Search

Background/aim: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition., Materials and Methods: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model., Results: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects., Conclusion: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Since the introduction of tyrosine kinase inhibitors (TKI), the prospects for patients with chronic myeloid leukemia (CML) have improved significantly. Herein we present the case of a patient with CML who experienced blast crisis and development of acute myeloid leukemia (AML) 10 years after presentation. The CML was characterized by the gene fusion of breakpoint cluster region BCR and tyrosine-protein kinase ABL1. During treatment different therapeutic protocols including imatinib, nilotinib, dasatinib and ponatinib were applied due to development of resistance or non-response. Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) were used to describe cytogenetic and molecular aberrations elucidating the development into AML: A loss of chromosome 7, as well as an arising frequency of variants in the gene met proto-oncogene MET (p.T110I) and tyrosine-protein phosphatase non-receptor type 11 PTPN11 (p.Q510L) was observed. This report describes the comprehensive characterization of a clinical case showing multiple therapeutic resistances correlated with genetic aberrations., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Canine lymphoma has lately drawn focus as a model of human non-Hodgkin's lymphoma due to its spontaneous occurrence and similar biological behavior. Cells with stem cell-like characteristics are believed to play a key role in therapeutic failure. Thus, an initial characterization and the possibility of specific detection of such cells could bear significant value., Materials and Methods: Expression of 12 stem cell markers were analyzed in two canine B-cell lymphoma cell lines, their generated spheres, and in primary lymphoma samples by quantitative real-time polymerase chain reaction and partially by flow cytometry and immunocytochemistry., Results: Expression of maternal embryonic leucine zipper kinase (Melk) was significantly higher in CLBL-1, CLBL-1M and in primary B-cell lymphoma samples compared to non-neoplastic lymph nodes. Spheres displayed a higher expression of v-myc myelocytomatosis viral oncogene homolog (Myc) and lower expression of Cd44 compared to original cell lines and primary B-cell lymphoma samples., Conclusion: The results suggest a potential interesting role of Melk in canine B-cell lymphoma. Furthermore, the up-regulation of Myc in serum-free-generated spheres offers interesting possibilities for functional assays characterizing the specific generated sub-population., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential., Materials and Methods: Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance., Results: Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable., Conclusion: ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Canine prostate cancer (PC) is a highly aggressive malignancy. However, in contrast to man, neither standard screening strategies nor curative therapeutic options exist for the companion animal. A prostate-specific membrane antigen (PSMA) screening as molecular marker akin to human PC is currently not available for dogs as data on specific canine PSMA detection are contradictory., Materials and Methods: To evaluate an antibody for specific canine PSMA detection by western blotting (WB), lysates of three canine prostatic cell lines (CT1258, DT08/40, DT08/46) were comparatively analyzed by WB and mass spectrometry (MS) to the human cell lines VCaP, LnCaP and PC-3., Results: MS analyses of the detected canine proteins confirmed cross reactivity of the antibody clone YPSMA-1 with canine PSMA., Conclusion: The MS analyses of the extracted canine protein bands proved that the YPSMA-1 clone is as well specific for canine PSMA in WB and, thus, represents a reliable tool for comparative PSMA studies., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: In human prostate cancer cells with a stem cell-like character (cancer stem cells, CSC) are considered to play a major role in disease development, progression and relapse. Aim of the study was to evaluate if similar cells are present and active in canine prostate cancer providing a naturally-occurring mammalian model for the development of therapeutic approaches targeting CSC., Materials and Methods: Stem cell marker expression of CD133, CD44, C-KIT, CD34, ITGA6, OCT4, DDX5 and MELK in canine prostate carcinomas and prostate cyst cell lines were screened by Polymerase Chain Reaction (PCR), quantitative Polymerase Chain Reaction (qPCR) and partially analysed by flow cytometry., Results: Marker analyses by PCR and qPCR, revealed a complex expression pattern for the analysed marker genes, providing a characteristic marker pattern for the studied cell lines. Thereby CD44, CD133, ITGA6 and DDX5 showed the most prominent expression in the analysed cell lines., Conclusion: The results revealed a characteristic stem cell marker expression in the analysed cell lines, indicating the presence of CSC in canine prostate cancer.

Background/aim: Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ)., Materials and Methods: Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples., Results: Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations., Conclusion: Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.

Background: Disorders of histiocytic origin affecting humans and dogs share various similarities. Canine disseminated histiocytic sarcoma (DHS) (formerly known as malignant histiocytosis) is an aggressive neoplasm of interstitial dendritic cells (DCs). The receptor for glycation end products (RAGE) and the high mobility group box1 protein (HMGB1) have been shown to be required for the maturation and migration of DCs. Thus, deregulation of the expression of these genes could have a major effect on the progression of histiocytic disorders., Materials and Methods: Neoplastic canine DHS samples and non-neoplastic control samples were analysed immunohistochemically and via real-time PCR., Results: Significant down-regulation of RAGE in the lung tumour samples and down-regulation of HMGB1 in the lung, lymph node and spleen tumour samples were detected compared to their non-neoplastic counterparts., Conclusion: RAGE and HMGB1 expression down-regulation in canine DHS points to a role in the progression of histiocytic disorders.

Background: Canine lymphoma is a commonly occurring, spontaneously developing neoplasia similar to human non-Hodgkin's lymphoma and, thus, is used as a valuable model for human malignancy. HMGB1 and RAGE are strongly associated with tumour progression and vascularisation. Consequently, deregulated RAGE and HMGB1 may play an important role in the mechanisms involved in lymphoma progression., Materials and Methods: Expression patterns of HMGB1 and RAGE were analysed in 22 canine lymphoma and three canine non-neoplastic control samples via real time PCR and canine beta-glucuronidase gene (GUSB) as endogenous control., Results: HMGB1 was up-regulated in the neoplastic samples, while RAGE expression remained inconspicuous., Conclusion: This study demonstrated similar mechanisms in lymphoma progression in humans and dogs due to overexpression of HMGB1, which was described in human lymphomas. RAGE remained stable in terms of expression indicating that the extracellular HMGB1-induced effects are regulated by HMGB1 itself.

In humans, malignant histiocytosis is a tumour-like disease characterised by increasing proliferation of macrophages and reinforced degradation of erythrocytes. High progression of this disease leads to an unfavourable prognosis for the patients, most of them children up to the age of three years. Histological and cytological findings have proposed an important role of aberrant expression of cytokines in histiocytosis. Due to the fact that Bernese Mountain Dogs (BMD) show a predisposition for spontaneously developing malignant histiocytosis, these dogs could possibly be used as a genetic model organism to elucidate the mechanisms of human malignant histiocytosis. Canine cytokine cDNA transcripts of TNFalpha, Interleukin-1-alpha (IL-1alpha) and Interleukin-1-beta (IL-1beta) were screened for single nucleotide polymorphisms (SNPs). SNP screening in canine cytokine transcripts for malignant histiocytosis has not been carried out before. Total RNA was isolated from tissue samples from lung, spleen, testis and skin of 17 different dogs (fifteen BMDs, one Collie and one West Highland Terrier). The corresponding cytokine cDNAs were amplified, sequenced and then screened for SNPs. The resulting effects on the protein sequence were analysed. Several BMDs and the West Highland Terrier showed SNPs in the coding sequences which led to missense mutations within the protein sequences of TNFalpha, IL1alpha and IL1beta.

Four cases of canine lymphoma are presented, including histological examination and cytogenetic investigation. The first case showed a derivative chromosome 13, the second case showed a clonal trisomy 8 and the third case showed a complex karyotype with a clonal trisomy 13 and additional clonal trisomies of the chromosomes 20, 30 and 37, as well as a non-clonal tetrasomy 9. Case four showed a single trisomy 2. Comparing these results with human hematopoietic malignancies, there are notable similarities between both species.

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

Background: The human high mobility group protein B1 (HMGB1) has attracted considerable interest among oncologists because it sensitises cancer cells to the anticancer drug cisplatin by shielding cisplatin-DNA adducts from nucleotide excision repair., Materials and Methods: Since cisplatin is the cornerstone of adjuvant systemic therapy for osteosarcomas, in both humans and dogs, the expression pattern of the HMGB1 gene in seven canine sarcomas was investigated by Northern blot analysis and semi-quantitative RT-PCR., Results: A strong intertumoural variation of HMGB1 expression was detected by Northern blot analysis and confirmed by the semi-quantitative RT-PCR established herein., Conclusion: The observed variations of HMGB1 expression in canine sarcomas emphasises the role of HMGB1 as a potential marker of clinical interest as its expression level may predict the clinical outcome of therapies based on cisplatin. The semi-quantitative RT-PCR established allows a quick and convenient determination of the HMGB1 expression level as necessary for clinical applications.