Investigating comparative polymerase chain reaction for antigen receptor rearrangement analysis in different types of feline lymphoma samples.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

Single nucleotide polymorphism profiles of canine T-cell and null-cell lymphomas.

Background: The histopathological classification of T-cell lymphoma (TCL) in humans has distinctive mutational genotyping that suggests different lymphomagenesis. A similar concept is assumed to be observed in dogs with different TCL phenotypes. Objective: This study aimed to identify the previously reported single-nucleotide polymorphisms (SNPs) in both human beings and dogs in canine TCLs and null-cell lymphomas (NCLs) and to design compatible oligonucleotides from each variant based on the multiplex polymerase chain reaction. Methods: Genomic DNA was extracted from 68 tumor specimens (62 TCLs and 6 NCLs) and 5 buffy coat samples from dogs with TCL. Four TCL subtypes and NCL were analyzed in 44 SNPs from 21 genes using the MassARRAY. Results: The greatest incidences of SNPs observed in all TCL subtypes and NCL ware SATB1 c.1259A > C, KIT c.1275A > G, SEL1L c.2040 + 200C > G, and TP53 c.1024C > T, respectively. Some SNP locations were statistically significant associated with NCL, including MYC p.S75F (p = 0.0003), TP53 p.I149N (p = 0.030), PDCD1 p.F37LX (p = 0.012), and POT1 p.R583* (p = 0.012). Conclusion: Each TCL histological subtype and NCL are likely to contain distinctive mutational genetic profiles, which might play a role in lymphoma gene-risk factors and might be useful for selecting therapeutic target drugs for each canine patient.

Post-Chemotherapy Canine Lymphomatous Lymph Node Observations on B-Mode and Strain Elastographic Ultrasound.

Canine multicentric lymphoma (CML) is a prevalent hematopoietic neoplasm that initially responds well to treatment but often relapses due to chemotherapy resistance. Evaluation of treatment response is essential for effective management. Ultrasound (US) can differentiate between benign and lymphomatous lymph nodes (LLNs). However, its utility in monitoring LLNs post chemotherapy is limited. This study aimed to compare US parameters of LLNs during the first 3 weeks post treatment and evaluate their diagnostic performance compared with the conventional method for assessing treatment response. This study included 95 LLNs from 15 dogs with CML and 60 normal lymph nodes (NLNs) from 15 healthy dogs. US, including B-mode and elastography, was performed pre-treatment and weekly for 3 weeks post treatment, and compared with the results of NLNs. LLNs were categorized into partial response and stable disease groups using the conventional method. US scores were established by combining B-mode and elastography parameters. The results showed significantly higher values of LLNs in the short-to-long axis ratio, elastographic scales, and blue-to-green color histogram compared with NLNs. Additionally, LLNs at pre-treatment had significantly higher values than LLNs post treatment. US scores significantly differed among the healthy, partial response, and stable disease groups. In conclusion, B-mode US, elastography, and US scores demonstrated changes during chemotherapy consistent with the conventional method and can be used in conjunction with the conventional method to evaluate the treatment response of CML.

Analysis of serum peptidome profiles of non-metastatic and metastatic feline mammary carcinoma using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Feline mammary carcinoma (FMC) is a common aggressive and highly metastatic cancer affecting female cats. Early detection is essential for preventing local and distant metastasis, thereby improving overall survival rates. While acquiring molecular data before surgery offers significant potential benefits, the current protein biomarkers for monitoring disease progression in non-metastatic FMC (NmFMC) and metastatic FMC (mFMC) are limited. The objective of this study was to investigate the serum peptidome profiles of NmFMC and mFMC using liquid chromatography-tandem mass spectrometry. A cross-sectional study was conducted to compare serum peptidome profiles in 13 NmFMC, 23 mFMC and 18 healthy cats. The liquid chromatography-tandem mass spectrometry analysis was performed on non-trypsinized samples. RESULTS: Out of a total of 8284 expressed proteins observed, several proteins were found to be associated with human breast cancer. In NmFMC, distinctive protein expressions encompassed double-stranded RNA-binding protein Staufen homolog 2 (STAU2), associated with cell proliferation, along with bromodomain adjacent to zinc finger domain 2A (BAZ2A) and gamma-aminobutyric acid type A receptor subunit epsilon (GABRE), identified as potential treatment targets. Paradoxically, positive prognostic markers emerged, such as complement C1q like 3 (C1QL3) and erythrocyte membrane protein band 4.1 (EPB41 or 4.1R). Within the mFMC group, overexpressed proteins associated with poor prognosis were exhibited, including B-cell lymphoma 6 transcription repressor (BCL6), thioredoxin reductase 3 (TXNRD3) and ceruloplasmin (CP). Meanwhile, the presence of POU class 5 homeobox (POU5F1 or OCT4) and laminin subunit alpha 1 (LAMA1), reported as metastatic biomarkers, was noted. CONCLUSION: The presence of both pro- and anti-proliferative proteins was observed, potentially indicating a distinctive characteristic of NmFMC. Conversely, proteins associated with poor prognosis and metastasis were noted in the mFMC group.

Retrospective Study of Clinicopathological Changes and Prediction Model for Canine Vascular Neoplasms.

Vascular neoplasms, including hemangiosarcoma (HSA) and hemangioma (HMA), are more common in dogs than other domestic animal species; however, comprehensive laboratory screening tests for early diagnosis are currently limited. The aims of this study were to investigate general signalments, anatomic locations, and clinicopathological abnormalities of dogs diagnosed with vascular neoplasms and to determine the diagnostic significance of these abnormalities. Retrospective data of dogs with HMA, HSA, and healthy dogs were analyzed. Dogs with HMA and HSA were seniors, with mixed breeds being most affected. HMA affected predominantly non-visceral sites, while HSA was more common in visceral sites, particularly the spleen. In multivariate model analyses, the odds of HMA diagnosis were 5.5 times higher in anemic dogs and 33.0 times higher in lymphopenic dogs compared to dogs without the abnormalities. The odds of HSA diagnosis were 42.5 times higher in anemic dogs, 343 times higher in lymphopenic dogs and 92.7 times higher in dogs with hyperfibrinogenemia compared to dogs without the abnormalities. The study suggested that these identified abnormalities were nonspecific and commonly observed in various chronic diseases, and hence their combination with clinical information, such as diagnostic imaging and histopathology, is important to facilitate a more precise diagnosis of canine vascular neoplasms.

Hematological and blood biochemistry parameters as prognostic indicators of survival in canine multicentric lymphoma treated with COP and L-COP protocols.

Background and Aim: Hematological and blood chemistry parameters are crucial for evaluating and monitoring canine multicentric lymphoma during chemotherapy. Pre-treatment hematological and blood chemistry parameters can be used as prognostic survival outcomes for this disease. Therefore, this study aimed to investigate the effect of hematological and blood chemistry parameters pre-treatment and 4 weeks post-treatment on the survival outcomes of dogs treated with either a combination of cyclophosphamide, vincristine, and prednisolone (COP) or a combination of COP with L-asparaginase (L-COP) protocols. Materials and Methods: We conducted a retrospective study. Medical records and hematological and blood chemistry parameters of 41 dogs with multicentric lymphoma treated with L-COP (n = 26) and the COP protocols (n = 15) were obtained from the hospital information system. Most cases were classified as high-grade lymphoma based on the Kiel cytological classification. The effects of hematological and blood chemistry parameters on survival outcomes were investigated using the Cox proportional hazard regression model. The median survival time (MST) for each hematological and blood chemistry parameter affecting survival outcome was established and compared using the Kaplan-Meier product limit method with the log-rank test. Results: Dogs with high-grade multicentric lymphoma that were treated with the COP protocol and had monocytosis at pre-treatment had a significantly shorter MST than dogs with normal monocyte counts (p = 0.033). In addition, dogs with azotemia, both pre-treatment and 4 weeks post-treatment, had a significantly shorter MST than dogs with normal serum creatinine levels (p = 0.012). Dogs with high-grade multicentric lymphoma treated with the L-COP protocol who had hypoalbuminemia (serum albumin concentration <2.5 mg/dL) at both pre-treatment and 4 weeks post-treatment had a significantly shorter MST than dogs with normal serum albumin levels (p < 0.001). Furthermore, dogs with leukocytosis at 4 weeks post-treatment had a significantly shorter MST than those with a normal total white blood cell count (p = 0.024). Conclusion: Serum albumin level can serve as a simple negative prognostic indicator of survival outcomes in dogs with high-grade multicentric lymphoma treated with the L-COP protocol. Dogs with hypoalbuminemia pre-treatment and 4 weeks post-treatment tended to have a shorter MST than those with normal serum albumin concentrations.

Proteomic analysis of the serum in dogs with pulmonary hypertension secondary to myxomatous mitral valve disease: the preliminary study.

Background: Pulmonary hypertension (PH) is a common complication in dogs with myxomatous mitral valve disease (MMVD), characterized by elevated blood pressure in pulmonary artery. Echocardiography is a reliable technique for PH diagnosis in veterinary medicine. However, it is limited to use as an early detection method. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has found extensive application in the discovery of serum protein biomarkers for various diseases. The objective of this study was to identify serum proteins in healthy control dogs and MMVD dogs both with and without PH using LC-MS/MS. Materials and methods: In this research, a total of 81 small-breed dogs participated, and they were categorized into three groups: the control (n = 28), MMVD (n = 24) and MMVD+PH (n = 29) groups. Serum samples were collected and analyzed by LC-MS/MS. Results: Differentially expressed proteins were identified, and the upregulated and downregulated proteins in MMVD+PH group including Myomesin 1 (MYOM1) and Histone deacetylase 7 (HDAC7), Pleckstrin homology domain containing M3 (PLEKHM3), Diacylglycerol lipase alpha (DAGLA) and Tubulin tyrosine ligase like 6 (TTLL6) were selected as proteins of interest in MMVD dogs with PH. Conclusion: Different types of proteins have been identified in healthy dogs and MMVD dogs with and without PH. Additional studies are needed to investigate the potential of these proteins as biomarkers for PH in dogs with MMVD.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model.

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti-tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti-proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

Domestic Cat Hepadnavirus Antigens in Lymphoma Tissues. Comment on Beatty et al. Domestic Cat Hepadnavirus and Lymphoma. Viruses 2023, 15, 2294.

We are addressing the comments made by Beatty et al [...].

Comparative E-cadherin and syndecan-1 protein expression in human and canine oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent form of oral cancer in humans and dogs. The altered expression of cell adhesion molecules, including E-cadherin (CDH1) and syndecan-1 (SDC1), is involved in cancer progression. This study aimed to investigate the protein expression of CDH1 and SDC1 in early and late clinical stages of human and canine OSCC (hOSCC and cOSCC, respectively), using immunohistochemistry. Formalin-fixed and paraffin embedded tissue blocks were obtained from 21 hOSCC, 8 human normal gingiva, 26 cOSCC, and 13 canine normal gingiva. Clinical stages and histological subtypes of samples were evaluated. The results indicated that both human and canine OSCC exhibited reduced levels of CDH1 and SDC1 expression at the cell membrane regardless of clinical stage or histological subtype. Additionally, decreased levels of total SDC1 expression were observed in hOSCC compared with normal controls. In conclusion, this study demonstrates a similarity in the immunohistochemical expression of CDH1 and SDC1 between humans and dogs with OSCC, lending support to the potential use of dogs as a model for studying human head and neck squamous cell carcinoma.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Domestic cat hepadnavirus detection in blood and tissue samples of cats with lymphoma.

Domestic cat hepadnavirus (DCH), a relative hepatitis B virus (HBV) in human, has been recently identified in cats; however, association of DCH infection with lymphoma in cats is not investigated. To determine the association between DCH infection and feline lymphoma, seven hundred and seventeen cats included 131 cats with lymphoma (68 blood and 63 tumor samples) and 586 (526 blood and 60 lymph node samples) cats without lymphoma. DCH DNA was investigated in blood and formalin-fixed paraffin-embedded (FFPE) tissues by quantitative polymerase chain reaction (qPCR). The FFPE lymphoma tissues were immunohistochemically subtyped, and the localization of DCH in lymphoma sections was investigated using in situ hybridization (ISH). Feline retroviral infection was investigated in the DCH-positive cases. DCH DNA was detected in 16.18% (11/68) (p = 0.002; odds ratio [OR], 5.15; 95% confidence interval [CI], 2.33-11.36) of blood and 9.52% (6/63) (p = 0.028; OR, 13.68; 95% CI, 0.75-248.36) of neoplastic samples obtained from lymphoma cats, whereas only 3.61% (19/526) of blood obtained from non-lymphoma cats was positive for DCH detection. Within the DCH-positive lymphoma, in 3/6 cats, feline leukemia virus was co-detected, and in 6/6 were B-cell lymphoma (p > 0.9; OR, 1.93; 95% CI, 0.09-37.89) and were multicentric form (p = 0.008; OR, 1.327; 95% CI, 0.06-31.18). DCH was found in the CD79-positive pleomorphic cells. Cats with lymphoma were more likely to be positive for DCH than cats without lymphoma, and infection associated with lymphoma development needs further investigations.

Development and characterization of mouse anti-canine PD-L1 monoclonal antibodies and their expression in canine tumors by immunohistochemistry in vitro.

Immune escape is the hallmark of carcinogenesis. This widely known mechanism is the overexpression of immune checkpoint ligands, such as programmed cell death protein 1 and programmed death-ligand 1 (PD-1/PD-L1), leading to T cell anergy. Therefore, cancer immunotherapy with specific binding to these receptors has been developed to treat human cancers. Due to the lack of cross-reactivity of these antibodies in dogs, a specific canine PD-1/PD-L1 antibody is required. The aim of this study is to develop mouse anti-canine PD-L1 (cPD-L1) monoclonal antibodies and characterize their in vitro properties. Six mice were immunized with recombinant cPD-L1 with a fusion of human Fc tag. The hybridoma clones that successfully generated anti-cPD-L1 antibodies and had neutralizing activity were selected for monoclonal antibody production. Antibody properties were tested by immunosorbent assay, surface plasmon resonance, and immunohistochemistry. Four hybridomas were effectively bound and blocked to recombinant cPD-L1 and cPD-1-His-protein, respectively. Candidate mouse monoclonal antibodies worked efficiently on formalin-fixed paraffin-embedded tissues of canine cancers, including cutaneous T-cell lymphomas, mammary carcinomas, soft tissue sarcomas, squamous cell carcinomas, and malignant melanomas. However, functional assays of these anti-cPD-L1 antibodies need further investigation to prove their abilities as therapeutic drugs in dogs as well as their applications as prognostic markers.

Isolation of Oral Bacteria, Measurement of the C-Reactive Protein, and Blood Clinical Parameters in Dogs with Oral Tumor.

Canine oral cancers have a poor prognosis and are related to chronic inflammation. This may pose a risk of secondary bacterial infection. This study aimed to compare the bacteria isolated from oral swab samples, values of C-reactive proteins (CRPs), and clinical blood profiles of dogs with and without oral mass. A total of 36 dogs were divided in three groups: no oral mass (n = 21), oral mass (n = 8), and metastasis groups (n = 7). Significantly, both the clinical groups (the oral mass group and metastasis group) showed anemia, a decrease in the albumin-to-globulin ratio (AGR), and an increase in the neutrophil-to-lymphocyte ratio (NLR), globulin-to-albumin ratio (GAR), CRP, and CRP-to-albumin ratio (CAR) compared to the normal group. CAR showed an increasing trend in the oral mass and metastasis groups (10 times and 100 times, respectively) compared to the no oral mass group (P < 0.001). Neisseria spp. (20.78%) was the main isolated bacteria in all groups. The main genera in the no oral mass group were Neisseria spp. (28.26%), Pasteurella spp. (19.57%), and Staphylococcus spp. (19.57%). Neisseria spp., Staphylococcus spp., Klebsiella spp., and Escherichia spp. were found equally (12.5%) in the oral mass group. Escherichia spp. (26.67%), Pseudomonas spp. (13.33%), and Staphylococcus spp. (13.33%) were the main genera in the metastasis group. Interestingly, Neisseria spp. decreased in the clinical groups (Fisher's exact = 6.39, P=0.048), and Escherichia spp. increased in the metastasis group (Fisher's exact = 14.00, P=0.002). The difference of oral bacteria in clinical dogs compared to healthy dogs may be related to microbiome alterations, and both the clinical groups showed the increment of inflammatory biomarkers. This suggested that further studies should be conducted on the correlation between the specific bacteria, CRP, blood clinical parameters, and type of canine oral mass.

Perivascular inflammatory cells and their association with pulmonary arterial remodelling in dogs with pulmonary hypertension due to myxomatous mitral valve disease.

Pulmonary hypertension (PH), an increase in pulmonary arterial pressure (PAP), may occur in dogs affected with myxomatous mitral valve disease (MMVD). Recent studies suggest that an accumulation of perivascular inflammatory cells may be involved with medial thickening which is a sign of the pulmonary artery remodelling in PH. The aim of this study was to characterise perivascular inflammatory cells in the surrounding pulmonary arteries of dogs with PH due to MMVD compared to MMVD dogs and healthy control dogs. Nineteen lung samples were collected from cadavers of small-breed dogs (control n = 5; MMVD n = 7; MMVD + PH n = 7). Toluidine blue stain and multiple IHC targeting α-SMA, vWF, CD20, CD68 and CD3 was performed to examine intimal and medial thickening, assess muscularisation of the small pulmonary arteries and characterise perivascular leucocytes. Medial thickening without intimal thickening of pulmonary arteries and muscularisation of normally non-muscularised small pulmonary arteries was observed in the MMVD and MMVD + PH groups compared with the control group. The perivascular numbers of B lymphocytes, T lymphocytes and macrophages was significantly increased in the MMVD + PH group compared with the MMVD and control groups. In contrast, the perivascular number of mast cells was significantly higher in the MMVD group compared with the MMVD + PH and control groups. This study suggested that pulmonary artery remodelling as medial thickening and muscularisation of the normally non-muscular small pulmonary arteries is accompanied by the accumulation of perivascular inflammatory cells.

Case report: Mature extragonadal teratoma at the proximal part of the tail in a kitten.

An 8-month-old, intact male, domestic shorthair cat was referred for a mass on the proximal ventral part of the tail which had been found since the animal was born, and due to the presence of a linear fissure with rows of ectopic teeth, the veterinarian suspected that the mass had recently ruptured. Tail amputation was elected and the entire mass was successfully surgically excised. From the gross examination, this mass had an open cyst-like structure with a prominent area composed of hair, teeth, and bone. Histopathology revealed two components of germinal layers including hair follicles, adnexal tissue, neural tissue, teeth, muscle, fat, bone, and lymphatic vessels. The histopathological diagnosis was consistent to mature teratoma. Although, complete excision could not be definitively confirmed histologically, this kitten is currently well and has not developed any recurrent mass at the surgical site after 2 years of post-operation.

Diagnostic utility of LDH measurement for determining the etiology of modified transudate pleural effusion in cats.

Fluid analysis is an initial approach for determining the underlying causes of body cavity effusions. Modified transudate is commonly diagnosed in pleural effusion in cats, however, it provides limited diagnostic information. Aims of this study were to investigate common etiologies causing different pleural fluid types and to evaluate the usefulness of lactate dehydrogenase (LDH) for differentiating the etiology in modified transudates in cats. Pleural effusion samples from 122 cats were analyzed and classified into three types: transudate, modified transudate, and exudate. Causes of pleural effusion were classified into four conditions: cardiac disease, neoplasia, feline infectious peritonitis (FIP), and pyothorax. The relationship of underlying etiology and fluid types was described. The LDH levels in pleural fluid and plasma were compared between the causes in the samples classified as modified transudate. The fluid analysis of pleural effusion showed that modified transudate was the most common fluid type (44.2%). Neoplasia was predominantly diagnosed (38.5%) as the etiology of pleural effusion. There was no significant correlation between pleural fluid and plasma LDH level in any type of pleural fluid, suggesting that pleural fluid LDH does not appear to be affected by plasma LDH. The occurrence of modified transudate was not associated to its etiologies, however, the LDH level in modified transudates showed significant differences between etiologic groups. The LDH level in modified transudate was excellent in separating cardiac from non-cardiac diseases with a cut-off value of <535 U/L and separating FIP from non-FIP diseases with a cut-off value of >641 U/L. Based on the current findings, pleural fluid LDH can be a useful adjunctive marker for differentiating some causes of modified transudate pleural effusion and should be added in the routine diagnostic work-up of feline patients with pleural effusions.

Case report: BCL-2 and CD31 immunoexpression related to clinical and histopathological evaluation of renal dysplasia in a Welsh Corgi Puppy.

A case of renal dysplasia (RD) in the Welsh Corgi dog has been reported. Clinically, the affected 3-month-old, female, Welsh Corgi dog showed unclear symptoms of chronic kidney disease. Grossly, both left and right kidneys revealed cystic hypoplasia. Histologically, the primary lesions included immature or fetal glomeruli/tubules, proliferative arterioles, persistent metanephric ducts, persistent mesenchyme, and atypical tubular epithelium were presented. A group of degenerative and inflammatory lesions consisting of interstitial nephritis, interstitial fibrosis, and mineralization of tubules were found. Immunohistochemically, the epithelial cells of immature (fetal) tubules had BCL-2 labeling whereas CD31 (PECAM-1) was labeled in the endothelial cells of the proliferative arterioles. The immunohistochemical findings were confirmed and consolidated with the routine histopathological findings. This study was the first demonstration of the clinical, histopathological, and immunohistochemical features of RD disease in a Welsh Corgi puppy.

Activating Mutation in the Receptor Tyrosine Kinase FLT3 with Clinicopathological Relevance in Canine Mast Cell Tumors.

Recent research has focused on the receptor tyrosine kinase (RTK) KIT which is involved in the pathogenesis of canine mast cell tumors (MCT). However, the role of other RTKs in this neoplasm remains unclear. The present study aimed to determine the frequency of FLT3 mutations and to evaluate the mutational status and clinicopathological relevance of canine MCT patients. There were a total of 20 cases that were cytologically and histopathological diagnosed as canine MCTs; genomic polymerase chain reaction (PCR) and Sanger sequencing were used to identify mutations. For the juxtamembrane (JM) domain, the FLT3 14/15 primer pair was used to investigate exon 14/15 loci. Based on genomic PCR amplification of exon 14/15 and 20 of the FLT3 gene and Sanger sequencing of 20 cases of canine MCTs, the overall frequency of FLT3 mutation in canine MCTs was 75%. The majority of FLT3 mutations (70%) were internal tandem duplications (ITD) of the JM domain, while one case arose from deletion mutations of the tyrosine kinase domain (TKD). However, double mutations were not observed in this study. Furthermore, there is also clinicopathological relevance to MCT dogs carrying FLT3-ITD mutations, showing a tendency toward leukocytosis due to neutrophilia, and resembling human acute myeloid leukemia (AML) with FLT3-ITD mutations. A subset of MCTs with FLT3-ITD mutations, showing an enhanced signal of phosphorylated ERK1/2 identified by immunoblotting, suggests that an activating mutation may be driven by a distinct signal of the ERK pathway. Our results indicate that FLT3-ITD mutation is an oncogenic driver of canine MCTs, and that it shares some clinicopathologic features with human AML. These findings may offer new opportunities for further studies on canine mast cell tumorigenesis and a novel therapeutic target for canine MCT cases harboring FLT3-ITD mutations.

Investigation of proteomic profiles in canine lymphoma using tandem mass tag-based quantitative proteomics approach.

Background and Aim: Specific tumor biomarkers are useful for the early diagnosis of cancer or can predict the recurrence of neoplastic disease in humans and animals. Lymphoma in dogs could be classified into B-, T-, and NK-cell origins. T-cell lymphoma has the worst prognosis with a shorter survival time and disease-free interval. This study aimed to identify the differential serum protein expressions of canine B- and T-cell lymphomas compared with healthy dogs using a tandem mass tag (TMT)-based quantitative proteomics. Materials and Methods: Serum samples were collected from 20 untreated canine lymphomas (14 B-cells and 6 T-cells) and four healthy control dogs. Sera peptides from each sample were processed for TMT 10-plex tagging and analyzed using liquid chromatography-mass spectrometry (MS). Differential proteome profiling was then compared between lymphoma and control. Results: We discovered 20 elevated and 14 decreased serum proteins in the lymphoma group relative to the healthy group. Six candidate increased proteins in canine lymphomas were beta-actin cytoplasmic 1 (ACTB, p=0.04), haptoglobin (p=0.002), beta-2 microglobulin (aaaaaaaa2M, p=0.007), beta-2 glycoprotein 1 (APOH, p=0.03), metalloproteinase inhibitor 1 (TIMP-1, p=0.03), and CD44 antigen (p=0.02). When compared between B- and T-cell lymphomas, B-cell phenotypes had upregulated immunoglobulin (Ig) heavy chain V region GOM (p=0.02), clusterin (p=0.01), apolipoprotein C1 (APOC1, p=0.05), and plasminogen (p=0.02). Conclusion: These findings were investigated quantitative serum proteomes between B- and T-cell lymphomas using TMT-based MS. ACTB, aaaaaaaa2M, APOH, TIMP-1, CD44 antigen, Ig heavy chain V region GOM, and APOC1 are novel candidate proteins and might serve as a lymphoma biomarker in dogs. However, evaluation with an increased sample size is needed to confirm their diagnostic and prognostic ability.