Over-expression of hsa-miR-150 induces re-differentiation of diffuse large B-cell lymphoma cell line OCI-Ly10

AIM:To investigate the mechanism that over-expression of hsa-miR-150 induces the re-differentiation of diffuse large B-cell lymphoma cell line OCI-Ly10. METHODS:The expression level of hsa-miR-150 in CD19+B and OCI-Ly10 cell lines was detected by real-time PCR. The expression level of c-Myb was detected by Western blotting and immunofluorescence cytochemistry methods. Lentiviral supernatant containing recombinant plasmids was transfected into OCI-Ly10 cells by LipofectamineTM 2000 and named Ly10-control and Ly10-miR-150. The biological functions of the 2 cell sublines were identified by MTT assay. The cell cycle and apoptotic rates were detected by flow cytometry. The expression levels of B-lymphocyte differentiation-related genes and c-myb in Ly10-control and Ly10-miR-150 cells were detected by real-time PCR and Western blotting. When c-myb was interfered in by interference fragment in OCI-Ly10 cells, the interference efficiency and the expression levels of BCL6 and PRDM1 were detected by real-time PCR and Western blotting. RESULTS:The expression level of hsa-miR-150 in CD19+ B cells was significantly higher than that in OCI-Ly10 cells. The expression level of c-Myb in OCI-Ly10 cells was higher than that in CD19+ B cells. The expression levels of B-lymphocyte differentiation-related genes were changed significantly in OCI-Ly10 cells after transfected with hsa-miR-150. The expression levels of PAX5, BCL6 and c-Myb in Ly10-miR-150 cells were lower than those in Ly10-control cells, but the expression levels of IRF4, PRDM1 and XBP1 were higher than those in Ly10-control cells. The expression level of BCL6 was lower and PRDM1 was higher after interference. CONCLUSION:Hsa-miR-150 plays a significant role in inhibiting proliferation and inducing apoptosis of OCI-Ly10 cells. The mechanism that over-expression of hsa-miR-150 induces OCI-Ly10 cell differentiation toward terminal B cells may be related to the down-regulation of c-myb.     

IMAGING DIAGNOSIS—VERTEBRAL POLYOSTOTIC LYMPHOMA IN A GERIATRIC DOG

A 9‐year‐old spayed female Scottish terrier presented with an 8‐day history of progressive paraparesis. Neurological examination suggested a painful T3‐L3 myelopathy. Multifocal uniform contrast‐enhancing masses involving the vertebral bodies, pedicles, laminae, and spinous processes of two vertebrae and compressing the spinal cord were present on MRI. Fluoroscopic‐guided fine needle aspiration of one of the vertebral lesions revealed a predominantly lymphoblastic population of cells compatible with a diagnosis of lymphoma. To the authors’ knowledge, this represents the first published case of canine lymphoma with vertebral involvement, characterized with MRI.     

VEGF and MMP‐9: biomarkers for canine lymphoma

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF‐β) stimulates VEGF and MMPs production. VEGF and TGF‐β plasma levels were tested by ELISA, MMP‐2 and MMP‐9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act‐MMP‐9 (P < 0.01) and VEGF (P < 0.05), and lower TGF‐β than controls, and a positive correlation between act‐MMP‐9 and VEGF (P < 0.001). Act‐MMP‐9 and VEGF were significantly higher in T‐cell lymphomas, and in stage V compared with stages III–IV disease, regardless of immunophenotype. VEGF was higher in high‐grade compared with low‐grade T‐cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act‐MMP‐9 and VEGF decreased in B‐cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.     

Unicentric extranodal lymphoma of the upper eyelid conjunctiva in a dog

A case of conjunctival lymphoma in a 4-year-old Siberian husky is reported. A large red mass protruding from the conjunctiva of the upper eyelid of the right eye was present. Irritation, blepharospasm and epiphora were revealed on initial ophthalmic examination. After anti-inflammatory treatment, surgery was performed. Histologically, the mass consisted of large polygonal cells with a high nucleus to cytoplasm ratio, moderate amounts of slightly eosinophilic cytoplasm, pleomorphic nuclei with vesicular chromatin and prominent multiple nucleoli. Mitotic figures were frequent. Approximately 70% of the neoplastic cells were CD3 positive and CD79alpha negative. On the basis of histologic and immunohistochemical findings, a diagnosis of intermediate-grade, diffuse large-cell lymphoma with T-cell immunophenotype was made. The surgical area healed uneventfully and, although chemotherapy was not received, after 12 months the dog exhibited no recurrence. To the authors' knowledge, this is the first report of unicentric extranodal conjunctival lymphoma in a dog.     

Mediastinal lymphoma in a young Turkish Angora cat

An 8-month old intact male Turkish Angora cat was referred to the Veterinary Medical Teaching Hospital (VMTH), Seoul National University, for an evaluation of anorexia and severe dyspnea. The thoracic radiographs revealed significant pleural effusion. A cytology evaluation of the pleural fluid strongly suggested a lymphoma containing variable sized lymphocytes with frequent mitotic figures and prominent nucleoli. The feline leukemia virus and feline immunodeficiency virus tests were negative. The cat was euthanized at his owner''s request and a necropsy was performed. A mass was detected on the mediastinum and lung lobes. A histopathology evaluation confirmed the mass to be a lymphoma. Immunohistochemistry revealed the mass to be CD3 positive. In conclusion, the cat was diagnosed as a T-cell mediastinal lymphoma.     

Rapid,effective and user-friendly immunophenotyping of canine lymphoma using a personal flow cytometer

Background

Widespread use of flow cytometry for immunophenotyping in clinical veterinary medicine is limited by cost and requirement for considerable laboratory space, staff time, and expertise. The Guava EasyCyte Plus (Guava Technologies, Hayward, CA, US) is the first, personal, bench-top flow cytometer designed to address these limitations.

Objective

The aim of this study was to adapt the immunohistochemical protocol used for immunophenotyping of canine lymphoma to the personal flow cytometer for rapid, effective and user-friendly application to the diagnosis and prognosis of canine lymphoma and to demonstrate its practicality for widespread veterinary application. Performance of the personal flow cytometer for immunophenotyping T and B lymphocytes in blood and lymph nodes from normal dogs and dogs with lymphoproliferative disease, was assessed using only two monoclonal antibodies (against CD3 and CD21), and by comparison with analysis using two conventional flow cytometers.

Methods

26 dogs with lymphoproliferative disease (23 with lymphoma, 3 with lymphocytic leukaemia) were studied along with 15 controls (2 non-lymphoma lymph nodes and 13 non-leukemic bloods. Lymphocytes were immunostained with fluorescent-labeled, monoclonal antibodies against CD3 and CD21. To assess the effectiveness of the personal flow cytometer in discrimination between T and B cell immunophenotypes, T and B cell counts for half the samples (14 blood and 11 lymph node) were also determined using the same method and conventional flow cytometers (FACSCalibur, Cyan Dako). To assess the effectiveness of the personal flow cytometer in discriminating between leukocyte types, lymphocyte differential counts were determined for 21 blood samples and compared with those from automated hematology analyzers (CELL-DYN 3500, n=11 and ADVIA 2120, n=10). Quality and sub-cellular distribution of immunostaining was assessed using fluorescence microscopy.

Results

The protocol for immunophenotyping took 2 to 3 hours to complete from the point of receipt of sample to reporting of immunophenotype. The personal flow cytometer differential lymphocyte counts correlated highly (n=20; r=0.97, p<0.0001) with those of automated haematology analyzers. The personal flow cytometer counts consistently, but mildly, underestimated the percentages of lymphocytes in the samples (mean bias of -5.3%.). The personal flow cytometer immunophenotype counts were indistinguishable from those of conventional flow cytometers for both peripheral blood samples (n=13; r=0.95; p<0.0001; bias of -1.1%) and lymph node aspirates (n=11,r=0.98; p<0.001; bias of 1%). All but one leukemic and one lymphomatous lymph node sample, out of 26 samples of dogs with lymphoproliferative disease analyzed, could be immunophenotyped as either B or T cells.

Conclusions

We conclude that use of only 2 monoclonal antibodies is sufficient for immunophenotyping most cases of canine lymphoma by flow cytometry and enables rapid immunophenotyping. The personal flow cytometer may be as effectively used for immunophenotyping canine lymphoma as conventional flow cytometers. However, the personal flow cytometer is more accessible and user-friendly, and requires lower sample volumes.     

Effects of IL-6 and AG490 on growth of diffuse large B-cell lymphoma and Burkitt lymphoma transplanted into nude mice

AIM: To observe the effects of interleukin-6 (IL-6) and AG490 on diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) transplanted into nude mice, and to explore the effects of STAT3 activation on growth of these kinds of lymphoma in nude mice and its related mechanisms. METHODS: The nude mouse models with DLBCL and BL were established by transplantation with OCI-LY8 cells and Raji cells, respectively, and were divided into 3 groups:control group, IL-6 group and AG490 group. The body weight of mice and tumor size were measured. Western blot and immunohistochemical staining were used to detect the protein levels of p-STAT3, survivin and vascular endothelial growth factor (VEGF), and real-time PCR was used to detect the mRNA expression of survivin and VEGF. RESULTS: The tumorigenic rate of 2 kinds of tumor cell lines in nude mice was 83.3% (25/30) totally. The tumorigenicity of OCI-LY8 cells (66.7%, 10/15) was significantly lower than that of Raji cells (100%, 15/15) (P<0.05). The tumor size and body weight on days 9 and 10 in IL-6 group increased as compared with the control group, and the total difference value of tumor size between day 1 and day 10 in IL-6 group was obviously larger than that in control group (P<0.05). The positive protein of p-STAT3 was found in the nucleus, while the positive expression of survivin and VEGF was found in the cytoplasm. As compared with control group, the expression of survivin and VEGF was significantly increased (P<0.05), while the protein level of p-STAT3 was not significantly increased in IL-6 group of DLBCL. The protein levels of p-STAT3 and VEGF were significantly decreased (P<0.05), while the expression of survivin did not significantly decreased in AG490 group of DLBCL. The p-STAT3 and VEGF levels significantly increased (P<0.05) in IL-6 group of BL, while the levels of 3 kinds of proteins significantly deceased (P<0.05) in AG490 group of BL, as compared with control group. No statistical difference of mRNA expression of survivin and VEGF among IL-6, AG490 and control groups was observed. CONCLUSION: IL-6 and AG490 affect the growth of DLBCL and BL through activation of STAT3 pathway. The activated STAT3 participates in pathogenesis and progress of DLBCL and BL by up-regulating the expression of survivin and VEGF.     

Cytosine arabinoside in addition to VCAA‐based protocols for the treatment of canine lymphoma with bone marrow involvement: does it make the difference?

Cytosine arabinoside (ara‐C) is a component of many protocols for the treatment of acute leukaemia and non‐Hodgkin lymphomas in humans. The aim of the study was to prospectively evaluate the efficacy of ara‐C in a myeloablative regimen in a cohort of canine lymphomas with bone marrow involvement. Seventeen dogs were enrolled. Eight were treated with a VCAA‐based protocol (Group 1) and nine with the same regimen added with ara‐C (Group 2). Ara‐C was administered on a 5‐day schedule as an i.v. continuous infusion at the dose of 150 mg m^?2 per day for five consecutive days. During treatment complete remission (CR) was achieved in two dogs in Group 1 and in eight dogs in Group 2. CR rate was significantly higher in Group 2 (P < 0.01). Median survival was 72.5 days (range 6–174) in Group 1 and 243 days (range 73–635) in Group 2. Survival was significantly longer in Group 2 (P < 0.001). Both protocols were well tolerated, with a low incidence of adverse events. Ara‐C added to a VCAA‐based protocol appears to be safe and beneficial in dogs with stage V lymphoma. Incorporation of the nucleoside analogue might be crucial for the development of future therapeutic strategies in dogs.     

Immunophenotypic comparison of blood and lymph node from dogs with lymphoma

Peripheral blood and lymph node tissue from 12 dogs with lymphoma was immunophenotyped. Additionally, the bone marrow was immunophenotyped in 6 dogs. The lymphomas were characterized as B-cell in 11 dogs and T-cell in 1 dog. Immunophenotypic patterns in the peripheral blood and bone marrow were variable. The trend in dogs with B-cell lymphoma was normal to increased percentage of IgG-positive cells, decreased percentage of pan-T-positive cells, decreased percentage of CD4-positive cells, and decreased CD4/CD8 ratio. Simultaneous immunophenotyping of lymph node, blood and bone marrow cannot be recommended routinely without further studies to document its value as an independent prognostic indicator. However, it is potentially useful for tumor staging and monitoring remission, especially in lymphoma patients with a leukemic phase.     

Electronic brachytherapy used for the successful treatment of three different types of equine tumours

An electronic form of interstitial brachytherapy was used to treat tumours on 3 different horses. One horse was treated for an ocular lymphoma, the second for a sarcoid, and the third for melanoma. This form of brachytherapy contains inherent advantages over previously used forms of brachytherapy and provides another treatment option for equine tumours that cannot be treated with, or do not respond to, conventional tumour therapies.