A clonality assay in canine B cell tumors targeting the immunoglobulin light chain lambda locus.

Clonality assays for antigen receptor rearrangement have been used as adjunct examinations of lymphoproliferative diseases. These assays have been useful for differentiation between inflammation and clonal expansion of lymphocytes. Whereas the immunoglobulin heavy chain (IGH) and immunoglobulin light chain kappa (IGK) loci have been targeted in canine clonality assays previously, the immunoglobulin light chain lambda gene (IGL) locus has not yet been investigated. This study aimed to evaluate the usefulness of clonality assays in dogs using IGL. Canine diffuse large B cell lymphomas (DLBCL), cutaneous plasmacytomas, and pathologically diagnosed lymph nodes without lymphoma, were used in this study. Genomic DNA was extracted from formalin-fixed paraffin embedded sections. Sequences of IGLV and IGLJ were obtained from the ImMunoGeneTics database. Several primers against IGLVs and IGLJs were designed in the regions showing homology, by alignment of the gene segments. Products of polymerase chain reaction were analyzed on a capillary electrophoresis. In total, 20 of 23 cases of DLBCL showed clonality (87.0 %), whereas 8 of 30 cutaneous plasmacytomas were clonal (26.7 %). One of 23 lymph nodes without lymphoma showed clonality, thus the specificity was 95.7 %. These data indicate that the IGL locus could be a target for canine clonality assays and that the sensitivity of IGL-based clonality assays in cutaneous plasmacytomas was lower than that in DLBCL.

A clonality analysis based on T-cell receptor beta and delta loci for high-grade gastrointestinal lymphoma in dogs.

Clonality assays based on antigen receptors are used as adjunct examinations in the diagnosis of lymphoproliferative diseases. We investigated the usefulness of the T-cell receptor beta (TRB) and T-cell receptor delta (TRD) loci in clonality assays for high-grade gastrointestinal (GI) lymphoma in dogs. For TRB, we used primers reported previously; for TRD, we designed primers for each of the V and J genes based on genomic sequences. Genomic DNA was extracted from 39 formalin-fixed, paraffin-embedded sections of high-grade GI lymphoma diagnosed histologically. The sensitivity of TRB and TRD primers for GI lymphoma was 41.0% and 38.5%, respectively, which was lower than the 82.1% sensitivity of T-cell receptor gamma (TRG) primers However, some cases that could not be detected using TRG primers had clonality with either TRB or TRD primers. We found the TRG locus to be more suitable as a first choice for the assay of canine lymphoma clonality than the TRB and TRD loci. However, the detection rate of T-cell clonality may be enhanced using TRB and TRD primers for lymphoma cases not detected using TRG primers.

Gastrointestinal stromal tumors with Kit gene mutation in 4 guinea pigs (Cavia porcellus).

Gastrointestinal stromal tumors (GISTs) have been rarely reported in guinea pigs. We aimed to characterize the clinical and pathological features of GISTs in 4 guinea pigs and investigate the presence of mutations in exon 11 of the KIT proto-oncogene receptor tyrosine kinase (Kit) gene. Two subjects were male and 2 were female; 2 were 6 years old, 1 was 7 years old, and 1 was of an unknown age. Three cases had primary gastric tumors, whereas 1 had a primary small intestinal tumor. All cases had tumors that extended from the submucosa to the serosa with extraluminal growth. A gastric tumor had gastric, pancreatic, and cecal metastases. Histologically, the tumors were sharply demarcated and composed of spindle cells arranged in bundles, intermixed with small amounts of collagenous stroma. The tumor cells had mild atypia with few mitotic figures (0-5/50 high power fields, 7.95 mm2) and were immunolabeled for KIT and Discovered-on-GIST 1 (DOG1). All cases had mutations in exon 11 of the Kit gene. These findings indicate that GISTs in guinea pigs are similar to those in humans and dogs. GISTs in guinea pigs are potentially malignant submucosal tumors with KIT- and DOG1-immunolabeling, exon 11 KIT mutations, and the possibility of metastasis.

Diagnosis of severe fever with thrombocytopenia syndrome (SFTS) in a cat with clinical findings resembling lymphoma.

A two-year-old male domestic cat showed lethargy, tonic-clonic convulsion, and mucosal jaundice. Upon admission, blood examination indicated severe neutropenia and thrombocytopenia, and ultrasonography revealed diffuse splenomegaly with a honeycomb appearance and abdominal lymph nodes enlargement in addition to a decrease in cardiac blood flow indicating a shock condition. Cytology of the spleen showed a cell population composed of immature large lymphoid cells with distinct nucleoli, suggesting lymphoma. The cat received symptomatic treatments but died four hours later. Reverse transcriptase polymerase chain reaction assay of the spleen sample indicated the presence of severe fever with thrombocytopenia syndrome (SFTS) virus S gene segment. Clinical features of this case that was diagnose as SFTS were similar to lymphoma. Therefore, pet owners and veterinary workers should be protected against infection of SFTS.

Severe osteogenesis imperfecta caused by CREB3L1 mutation in a cat.

We examined the clinical features and pathology, and identified the causative mutation, of osteogenesis imperfecta in a 2-mo-old kitten with growth retardation and abnormal gait. Blood and radiographic examinations were performed on presentation. Radiographs revealed decreased opacity of numerous bones. Fractures were observed in some long bones, including femur and tibia. Histologic examination of the tibia showed decreased osteoid and osteoblasts at the primary spongiosa extending from the growth plate. The periosteum was thickened, and cortical bone and osteoblasts were decreased. Consequently, osteogenesis imperfecta was diagnosed. Genomic DNA and total RNA were extracted from the skin and used for PCR. Whole-genome sequencing identified a 2-bp deletion (c.370_371delTG; p.C124fs), which resulted in a homozygous frameshift mutation on exon 3 of CREB3L1. This mutation introduced a premature stop codon, suggesting production of the truncated protein without a functional domain as a transcription factor for expression of COL1A1 mRNA. This error may have affected collagen fibril formation, leading to the development of osteogenesis imperfecta.

Genotype frequency of ATP7A and ATP7B mutation-related copper-associated hepatitis in a Japanese guide dog Labrador retriever population.

The incidence of copper-associated hepatitis in Labrador retriever in Japan has not been examined. This study examined the genotype frequencies of ATP7B:c.4358G>A, a mutation responsible for copper-associated hepatitis, and ATP7A:c.980C>T, a modifier of this disease, in Labrador retrievers of guide dog associations in Japan. Genetic material was collected by buccal swabs from 253 Labrador retrievers and genotyping was performed for the ATP7B and ATP7A mutations. The gene frequency was 0.107 for ATP7B:c.4358A. For ATP7A:c.980C, the gene frequencies were 0.703 in females and 0.368 in males. In this study, we established genotyping methods for the ATP7B:c.4358G>A and ATP7A:c.980C>T mutations. Based on the genotyping results, the risk of copper-associated hepatitis in the study population was 0.80% in males and 1.05% in females.

Osteochondrodysplasia in Scottish Fold cross-breed cats.

Two Scottish Fold mixed cats are described in this report. Case 1 is a mixed Scottish Fold and Munchkin cat. Extremities of this cat resembled the Munchkin cat, while the ear pinna were folded forward like the Scottish Fold cat. Case 2 is a mixed Scottish Fold and American Curl cat. The ear pinna were curled caudally like the American Curl. Severe exostosis in the hind leg was observed in radiographs taken around one year of age in both cats. Both cats were dominant homozygous for c.1024G>T of the transient receptor potential vanilloid 4 gene, responsible for osteochondrodysplasia in the Scottish Fold cat. Cross breeding with Scottish Fold cats could produce unknown phenotypes, and should be avoided.

The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs.

BACKGROUND: Gain-of-function mutations in KIT are driver events of oncogenesis in mast cell tumours (MCTs) affecting companion animals. Somatic mutations of KIT determine the constitutive activation of the tyrosine kinase receptor leading to a worse prognosis and a shorter survival time than MCTs harbouring wild-type KIT. However, canine MCTs carrying KIT somatic mutations generally respond well to tyrosine kinase inhibitors; hence their presence represents a predictor of treatment effectiveness, and its detection allows implementing a stratified medical approach. Despite this, veterinary oncologists experience treatment failures, even with targeted therapies whose cause cannot be elucidated. The first case of an MCT-affected dog caused by a secondary mutation in the tyrosine kinase domain responsible for resistance has recently been reported. The knowledge of this and all the other mutations responsible for resistance would allow the effective bedside implementation of a deeply stratified and more effective medical approach. CASE PRESENTATION: The second case of a canine MCT carrying a different resistance mutation is herein described. The case was characterised by aggressive behaviour and early metastasis unresponsive to both vinblastine- and masitinib-based treatments. Molecular profiling of the tumoural masses revealed two different mutations; other than the already known activating mutation p.Asn508Ile in KIT exon 9, which is tyrosine kinase inhibitor-sensitive, a nearly adjacent secondary missense mutation, p.Ala510Val, which had never before been described, was detected. In vitro transfection experiments showed that the secondary mutation did not cause the constitutive activation by itself but played a role in conferring resistance to masitinib. CONCLUSIONS: This study highlighted the importance of the accurate molecular profiling of an MCT in order to improve understanding of the molecular mechanism underlying tumourigenesis and reveal chemoresistance in MCTs for more effective therapies. The detection of the somatic mutations responsible for resistance should be included in the molecular screening of MCTs, and a systematic analysis of all the cases characterised by unexpected refractoriness to therapies should be investigated in depth at both the genetic and the phenotypic level.

Improved clonality analysis based on immunoglobulin kappa locus for canine cutaneous plasmacytoma.

Sensitivity of clonality analysis based on immunoglobulin heavy chain (IGH) in canine cutaneous plasmacytoma is lower than that in diffuse large B cell lymphoma (DLBCL) because of somatic hypermutation occurring at the IGH locus. Therefore, this study aimed to improve the sensitivity of clonality analysis for canine cutaneous plasmacytoma. To achieve this, clonality analysis based on the immunoglobulin kappa chain (IGK) locus was established. Sensitivity and specificity were examined in genomic DNA extracted from formalin-fixed paraffin-embedded sections of cutaneous plasmacytomas, DLBCLs, and lymph nodes without lymphoma. Forward primers were designed based on the IGKV genes, and reverse primers were designed based on the IGKJ genes and kappa deleting element (Kde). Analysis using IGKV and IGKJ primers demonstrated clonality in 24 of 29 cutaneous plasmacytomas (82.8%), while analysis with primers for IGKV and Kde showed clonality in 16 of 29 cases (55.2%). In DLBCL, the IGKV and IGKJ primer set yielded clonality in 18 of 23 cases (78.3%), and the IGKV and Kde primer set yielded 9 of 23 cases (39.1%). No clonal results were obtained from 23 lymph nodes without lymphoma. Sensitivity of the IGKV and IGKJ primer set was significantly higher than that of the IGH primers reported previously. Thus, clonality analysis based on the IGK locus can be utilized for canine B cell tumors. In conclusion, clonality testing based on IGH and IGK may be beneficial as an adjunct tool for diagnosis of canine B cell tumors including cutaneous plasmacytoma.

Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan.

Progressive rod-cone degeneration (PRCD) is an autosomal recessive disease caused by c.5G>A mutation of the PRCD exon 2. This mutation has been identified in various breeds, including Labrador Retriever. The present study aimed to examine the allelic frequency of PRCD in Labrador Retrievers in Japan. A domestic and a guide dog population were genotyped for PRCD using polymerase chain reaction-restriction fragment length polymorphism. The allelic frequency of c.5G>A in domestic and guide dog populations (0.114 and 0.026, respectively) differed significantly. The allele with c.5G>A mutation appeared to spread widely in the domestic population as compared to that in the guide dog population. This might be the result of mating control for PRCD in the guide dog population.

Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor.

Imatinib-resistance is a major therapeutic problem in human chronic myeloid leukemia, human gastrointestinal stromal tumors, and canine mast cell tumors. In the present study, we identified the secondary mutation c.2006C>T in c-KIT exon 14 in a mast cell tumor obtained from a dog carrying c.1663-1671del in exon 11 and showing resistance to imatinib. The mutation in exon 14 resulted in substitution of threonine with isoleucine at position 669, which was located at the center of the ATP binding site as a gatekeeper and played an important role in binding to imatinib. Transfectants were constructed to survey the functions of these mutations in exons 11 and 14. The transfectant with mutant KIT encoded by c-KIT carrying c.1663-1671del showed constitutive ligand-independent phosphorylation that was suppressed by imatinib, indicating a gain-of-function mutation. Furthermore, the transfectant with mutant KIT encoded by c-KIT carrying both c.1663-1671del and c.2006C>T caused ligand-independent phosphorylation, which was not suppressed by imatinib. From these results, we concluded that the mutation c.2006C>T in c-KIT exon 14 was an imatinib-resistance mutation in a canine mast cell tumor. These findings revealed, for the first time, a mechanism of imatinib resistance in a clinical case of canine mast cell tumor.

Comparison of primer sets for T-cell clonality testing in canine intestinal lymphoma.

Clonality testing based on polymerase chain reaction is an important tool for diagnosis of lymphoproliferative diseases. Many primers have been designed and used for canine clonality testing. Canine intestinal lymphoma is usually diagnosed pathologically by examination of excised intestinal or endoscopic biopsy tissues, and clonality testing is sometimes used to support the pathological diagnosis if this examination is inconclusive. In the present study, the sensitivity of each previously published primer set for clonality testing was examined by using formalin-fixed, paraffin-embedded sections from 39 cases pathologically diagnosed as canine intestinal lymphoma (large-cell type). All 39 cases were immunohistochemically positive for cluster of differentiation (CD)3. Thirty-two out of the 39 cases showed clonality in the T-cell receptor gamma (TRG) with at least 1 of the tested primers. The primer set with the highest sensitivity detected all 32 cases with TRG clonality, with a sensitivity of 82.1%. These results provide useful evidence for the selection of primer sets for clonality testing of canine intestinal lymphoma.

Imatinib responsiveness in canine mast cell tumors carrying novel mutations of c-KIT exon 11.

In 2 individual cases of canine mast cell tumors, we identified 2 novel c-KIT mutations in exon 11: a 9-base pair (bp) deletion (c.1663-1671del) and a point mutation (c.1676T>A). The 9-bp deletion mutation caused a loss of 3 amino acids, corresponding to p.Gln555_Lys557del, and the point mutation resulted in the substitution of valine by aspartic acid (p.Val559Asp) in the juxtamembrane domain of the protein. Imatinib mesylate, a therapeutic agent for canine mast cell tumors, was used to treat both tumors. Complete remission was achieved at 33 and 14 days after administration, respectively. However, in both cases, the therapeutic response subsequently tapered with the duration of remission lasting 66 and 255 days, respectively. Although these 2 novel c-KIT mutations in exon 11 were not confirmed to be gain-of-function mutations, a further study may help clarify relevance between mutations identified in this report and responsiveness.

The vitreous glycoprotein opticin inhibits preretinal neovascularization.

PURPOSE: Opticin is an extracellular matrix glycoprotein that the authors discovered in the vitreous humor of the eye. It is synthesized by the nonpigmented ciliary epithelium and secreted into the vitreous cavity and, unusually for an extracellular matrix molecule, high-level synthesis is maintained into adult life. Here the authors investigated the hypothesis that opticin influences vascular development in the posterior segment of the eye and pathologic angiogenesis into the normally avascular, mature (secondary) vitreous. METHODS: Opticin was localized in murine eyes by immunohistochemistry. An opticin knockout mouse was established and vascular development was compared between knockout and wild-type mice. Wild-type and opticin null mice were compared in the oxygen-induced retinopathy model, a model of pathologic angiogenesis, and this model was also used to assess the effects of intravitreal injection of recombinant opticin into eyes of wild-type mice. RESULTS: Opticin colocalizes with the collagen type II-rich fibrillar network of the vitreous, the inner limiting lamina, the lens capsule, the trabecular meshwork, and the iris. Analyses of the hyaloid and retinal vasculature showed that opticin has no effect on hyaloid vascular regression or developmental retinal vascularization. However, using the oxygen-induced retinopathy model, the authors demonstrated that opticin knockout mice produce significantly more preretinal neovascularization than wild-type mice, and the intravitreal delivery of excess opticin inhibited the formation of neovessels in wild-type mice. CONCLUSIONS: A lack of opticin does not influence vascular development, but opticin is antiangiogenic and inhibits preretinal neovascularization.

Genotyping of exercise-induced collapse in Labrador retrievers using an allele-specific PCR.

Exercise-induced collapse (EIC) is an autosomal recessive disorder in Labrador retrievers. In this study, an allele-specific PCR was developed to detect the point mutation G767T in exon 6 of canine DNM1, previously shown to be responsible for canine EIC. Of 133 Labrador retrievers tested in Japan, 6 (4.5%) were homozygous (EIC) and 50 (37.6%) were heterozygous (carriers) for the G767T mutation.

Heteroduplex polymerase chain reaction is essential for canine receptor rearrangement analysis.

Polymerase chain reaction (PCR) for antigen receptor rearrangement is a sensitive technique for detecting lymphocyte-proliferative disorders, but it tends to produce false-positive results, a phenomenon termed pseudoclonality. Heteroduplex analysis, which is useful to distinguish clonal reactions from pseudoclonal ones in dogs, can be applied to avoid misdiagnosis and determine the reliability of results. In the current study, PCR for antigen receptor rearrangement was used to identify clonal proliferation of lymphocytes in duodenal and lymphoid tissue from dogs presenting with chronic vomiting and enlarged peripheral lymph nodes typical of multicentric lymphoma, and the test results were verified with heteroduplex analysis. In the case of almost all of the duodenal samples, even without a histologic diagnosis of lymphoma, a distinct band similar to that observed in the case of lymphoma was obtained for both B- and T-cell clonality. All of the bands obtained from the nonneoplastic duodenum disappeared following heteroduplex analysis of the PCR product, whereas the distinct bands from the lymphoma remained. In the lymph node samples, the pseudoclonal bands that disappeared in the heteroduplex analysis were detected mainly in B cells. In conclusion, heteroduplex analysis with PCR for antigen receptor rearrangement is a suitable tool for diagnosing canine lymphoma and decreasing the possibility of misdiagnosis of pseudoclonality.