected dogs die from acute internal haemorrhage
secondary to rupture of the tumour. Despite surgical
and chemotherapeutic management, the median sur-
vival time for dogs diagnosed with HSA is little more
than 6 months (Hammer et al., 1991; Clifford et al.,
2000; Sorenmo et al., 2000).
Canine HSA resembles human angiosarcoma, a tu-
mour that also carries an unfavourable prognosis
(Timaran et al., 2000; Budd, 2002). For both tumours
there have been relatively few studies of the cellular
and molecular features, which means that there are
few markers that can be applied to the detection of
early lesions or identification of potential targets for
the development of novel immunotherapeutic ap-
proaches (Hoover et al., 1993; Masuzawa et al.,
1999; Krump-Konvalinkova et al., 2003). Previous
studies have described the expression of von Wille-
brand factor (vWF) (von Beust et al., 1988) and
CD31 (Ferrer et al., 1995) by canine HSA. More re-
cently, the expression of CD117 (Fosmire et al.,
2004), cyclooxygenase-2 (Heller et al., 2005), vascular
endothelial growth factor-A and its receptors, basic fi-
broblastic growth factor and its receptor (Yonemaru
et al., 2006), bcl-2 and survivin (Murakami et al.,
2008) has also been investigated. The aim of the pres-
ent study was to examine the expression of an ex-
panded panel of immunohistochemical markers in
canine HA and HSA in order to further define the bi-
ological characteristics of these tumours.
Materials and Methods
Cases of HA (n ¼ 29) and HSA (n ¼ 40) were selected
from the archives of the Pathology Unit of theCorrespondence to: G. Bettini (e-mail: giuliano.bettini@unibo.it).
0021-9975/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcpa.2008.10.006
J. Comp. Path. 2009, Vol. 140, 158e168 Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
2. Department of Veterinary Public Health and Animal
Pathology, Faculty of Veterinary Medicine, Univer-
sity of Bologna, Italy. These samples had been sub-
mitted over a 10 year period (1998e2008).
Additionally, 10 samples of cutaneous granulation tis-
sue (GT) were selected for comparative analysis.
Samples had been fixed in 10% neutral buffered for-
malin, embedded in paraffin wax, sectioned (4 mm)
and stained with haematoxylin and eosin (HE).
HSAs (29 visceral and 11 cutaneous) were graded
for overall differentiation and nuclear pleomorphism
as described in Table 1, whereas HAs (4 visceral
and 25 cutaneous) were classified by dominant histo-
logical pattern as cavernous, capillary or mixed. The
level of angiogenesis was evaluated in the samples of
GT and scored as mild, moderate or marked accord-
ing to the number and thickness of the wall of newly
formed blood vessels and the immaturity of the lining
endothelial cells.
A tissue array technique was employed in order to
limit the duration of the experiment, reduce reagent
waste and minimize tissue damage. The most repre-
sentative areas in each section were defined by study
of the HE-stained sections, and then 3 mm tissue cores
were punched from the associated tissue blocks. These
cores were transferred to a recipient block with pre-
formed holes to form a composite block containing
up to 20 samples in the same section (Nocito et al.,
2001; Hidalgo et al., 2003). Replicate sections
(4 mm) of tissue arrays were stained with HE and to-
luidine blue and processed for immunohistochemistry
(IHC). The panel of antibodies used in IHC is de-
scribed in Table 2.
IHC was performed with a streptavidin-biotin-per-
oxidase technique. Slides were initially incubated
with hydrogen peroxide 0.3% in methanol for
20 min to block endogenous peroxidase activity and
were then microwaved in citrate buffer (pH 6.0), for
two cycles of 5 min, in a 750 Watt microwave oven
for antigen retrieval (for all antibodies except that
specific for VEGFC). The sections were incubated
overnight at 4
C in a humid chamber with the pri-
mary antibody diluted in phosphate-buffered saline
(PBS; pH 7.4, 0.01 M). Following washing in PBS,
sections were then incubated with secondary biotiny-
lated anti-rabbit IgG, anti-mouse IgG and anti-goat
IgG (LSAB, Dako Cytomation, Glostrup, Denmark)
for 30 min at room temperature, and then subse-
quently with streptavidin-peroxidase complex for
25 min at room temperature. After incubation in
DAB chromogenic substrate solution (diaminobenzi-
dine 0.02% and H2O2 0.001% in PBS) for 12 min,
sections were immediately rinsed in PBS and in run-
ning tap water, counterstained with haematoxylin,
dehydrated and mounted under DPX (Fluka, Rie-
del-de Hae¨ n, Germany). Appropriate positive con-
trols were used throughout to assess the specificity of
the reactions. As negative control, an isotype-
matched antibody of irrelevant specificity (Neo-
Markers, Fremont, CA) was used in place of the
primary antibody.
The intensity of immunolabelling for each marker
was graded as absent (0), weak (1), moderate (2) or
strong (3). Cellular proliferation was assessed using
the MIB-1 antibody, which recognizes the Ki67 anti-
gen, a nuclear protein expressed in all of the active
phases of the cell cycle (G1, S, G2, M), but not in rest-
ing cells (G0). The labelling index (MIB-1 index) was
subjectively estimated as the percentage of positively
labelled nuclei and scored as 0 (less than 5% of nuclei
labelled), 1 (5e15% of nuclei labelled), 2 (15e30%
of nuclei labelled) or 3 (more than 30% of nuclei la-
belled). The number of tumour-infiltrating mast cells
(MCs) was graded in toluidine blue-stained sections
as 0 (no mast cells), 1 (occasional mast cells), 2 (mod-
erate number of scattered MCs or clusters of at least
three cells) or 3 (MCs present in a high number). Cor-
relations between considered variables were evalu-
ated by Spearman’s rank analysis and Kruskale
Wallis nonparametric analysis of variance.
Results
Haemangiosarcomas
The mean age of dogs with HSA was 9.3 years
(n ¼ 40, range 3e15 years). Breed was recorded in
37 cases and there were 12 German shepherd dogs.
The ratio of male to female dogs was 2.1:1 with
67.6% of dogs with HSA being male. It was not pos-
sible to assess accurately the proportion of animals
that had been neutered.
Table 1
Histological scoring system for canine HSA
Score Overall
differentiation
Nuclear variation
1 Well-differentiated tumour
with numerous irregular
vascular channels
Minimal variation in nuclear
size and shape
2 Moderately differentiated
neoplasm with at least
50% of the tumour
showing well-defined
vascular channels
Moderate variation in
nuclear size and shape
3 Poorly differentiated,
solid tumour with few
vascular channels
Marked variation in nuclear
size and shape. Nuclear
size often differs by
twofold or more between
tumour cells
Immunohistochemistry of Canine Vascular Tumours 159
3. Of the 29 visceral tumours, the primary site of ori-
gin could not be determined in 7 cases due to the pres-
ence of multicentric lesions. The remaining 22 cases
had primary origin in the spleen (n ¼ 16), right
atrium (n ¼ 3), liver (n ¼ 1), omentum (n ¼ 1), retro-
peritoneal area and the urinary bladder (n ¼ 1).
Microscopically, the HSA cells had highly heteroge-
neous morphology, ranging from spindle-shaped to po-
lygonal to ovoid, with vasoformative to solid growth
patterns. Eleven of the 40 HSAs (27.5%) were catego-
rized as well-differentiated, with numerous, irregular
vascular channels and large areas of haemorrhage with
few cells, mimicking haematomas (Fig. 1D). Twenty-
four HSAs (60%) were characterized by areas of moder-
ate differentiation, where neoplastic tissue was more
compact but still showing, at least focally, a vasoforma-
tive component. Five samples (12.5%) were poorly dif-
ferentiated and were difficult to distinguish from
fibrosarcoma or other poorly differentiated sarcoma.
These tumours comprised solid sheets of cells with few
vascular channels. Nuclear variation was minimal in
12.5% (n ¼ 5) of specimens, moderate in 40%
(n ¼ 16) and marked in 47.5% (n ¼ 19). There was no
relationship between these different morphological pat-
terns and the anatomical location of the tumour.
The immunohistochemical expression of the panel
of markers by the HSAs is summarized in Table 3.
All HSA tumour cells exhibited strong and diffuse cy-
toplasmic expression of vimentin and actin, whereas
there was more variable expression of vWF, this rang-
ing from weak and inconsistent (n ¼ 15, 60%) to
strong (n ¼ 3, 12%). CD117 expression was detected
in the cytoplasm of 76.3% of HSAs, and the intensity
of expression ranged from low (n ¼ 16, 42.1%), to
moderate (n ¼ 9, 23.7%) to strong (n ¼ 4, 10.5%)
(Fig. 2C, D). Only 7 cases of HSA displayed cytoplas-
mic expression of VEGFC, while 36% (n ¼ 14) of
samples showed mild to intense cytoplasmic expres-
sion of VEGFR-3. Fifteen tumours had cytoplasmic
expression of CD44 with moderate (34.6%) to strong
(23.1%) intensity. Between 5 and 40% of nuclei ex-
pressed MIB-1 and the mean MIB-1 index was
16.7%.
Ten of the HSAs had a low to moderate number of
infiltrating MCs and in one tumour there were nu-
merous MCs. No MCs were detected in the remainder
of the samples (72.5%).
Haemangiomas
The mean age of dogs with HA was 9.3 years (range
4e15 years). There were almost equal numbers of
males and females and the German shepherd dog
was the most represented breed (n ¼ 8, 27.6%).
Twenty-four HAs arose within the skin and five
were splenic in origin.
Microscopically, most of the samples were of the
cavernous subtype, showing large, regular and well
defined vascular spaces filled with erythrocytes, com-
pletely enclosed by a single layer of endothelial cells
aligned on thin collagenous septa (Fig. 1B). Five spec-
imens (17.2%) were mixed capillary-cavernous in
which, focally, the vascular structures were smaller
and lined by slightly plump endothelial cells protrud-
ing into vessel lumens (Fig. 1C). Cellularity and nu-
clear pleomorphism in these areas were usually
higher than in cavernous HA.
The immunohistochemical expression of the panel
of markers by HAs is summarized in Table 3. Neo-
plastic cells were strongly labelled by antibodies spe-
cific for vimentin and actin in all samples. Low to
moderate expression of vWF and VEGFR-3 was con-
sistently detected, whilst CD44, CD117 and VEGFC
were not expressed in the majority of samples
(Fig. 2B). Conversely, there was strong cytoplasmic
labelling for CD117 and VEGFC in the areas of cap-
illary differentiation of two mixed HAs. MIB-1 ex-
pression was detected in only occasional endothelial
nuclei and the MIB-1 index was less than 5% in all
cases.
Infiltrating MCs were seen in the stromal compart-
ment of all HAs with the single exception of one
splenic HA in which no MCs were detected. The
Table 2
Primary antibodies used in IHC
Marker Type of antibody Source Dilution
Vimentin Mouse monoclonal Dako, Glostrup, Denmark 1 in 100
Actin Mouse monoclonal Dako 1 in 100
von Willebrand factor Rabbit polyclonal Dako 1 in 1500
CD117 Rabbit polyclonal Dako 1 in 100
VEGFR-3 Rabbit polyclonal Alpha Diagnostic International, San Antonio, USA 1 in 350
VEGFC Rabbit polyclonal Zymed laboratories Inc., San Francisco, USA 1 in 40
CD44 Mouse monoclonal Bender MedSystems, Vienna, Austria 1 in 12.5
MIB-1 Mouse monoclonal Dako 1 in 30
VEGFR-3, vascular endothelial growth factor receptor-3; VEGFC, vascular endothelial growth factor-C.
160 S. Sabattini and G. Bettini
4. number of MCs ranged from low (n ¼ 8, 33.3%) to
moderate (n ¼ 9, 37.5%) to high (n ¼ 6, 25%)
(Fig. 3AeF).
Granulation Tissue
Five of ten samples of GT had evidence of fibroplasia
and intense angiogenesis; three had fibrosis and mild
angiogenesis, one had non-recent angiogenesis and
one had low angiogenic activity (Fig. 1A). The immu-
nohistochemical expression of the panel of markers by
GT is summarized in Table 3. In all cases endothelial
cells expressed vimentin, actin and vWF but did not
label for VEGFC. Reactive endothelial cells had
mild expression of CD44 and VEGFR-3 and low,
yet consistent, expression of CD117 (Fig. 2A). Con-
versely, in the sample with a low level of angiogenesis,
the ECs did not express CD44, VEGFR-3 or CD117.
Five to 40% of nuclei were MIB-1-positive with
a mean MIB-1 index of 11.4%. A low to moderate
number of infiltrating MCs was detected in 50% of
samples.
Statistical Analysis
In HSA there was significant correlation (Spearman’s
method) between overall differentiation and the de-
gree of nuclear variation (P ¼ 0.0042), but there
was no significant correlation between MIB-1 index
and nuclear variation, between MIB-1 index and
overall differentiation, or between MIB-1 index and
CD117 expression.
There was significantly greater expression of
CD117 by HSA (average score 1.2) than HA (average
score 0.1) (P ¼ 0.0000) and GT (average score 0.9)
(P ¼ 0.037). The same trend was noted for CD44,
with a significantly greater expression in HSA (aver-
age score 1.7) than in HA (average score 0.1)
Fig. 1. Microscopical features of canine vascular lesions. (A) Granulation tissue showing fibroplasia and endothelial cells of an immature
phenotype engaged in angiogenesis. HE. Bar, 50 mm. (B) Cavernous haemangioma showing large, uniform blood-filled vascular
spaces lined by a single layer of endothelial cells aligned on thin collagenous septa. HE. Bar, 100 mm. (C) Haemangioma with cap-
illary differentiation showing narrow and irregular vascular structures lined by slightly plump endothelial cells with minimal stro-
mal interposition. HE. Bar, 50 mm. (D) Well-differentiated haemangiosarcoma showing plump endothelial cells aligned on delicate
collagen trabeculae creating an anastomosing meshwork of blood-filled channels of varying size. HE. Bar, 50 mm.
Immunohistochemistry of Canine Vascular Tumours 161
5. Table 3
Summary of immunohistochemical labelling of canine vascular tumours and granulation tissue
Marker Hemangiosarcoma Mean score Hemangioma Mean score Granulation tissue Mean score
Number positive
(% total positive)
Number positive
(% total positive)
Number positive
(% total positive)
0 1 2 3 0 1 2 3 0 1 2 3
Vimentin 0/39 (0%) 0/39 (0%) 17/39
(43.6%)
22/39
(56.4%)
2.5 0/29 (0%) 0/29 (0%) 16/29
(55.2%)
13/29
(44.8%)
2.4 0/10 (0%) 0/10 (0%) 3/10
(30%)
7/10
(70%)
2.7
Actin 1/37
(2.7%)
11/37
(29.7%)
11/37
(29.7%)
14/37
(37.8%)
2.6 0/29 (0%) 4/29
(13.8%)
18/29
(62.1%)
7/29
(24.1%)
2.1 0/10 (0%) 1/10
(10%)
2/10
(20%)
7/10
(70%)
2.6
vWF 3/25
(12%)
15/25
(60%)
4/25
(16%)
3/25
(12%)
1.3 22/25
(88%)
3/25
(12%)
0/25 (0%) 0/25 (0%) 1.1 0/10 (0%) 2/10
(20%)
5/10
(50%)
3/10
(30%)
2.1
CD117 9/38
(23.7%)
16/38
(42.1%)
9/38
(23.7%)
4/38
(10.5%)
1.2 27/29
(92.1%)
2/29
(6.9%)
0/29 (0%) 0/29 (0%) 0.1 2/10
(20%)
7/10
(70%)
1/10
(10%)
0/10 (0%) 0.9
VEGFR-3 10/39
(25.6%)
15/39
(38.5%)
12/39
(30.8%)
2/39
(5.1%)
1.6 2/29
(6.9%)
27/29
(93.1%)
0/29 (0%) 0/29 (0%) 0.9 1/10
(10%)
4/10
(40%)
5/10
(50%)
0/10 (0%) 1.4
VEGFC 33/40
(82.5%)
4/40
(10%)
2/40 (5%) 1/40
(2.5%)
0.3 27/29
(93.1%)
0/29 (0%) 0/29 (0%) 2/29
(6.9%)
0.2 10/10
(100%)
0/10 (0%) 0/10 (0%) 0/10 (0%) 0
CD44 4/26
(15.4%)
7/26
(26.9%)
9/26
(34.6%)
6/26
(23.1%)
1.7 25/28
(89.3%)
3/28
(10.7%)
0/28 (0%) 0/28 (0%) 0.1 1/10
(10%)
5/10
(50%)
3/10
(30%)
1/10
(10%)
1.4
MIB-1 12/40
(30%)
9/40
(22.5%)
13/40
(32.5%)
6/40
(15%)
16.7% 29/29
(100%)
0/29 (0%) 0/29 (0%) 0/29 (0%) 5% 3/10
(30%)
5/10
(50%)
2/10
(20%)
0/10 (0%) 11.4%
Mast cells 29/40
(72.5%)
5/40
(12.5%)
5/40
(12.5%)
1/40
(2.5%)
0.4 1/24
(4.2%)
8/24
(33.3%)
9/24
(37.5%)
6/24
(25%)
1.9 5/10
(50%)
3/10
(30%)
2/10
(20%)
0/10 (0%) 0.7
vWF, von Willebrand Factor; VEGFR-3, vascular endothelial growth factor receptor-3; VEGFC, vascular endothelial growth factor-C; MIB-1 mean score is the MIB-1 index expressed as a percentage.
For each lesion type (haemangiosarcoma, haemangioma, granulation tissue) the number of samples (and % of total) expressing a particular marker with intensity 0e3 is given. MIB-1 expression and
the number of mast cells are also assigned to a four point scale.
162S.SabattiniandG.Bettini
6. (P ¼ 0.0000), although there was no significant differ-
ence between HSA and granulation tissue (average
score 1.4).
Cellular proliferation was significantly lower
(P ¼ 0.0000) in HA (mean MIB-1 index 5%)
than in HSA (mean MIB-1 index 16.7%), whilst
the difference between HSA and GT (mean MIB-1
index 11.4%) was not significant (P ¼ 0.3).
MCs were more prominent in HA (average score 1.9)
than in HSA (average score 0.4) (P ¼ 0.0001), but the
number of mast cells was not related to the anatomical
location of the tumour (i.e. cutaneous or visceral).
Discussion
The distribution of age, breed and sex in this population
of 69 dogs with vascular tumours is consistent with that
described previously (Prymak et al., 1988; Srebernik
and Appleby, 1991). Dogs with both HA and HSA
hadameanageofover9yearsandtheGermanshepherd
dog was the breed most represented for both tumour
types.Althoughnocleargenderpredilectionhasbeenre-
portedforcanineHSA,inourstudy,asinothers(Sreber-
nik and Appleby, 1991; Bettini et al., 2001), males were
overrepresented. Nevertheless, these findings should be
interpreted cautiously due to the relatively small size of
the population examined.
There is continued controversy as to whether
multi-organ involvement in canine HSA represents
true multicentric origin or reflects the development
of one primary tumour with metastasis (Waters
et al., 1988; Ward et al., 1994; Goldschmidt and Hen-
drick, 2002). In 26.9% of the cases in the present se-
ries, two or more sites were involved in the same
animal and, based on knowledge of the common
metastatic patterns in sarcomas, neither would be
considered as a likely metastatic site. However, we
found no evidence that this subset of cases had dis-
tinct morphological or immunohistochemical
features.
Fig. 2. CD117 expression in canine vascular lesions. (A) Immature ECs in active GT showing consistent cytoplasmic labelling. (B) CD117-
negative HA with infiltrating MCs that exhibit strong labelling. (C) HSA cells with moderate expression of CD117. (D) Subcuta-
neous HSA with strong cytoplasmic immunoreactivity for CD117. IHC. Bars, 100 mm.
Immunohistochemistry of Canine Vascular Tumours 163
7. Unlike for other canine tumour types, a histological
grading system has not been widely applied to HSA.
In the present study, overall differentiation and nu-
clear variation were scored for each sample in order
to define the correlation between morphological fea-
tures and proliferative activity or immunophenotypic
characteristics. Other parameters tested in previous
studies were not estimated (Ogilvie et al., 1996). These
have included parameters such as number of mitoses
and amount of necrosis; however, we believe that pro-
liferative activity is more accurately assessed by eval-
uating the expression of the nuclear antigen Ki67, and
Fig. 3. Cutaneous haemangiomas showing interstitial infiltration of mast cells at low (A and B), moderate (C and D) and high (E and F)
number. Toluidine blue. Bars in A, C and E 200 mm, Bars in B, D and F 50 mm.
164 S. Sabattini and G. Bettini
8. the presence of necrosis is entirely dependent on the
sampling site. A positive correlation was found be-
tween differentiation and nuclear variation, whilst
there was no correlation between these parameters
and MIB-1 index or the expression of immunohisto-
chemical markers, with the exception of lower vWF
expression and higher VEGFC expression in poorly
differentiated HSA.
Previous immunohistochemical studies of canine
HSA have been restricted to the use of endothelial
cell-specific markers such as vWF and CD31 to distin-
guish poorly differentiated examples from other mes-
enchymal tumours. In the present study we applied
an extended panel of antibodies in order to test their
diagnostic utility and contribute to the understanding
of the biological characteristics of these tumours.
Vimentin is an intermediate filament protein that is
part of the cytoskeleton of mesenchymal cells and was
widely expressed in the cytoplasm of both neoplastic
and non-neoplastic ECs. Immunohistochemical la-
belling for vimentin could therefore be of value in
the diagnosis of atypical variants of these tumours
such as epithelioid HSA.
The strong expression of smooth muscle actin by the
endothelial cells from all of the vascular lesions studied
here reflects the contractile ability of these cells. There
was, however, no significant difference in expression be-
tween GT, HA and HSA, suggesting that actin expres-
sion holds no prognostic value for this group of lesions.
Similar to earlier observations (Ferrer et al., 1995),
all HA and GT samples were consistently positive for
the vascular marker vWF, whilst labelling was often
focal and weak in malignant ECs. This pattern of ex-
pression limits the utility of this antibody in the diag-
nosis of HSA, especially where the tumours are poorly
differentiated.
C-kit proto-oncogene product (KIT, CD117)is a ty-
rosine kinase growth factor receptor for stem cell fac-
tor (SCF, mast cell growth factor) involved in the
development and maintenance of haematopoietic
stem cells, mast cells, germ cells, melanocytes and in-
terstitial cells of Cajal. Mutations in the tyrosine ki-
nase or juxtamembrane domains of the c-kit gene
have been detected in mastocytoma, seminoma and
gastrointestinal stromal tumours (Miettinen et al.,
2000). Greater than half of human angiosarcomas ex-
press CD117, but KIT was not detected in the major-
ity of benign vascular tumours. KIT-immunoreactive
HSAs do not have mutations of c-kit, so such positivity
is more likely related to an immature phenotype of the
neoplastic cells (Miettinen et al., 2000). CD117 ex-
pression has also been reported in 7 canine splenic
HSAs (Fosmire et al., 2004).
These findings are consistent with those of the pres-
ent study in which there was low to intense cytoplas-
mic expression of CD117 in most HSAs. In contrast,
the benign vascular tumours were consistently
CD117-negative, except for two HAs with mixed cap-
illary-cavernous pattern. These observations may be
taken to support the theory whereby canine HSA
and human angiosarcoma originate from primitive,
poorly differentiated endothelial cells identified by
the expression of CD117 and other surface proteins re-
stricted to bone marrow precursor cells (EPC),
whereas HA would originate from mature, well differ-
entiated endothelial cells. This may provide a theoret-
ical explanation for multicentric involvement and
represents not only a major diagnostic advancement,
but also an appealing target for the development of
novel therapy based on kinase-inhibitors (Fosmire
et al., 2004; Lamerato-Kozicki et al., 2006). CD117
was also expressed, albeit weakly, by endothelial cells
from all sections of granulation tissue, except for the
sample with a low level of angiogenesis. This finding
is consistent with the immature phenotype of angio-
blasts, and precludes the use of KIT as a marker to
distinguish HSA from GT.
The VEGFR family comprises a group of three ty-
rosine kinase receptors for vascular endothelial
growth factors. VEGFR-3 (the receptor for VEGFC
and VEGFD) has been found in most endothelia dur-
ing embryogenesis, whilst later in development ex-
pression of this molecule is restricted to lymphatic
endothelium in most tissues. Recently, VEGFR-3 ex-
pression has been shown to be up-regulated in the en-
dothelial cells of areas of tumour neovascularization,
and this molecule is expressed by human vascular
neoplasms (Lymboussaki et al., 1999; Partanen et al.,
1999; Neuhauser et al., 2000; Laakkonen et al., 2007;
Petrova et al., 2008). This pattern of expression sug-
gests that antagonists or inhibitors of this protein
may be promising candidates for use in molecular-tar-
geted therapies.
In the present study, the majority of canineHSAs ex-
pressed VEGFR-3, suggesting that this marker would
not be suitable for the immunohistochemical discrimi-
nation between lymphangiosarcoma and HSA (Neu-
hauser et al., 2000). In contrast, expression of the
VEGFR-3 ligand (VEGFC) was limited to foci within
two HAs with capillary differentiation and seven
poorly differentiated HSAs. The greater expression of
VEGFR-3 in HSA compared with HA suggests that
HSA may be more influenced by VEGFC. As VEGFC
was not over-expressed in HSA, other growth promo-
tion mechanisms rather than autocrine may be impor-
tant in this tumour. No correlation was found between
VEGFR-3 expression and cell proliferation activity.
CD44 is a ubiquitous multi-structural and multi-
functional cell surface adhesion molecule involved in
cell-to-cell and cell-to-matrix interactions, cell traffic,
Immunohistochemistry of Canine Vascular Tumours 165
9. lymph node homing, transmission of growth signals
and signals mediating haematopoiesis and apoptosis.
Hyaluronic acid, an important component of the ex-
tracellular matrix (ECM), is the principal ligand for
CD44; others include collagen, fibronectin, laminin
and chondroitin sulphate (Naor et al., 1997; Hidalgo
et al., 2002). Many malignant cell types express high
levels of CD44 and it has been shown in animal models
that injection of reagents interfering with CD44-li-
gand interaction can inhibit local tumour growth
and metastatic spread (Kajita et al., 2001). These ob-
servations suggest that CD44 may confer a growth ad-
vantage on some neoplastic cells and, therefore, that
this molecule may be used as a target for cancer ther-
apy (Naor et al., 1997). The results of the present study
are consistent with these concepts as all HAs failed to
express this marker, whilst more than half of HSAs
were immunoreactive. CD44 was also expressed by
the proliferating ECs of GT, confirming their molecu-
lar similarity with malignant ECs.
Proliferative activity, as determined by the MIB-1 in-
dex, was markedly variable in HSA and not correlated
to differentiation and nuclear pleomorphism, suggesting
thattumourswithsimilarmorphologymayhaveadiffer-
entkineticbehaviour. TheMIB-1indexofHSAsdidnot
differ significantly from that of granulation tissue,
whereas almost no cycling cells were identified in HAs.
During the course of these immunohistochemical
studies, we often identified isolated VEGFC,
VEGFR-3 and CD117-positive cells in the interstitial
stroma of HAs. Morphologically, these were oblong to
round cells with round nuclei that were consistent
with either angioblasts or infiltrating mast cells. In or-
der to further identify these cells, toluidine blue stain-
ing was performed on replicate sections of the tissues
examined. This staining confirmed that these were
mast cells and that these were numerous in the major-
ity of HAs but less commonly found in GT and HSA.
There is evidence that several mast cell mediators
may have angiogenic activity by regulating EC prolif-
eration (e.g. vascular endothelial growth factor, fibro-
blast growth factor, histamine, heparin, interleukin-8,
tumour necrosis factor-a, platelet-derived growth fac-
tor, and hepatocyte growth factor), inducing vasodi-
latation, increasing vascular permeability and
degrading the extracellular matrix (e.g. chymase,
tryptase, matrix metalloproteinases, urokinase, inter-
leukins-3, -4 and -8). It has also been postulated that
MCs may play both pro-angiogenic and anti-angio-
genic roles in the proliferative and involuting phases
of infantile haemangioma (Marks et al., 1986; Yama-
moto et al., 2000; Sun et al., 2007).
Mast cells and their precursors are known to ex-
press the KIT receptor. The ligand for this receptor
is SCF, which acts to promote the proliferation, differ-
entiation, migration and secretory activity of these
cells. Endothelial cells in murine are known to secrete
SCF, which may act to recruit MCs into the neoplas-
tic microenvironment (Meininger et al., 1995). Yama-
moto et al. (2000) detected a significantly increased
number of MCs in the lesions of human angiosarcoma
compared with normal skin, additionally demonstrat-
ing the presence of SCF-positive cells in these neoplas-
tic tissues.
The above findings may be interpreted to suggest
that tumour cell-derived SCF might recruit infiltrat-
ing MCs, which may further contribute to the prolif-
eration and progression of tumour cells (Yamamoto
et al., 2000). The fact that fewer MCs were detected
in canine HSA compared with HA is at odds with
this hypothesis, but could be explained if there were
alternative proliferative mechanisms of an autocrine
nature that meant that canine HSA cells were inde-
pendent of external stimuli (Yonemaru et al., 2006).
In conclusion, the present study has shown that the
panel of markers employed here is able to distinguish be-
nign from malignant vascular tumours, but not discrim-
inate between neoplastic ECs and normal ECs
proliferating in GT. This finding is consistent with the
ontogenetic hypothesis that states that HSA originates
from incompletely differentiated, bone marrow-derived
stemcellsthatarenearoratthestageofendothelialcom-
mitment (haemangioblasts) (Schatteman and Awad,
2004). The significantly greater expression of CD117,
VEGFR-3 and CD44 by HSAs suggests that these mol-
ecules might be further explored as potential targets for
molecular interventional therapy for this tumour. Fi-
nally, the high number of mastcells infiltrating HAs sug-
geststhatthesecellsmighthavearoleinthepathogenesis
of benign vascular tumours.
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½ Received, August 8th, 2008
Accepted, October 31st, 2008 Š
168 S. Sabattini and G. Bettini