Asparaginase and MOPP Treatment of Dogs with Lymphoma

Background: Dogs with multicentric lymphoma are treated with various cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based chemotherapy protocols with variable success.
Objectives: To describe the progression-free survival (PFS) time and overall survival time (OST) of dogs with T-cell lymphoma or hypercalcemic lymphoma treated with l -asparaginase and mechlorethamine, vincristine, prednisone, procarbazine (MOPP).
Animals: Fifty dogs with T-cell lymphoma, hypercalcemic lymphoma, or both treated at 3 referral veterinary hospitals.
Methods: Retrospective study. Case were selected based on histologic or cytologic diagnosis of lymphoma; presence of the T-cell phenotype, presence of hypercalcemia or both; and absence of previous chemotherapy. The T-cell phenotype was determined by flow cytometry, immunocytochemistry, immunohistochemistry, or polymerase chain reaction of antigen receptor rearrangement.
Results: The overall response rate was 98% (78% complete response, 20% partial response). The median PFS for the entire study population was 189 days with 25% PFS at 939 days. The median OST for the entire study population was 270 days with 25% surviving 939 days. Twenty percent of the dogs required hospitalization for treatment related complications.
Conclusions and clinical importance: l -Asp/MOPP chemotherapy might result in longer PFS and OST for dogs with multicentric T-cell lymphoma, dogs with hypercalcemic lymphoma or both, than achieved with CHOP.     

Monitoring of Minimal Residual Disease (MRD) after Multidrug Chemotherapy and Its Correlation to Outcome in Dogs with Lymphoma: A Proof‐of‐Concept Pilot Study

Background: Tumor cell burden in dogs with lymphoma cannot be assessed accurately by diagnostic evaluation during clinical complete remission (CR). Recent advances in polymerase chain reaction (PCR)‐based methods enabled us to quantify minimal residual disease (MRD) in canine lymphoma. Hypothesis/Objectives: To quantify MRD in dogs with lymphoma treated with multidrug chemotherapy and to correlate it with remission duration after chemotherapy. Animals: Seventeen dogs with lymphoma that achieved CR by multidrug chemotherapy. Methods: Rearranged immunoglobulin heavy chain or T‐cell receptor γ chain gene fragments from lymphoma cells were PCR amplified and sequenced to prepare clone‐specific primers and probes for real‐time PCR to quantify MRD. MRD in the peripheral blood was monitored during and at the end of a 25‐week multidrug chemotherapy protocol. Correlation between MRD at the end of chemotherapy and remission duration after chemotherapy was analyzed. Results: MRD gradually decreased after initiation of multidrug chemotherapy, reached a nadir as low as <0.019–1.0 cells/μL at weeks 4–17, and remained low or slightly increased until week 25. MRD at the end of chemotherapy was negatively correlated with remission duration from the end of chemotherapy to relapse. Conclusion and Clinical Importance: MRD could be an objective marker to indicate tumor cell burden in dogs with lymphoma even in clinical CR. MRD at the end of chemotherapy could be a prognostic factor to predict remission duration after chemotherapy.     

Antioxidant Status and Biomarkers of Oxidative Stress in Dogs with Lymphoma

Background: Oxidative stress might play a role in carcinogenesis, as well as impacting morbidity and mortality of veterinary cancer patients. The purpose of this study was to evaluate antioxidant concentrations and biomarkers of oxidative stress in dogs with newly diagnosed lymphoma before treatment and once in remission, with comparison with healthy controls.
Hypothesis: Dogs with lymphoma have increased oxidant and reduced antioxidant concentrations compared with healthy controls, and that these abnormalities normalize once remission is achieved.
Animals: Seventeen dogs with lymphoma and 10 healthy controls.
Methods: Prospective, observational study. Measures of oxidative stress [malondialdehyde and total isoprostanes (isoP)] and antioxidants [α-tocopherol, γ-tocopherol, oxygen radical absorbance capacity (ORAC), and glutathione peroxidase (GSHPx)] were assessed in dogs with newly diagnosed lymphoma before treatment compared with healthy control dogs. The same parameters were measured in the dogs with lymphoma on week 7 of the chemotherapy protocol when all dogs were in remission.
Results: At baseline, dogs with lymphoma had significantly lower α-tocopherol ( P <.001) and γ-tocopherol ( P = .003) but higher GSHPx ( P = .05), ORAC ( P = .001), and isoP ( P < .001) compared with healthy controls. In the dogs with lymphoma, α-tocopherol concentrations were higher ( P = .005) and ascorbic acid were lower ( P = .04) after treatment.
Conclusions and Clinical Importance: Results suggest that dogs with lymphoma have alterations in oxidant and antioxidant concentrations and that the status of some of these biomarkers normalize after remission. Further studies are warranted to determine whether antioxidant interventions to correct these are beneficial in the treatment of canine lymphoma.     

Association of p53 Gene Mutation with the Prognosis of Canine Lymphoid Tumors

Introduction:  Many dogs with lymphoid tumors develop resistance to chemotherapy. As a mechanism of drug resistance in canine lymphoma, ATP‐dependent drug efflux by P‐glycoprotein was reported, however, inhibition of apoptosis mediated by P53 inactivation has not been investigated. In this study, we investigated the relationship between p53 gene mutation and clinical drug resistance in canine lymphoid tumors.
Methods:  Tumor specimens were obtained from 44 dogs with lymphoid tumors. Mutations of p53 gene at exon 4–8 of these tumor tissues were examined by PCR‐SSCP (single strand conformational polymorphism) analysis, followed by nucleotide sequencing of the abnormal bands. The cases were treated with UW‐Madison protocol, and its response was evaluated by the tumor size or the number of peripheral leukemic cells.
Results:  Of the 44 dogs, 15 dogs (34%) had p53 mutation, whereas 29 dogs (66%) were devoid of p53 mutation, before or during the chemotherapeutic protocol. Rate of good response (CR and PR) to chemotherapy was significantly lower in the dogs with p53 mutation (20%) than those without p53 mutation (55%)(p = 0.022). Median overall survival duration after examination of p53 mutation was significantly shorter in dogs with p53 mutation (101days) than those without p53 mutation (223days)(p = 0.008).
Conclusions:  Lymphoid tumors with p53 mutations were shown to have worse prognosis than those without p53 mutation.     

Lomustine (CCNU) for the Treatment of Resistant Lymphoma in Dogs

Forty-three dogs with lymphoma that had relapsed or had failed to achieve complete remission to previous chemotherapy were treated with lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea [CCNU]) at a dosage of 90-100 mg/m2 body surface area p.o. every 3 weeks. Durable complete or partial responses occurred in 11 dogs for a median of 86 days. The acutely dose-limiting toxicosis was neutropenia 7 days after administration, resulting in a recommended dosage of 90 mg/m2. Cumulative thrombocytopenia occurred in dogs receiving continued CCNU treatment, and a dose interval of 3 weeks may be too short for continued administration of this drug. Toxicoses evident as fever or central nervous system signs or renal damage were uncommon or rare. CCNU is effective in the treatment of relapsed lymphoma.     

Association of Vitamin D Status and Clinical Outcome in Dogs with a Chronic Enteropathy

Background

Dogs with a chronic enteropathy (CE) have a lower vitamin D status, than do healthy dogs. Vitamin D status has been associated with a negative clinical outcome in humans with inflammatory bowel disease.

Objectives

To examine the relationship between serum 25 hydroxyvitamin D (25(OH)D) concentrations at diagnosis and clinical outcome in dogs with a CE.

Animals

Forty‐one dogs diagnosed with CE admitted to the Royal Dick School of Veterinary Studies, Hospital for Small Animals between 2007 and 2013.

Methods

Retrospective review. Serum 25(OH)D concentrations were compared between dogs which were alive at follow up or had died because of non‐CE‐related reasons (survivors) and dogs which died or were euthanized due to their CE (non‐survivors). A binary logistic regression analysis was performed to determine significant predictors of death in dogs with CE.

Results

Serum concentrations of 25(OH)D at the time a CE was diagnosed were significantly lower in nonsurvivors (n = 15) (median nonsurvivors 4.36 ng/mL, interquartile range 1.6–17.0 ng/mL), median survivors (n = 26) (24.9 ng/mL interquartile range 15.63–39.45 ng/mL, P < .001). Serum 25(OH)D concentration was a significant predictor of death in dogs with CE (odds ratio 1.08 [95% CI 1.02–1.18)]).

Conclusions

Serum 25(OH)D concentrations at diagnosis are predictive of outcome in dogs with CE. The role of vitamin D in the initiation and outcome of chronic enteropathies in dogs is deserving of further study.     

马传染性贫血病毒疫苗株EIAV_(FDDV)穿膜蛋白GP45的截短突变

为研究中国马传染性贫血病毒(EIAV)弱毒疫苗致弱过程中基因组的进化特征,作者对EIAVFDDV前病毒的囊膜基因env进行了分析。使用PCR方法体外扩增EIAVFDDV前病毒DNA的env,并随机挑取PCR的阳性克隆子进行测序和序列比对分析。结果表明,随机挑取的PCR阳性克隆中,29/30的克隆子在第2 128-2 130位核苷酸处存在TGG→TGA(翻译中止密码)的突变。该基因对应的EIAV穿膜蛋白gp45的氨基酸数在突变株中较未截短株中减少154,变成259个aa。对EIAVFDDV进行Western blot分析时发现,EIAVDLV45 ku的gp45条带被约35 ku的条带取代。由此推测,EIAVFDDV疫苗株绝大多数病毒颗粒的gp45是截短型。对该代次疫苗株免疫马匹第15和40天体内EIAV前病毒DNA和基因组RNA的序列进行分析,结果表明该截短毒株具有在体内和体外进行复制的能力。由于已有研究表明细胞嗜性改变与gp45的截短有关,因此,推断EIAVFDDV的gp45的截短是其适应在体外培养的驴胎皮肤细胞上复制传代的结果。综上所述,本研究发现EIAVFDDV的gp45存在高比例截短突变,证明该截短突变毒株具有在体内和体外进行复制的能力。但该截短突变是否可通过改变EIAVFDDV在免疫马体内的细胞嗜性,进而影响其毒力和免疫原性,还有待进一步验证。     

J亚群禽白血病病毒gp37基因的克隆和序列分析

近两年我们从国内不同省份先后分离和鉴定了8株J亚群禽白血病病毒（ALV-J），并对其中的5株进行了gp37基因的克隆和序列分析，结果表明，我们分离的5株ALV-J之间的同源性为94.6%-99.0%；与国内最早分离的SD9901，SD9902和YZ9901等毒株之间的同源性分别为95.3%-98.5%,95.6%-99.0%和94.9%-98.6%；与国际最先报道的原型株HPRS-103之间的同源性为93.4%-95.1%；与其它国外毒株的同源性为92.7%-96.8%。这表明我国ALV-J的gp37基因正在不断发生变异，但相对于gp85基因的变异性要小。     

牛UCP3基因多态性及其与血清生化指标的相关性研究

解耦联蛋白3(UCP3)是位于线粒体内膜的质子转运体,其在适应性产热、体重调节和能量代谢中发挥重要作用。本试验利用PCR-RFLP技术研究了鲁西牛、西门塔尔牛、摩拉水牛和尼里-拉菲水牛4个品种151头牛的UCP3基因的多态性及其与血清生化指标的相关性,为研究牛的能量代谢及耐热、耐寒性提供参考依据。结果表明:牛UCP3基因的第3外显子存在限制性内切酶BglⅠ多态。4个群体中的优势等位基因均为A,鲁西牛和西门塔尔牛的优势基因型均为AB型,在尼里水牛和摩拉水牛中AA基因型略占优势。在所研究的群体中,该多态基因座均处于Hardy-Weinberg平衡状态(P>0.05)。对遗传多态性与血清生化指标进行相关性分析后,发现鲁西牛的β球蛋白含量AA基因型个体显著高于AB型(P<0.05),尼里水牛的血清钾和α2球蛋白含量AA型个体显著高于AB型(P<0.05),水牛品种白蛋白含量和α2球蛋白含量与鲁西牛和西门塔尔牛差异显著,为进一步研究水牛与黄牛的差异提供了参考。     

Canine Tumour Suppressor Gene p53 - Mutation in a Case of Adenoma of Circumanal Glands

Highly conserved regions of the tumour suppressor gene p53, including the typical human tumour hot spots (codons 175, 245, 248, 249, 273 and 282), were investigated in various canine neoplasms. A mutation CGG TGG (arginine tryptophan) was detected in codon 249 in an adenoma of the circumanal gland.     

Clinical Features and Outcome of Heterobilharzia americana Infection in Dogs

Background: Heterobilharzia americana (HA), the causative agent of canine schistosomiasis, is a flatworm with a freshwater snail as an intermediate host. Only case reports or small case series evaluating naturally infected dogs have been published.
Objective: Describe clinical signs in dogs naturally infected with HA.
Animals: Twenty-two dogs naturally infected with HA from 1985 to 2009.
Methods: Retrospective study. All medical records were searched for HA and schistosomiasis. Only dogs with a diagnosis based on identification of ova on histopathology or fecal saline sedimentation were included.
Results: The median age was 3.1 years (1–12). The median duration of clinical signs before diagnosis was 0.63 months (0.03–12). The most common clinical signs were lethargy (91%), weight loss (77%), hyporexia (68%), vomiting (59%), and diarrhea (55%). Eleven of the 22 dogs were hypercalcemic. Hypercalcemia did not resolve without definitive treatment with praziquantel. HA infection was an incidental diagnosis in 7/22 dogs. Diagnosis was obtained via necropsy (4), histopathology (9), and fecal examination (9). Definitive treatment included praziquantel and fenbendazole. Eighteen dogs were diagnosed antemortem and 17 were treated. Twelve dogs were alive for 6 months to 3 years after diagnosis.
Conclusions and Clinical Importance: HA infection occurs in younger, larger breed, indoor dogs. Hypercalcemia does not resolve without praziquantel treatment. Prognosis is good and neither hypercalcemic-induced renal failure nor ascites appears to worsen prognosis. Dogs in affected areas or that have traveled to affected areas that present for weight loss, gastrointestinal or liver disease, and hypercalcemia, should be tested.     

绵羊瘦素受体基因部分序列测定及其变异位点分析

试验旨在检测绵羊瘦素受体(leptin receptor,LEPR)基因序列突变位点,为进一步分析LEPR基因多态性与绵羊生长性状的关系奠定基础。选用美利奴羊与阿华西羊杂交群体作为试验材料,利用RT-PCR、测序等方法测定了4只绵羊的LEPR基因cDNA部分序列及内含子7序列,同时利用PCR-RFLP分析两个位点在群体(229只)中的多态性。测定出LEPR基因cDNA序列长2608 bp(包含外显子2-16完整序列及外显子1和17的部分序列)和LEPR第7内含子序列全长160 bp,共发现5个核苷酸变异位点,第2外显子中2个(T240C和A279G),第10、14外显子中各1个(A1683G,T2373C),内含子7中1个(1285+A73G),外显子中的4个变异位点均未引起编码氨基酸的改变。在研究群体中,A279G与A1683G中A的频率分别为0.415、0.467,后者处于非平衡状态,GG和AA是主要的单倍型。不同物种间序列一致性分值均比较高,但LEPR mRNA区序列一致性比内含子高,基于LEPR mRNA序列构建的系统发育树更符合实际情况,且可靠性值更高。结果表明,绵羊LEPR基因的保守性较强,突变形式主要是转换,A279G和A1683G可能是突变热点。     

山羊CMKLR1基因的克隆、表达及其与肌内脂肪的相关性研究

试验旨在克隆山羊CMKLR1基因序列并进行生物信息学分析,研究其表达与肌内脂肪（IMF）沉积的相关性。采集山羊皮下脂肪组织,应用RT-PCR克隆山羊CMKLR1基因序列;以GAPDH（GenBank登录号：AJ431207.1）为内参基因,实时荧光定量PCR检测其在山羊不同组织中的表达量及在肌肉组织中的时序表达谱（以肝脏表达量为基准）;测定肌肉组织中的IMF含量,分析其与CMKLR1 mRNA表达量的相关性。结果显示,克隆获得了山羊CMKLR1基因,1 089 bp的CDS区,GenBank登录号：KT165374。实时荧光定量PCR分析显示,CMKLR1基因在24月龄山羊心脏、肝脏、脾脏、肺脏、肾脏、脂肪、背最长肌、股二头肌和臂三头肌中均存在不同程度的表达,其中在肺脏中表达量显著高于其他组织（P< 0.05）;CMKLR1基因在1~3和24月龄的山羊背最长肌、臂三头肌和股二头肌中表达趋势相同,均是在股二头肌中表达量最高,而8~10月龄则是在臂三头肌中表达量最高。山羊IMF含量以24月龄背最长肌含量最高。山羊背最长肌、股二头肌和臂三头肌中CMKLR1 mRNA表达与IMF含量相关性不显著（P> 0.05）。CMKLR1基因不参与山羊IMF沉积作用,试验结果为进一步研究CMKLR1基因奠定理论基础。     

Cloning and Expression of CMKLR1 Gene and Its Correlation with Intramuscular Fat in Goat (Capra hirus)

In order to enrich basic date in goat (Capra hirus) CMKLR1 gene and investigate the correlation between CMKLR1 gene expression and intramuscular fat (IMF) content in muscles, the CMKLR1 gene was cloned from the goat of subcutaneous adipose tissue by RT-PCR, characterized by bioinformatics methods. The expression profiles of CMKLR1 gene of goat in various tissues were constructed liver was benchmark, the GAPDH (GenBank accession No.: AJ431207.1) as a reference gene.Then, the correlation between CMKLR1 mRNA expression and IMF content in muscles was analyzed. The results showed that the CMKLR1 gene CDS of goat (GenBank accession No.: KT165374) was 1 089 bp. Real-time PCR indicated that CMKLR1 was widely expressed in various tissues, including heart, liver, spleen, lung, kidney, adipose, longissimus dorsi, biceps femoris and triceps brachii, and was the highest in lung (P< 0.05). Similar expression variation trend of CMKLR1 was observed between the muscles from 1 to 3 and 24 months old goat, and was the highest in biceps femoris. Reversely, in 8 to 10 months old CMKLR1 was the highest in triceps brachii. The IMF of longissimus dorsi from 24 months old was the highest. Correlation analysis demonstrated that different correlations were observed between expression of CMKLR1 mRNA and IMF content in longissimus dorsi, biceps femoris, and triceps brachii in goat. The CMKLR1 gene did not participate in the deposition of IMF in goats. The research built the theoretical basis for further studies about the CMKLR1 gene.     

c‐Kit Mutation and Localization Status as Response Predictors in Mast Cell Tumors in Dogs Treated with Prednisone and Toceranib or Vinblastine

Background

KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL or TOC is superior for treating MCT without c‐kit mutations.

Hypothesis/Objectives

To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c‐kit mutated MCT would have a better response to TOC than VBL.

Animals

Eighty‐eight client‐owned dogs with macroscopic MCT.

Methods

Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m² weekly × 4 then EOW) by KIT localization and c‐kit mutation status using an adaptive randomization scheme.

Results

Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c‐kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 [0.62–3.92]; P = 0.28). Median progression‐free survival (PFS) for dogs receiving VBL was 78 days (7–1,521) and for TOC 95.5 (14–990); hazard ratio (HR) = 1.34 [0.72–2.50]; P = 0.36. Median overall survival (OS) was 241.5 days (10–1,521) for the VBL group and 159 (20–990) for the TOC group; HR = 0.80 ([0.45–1.41]; P = 0.44).

Conclusions and Clinical Importance

Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c‐kit mutations was not different between treatment groups in this population of dogs, c‐kit mutation status did not predict treatment response.     

安卡鸡TLR4基因外显子2多态性及其与经济性状的关联分析

研究采用PCR-SSCP对安卡鸡TLR4基因外显子2多态性进行检测,并分析其多态性对经济性状的影响。结果表明:安卡鸡TLR4基因存在AA、AB和BB 3种基因型,基因型频率分别为0.3143、0.4000和0.2857,等位基因A、B的频率分别为0.5143和0.4857。χ2适合性检验表明,安卡鸡TLR4基因外显子2等位基因的分布处于Hardy-Weinberg平衡状态。序列分析结果显示,TLR4基因外显子2的114 bp和142 bp处各有1个G/A突变,G114A为沉默突变,没有引起氨基酸改变,G142A为错义突变,氨基酸由谷氨酸转变为赖氨酸。分析这3种基因型与安卡鸡一些重要经济性状间的关联性,表明,3种基因型个体间的生长发育性状、屠宰性状以及常规肉质等重要经济指标差异不显著,安卡鸡TLR4基因外显子2对重要经济性状没有显著的影响。