Abstract: Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.

Abstract: The dorsal hair ridge in Rhodesian and Thai Ridgeback dogs is caused by a dominant mutation that also predisposes to the congenital developmental disorder dermoid sinus. Here we show that the causative mutation is a 133-kb duplication involving three fibroblast growth factor (FGF) genes. FGFs play a crucial role in development, suggesting that the ridge and dermoid sinus are caused by dysregulation of one or more of the three FGF genes during development.

Abstract: The objectives of this study were to describe the incidence of, survival until, and survival after the diagnosis of canine bone tumors by breed, sex, age, and geographic location of residence. Dogs under 10 y old and insured by a Swedish insurance company between 1995 and 2002 were studied. In total, 764 dogs had claims for bone tumors, and the incidence rate was 5.5 cases per 10 000 dog-years at risk (DYAR). At ages 6, 8, and 10 y, the proportions of dogs with bone tumors were 0.13%, 0.30%, and 0.64%. The top 3 breeds at risk were Irish wolfhound, St. Bernard, and leonberger (incidence rates 99, 78, and 53 cases per 10 000 DYAR, respectively). Median survival time after diagnosis was 56 d in the 419 dogs that survived > or = 1 d. With a Cox regression model controlling for breed and age, females were shown to be at decreased risk of bone tumors, with a hazard ratio of 0.71 (99% confidence interval 0.58 to 0.87).

Abstract: With several hundred genetic diseases and an advantageous genome structure, dogs are ideal for mapping genes that cause disease. Here we report the development of a genotyping array with approximately 27,000 SNPs and show that genome-wide association mapping of mendelian traits in dog breeds can be achieved with only approximately 20 dogs. Specifically, we map two traits with mendelian inheritance: the major white spotting (S) locus and the hair ridge in Rhodesian ridgebacks. For both traits, we map the loci to discrete regions of <1 Mb. Fine-mapping of the S locus in two breeds refines the localization to a region of approximately 100 kb contained within the pigmentation-related gene MITF. Complete sequencing of the white and solid haplotypes identifies candidate regulatory mutations in the melanocyte-specific promoter of MITF. Our results show that genome-wide association mapping within dog breeds, followed by fine-mapping across multiple breeds, will be highly efficient and generally applicable to trait mapping, providing insights into canine and human health.

Abstract: The aim of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA), for determination of serum thymidine kinase 1 (sTK1) activity in dogs with malignant lymphoma (ML) and compare it with a thymidine kinase (TK) radioenzymatic assay (TK-REA). The TK-REA has recently been shown to be useful in determining the clinical stage and prognosis in canine ML. In addition, serum lactate dehydrogenase (LDH) was measured. Forty-five dogs were included in the study. Sixty serum samples from these dogs, stored in a tumour serum sample bank (stored at -20 degrees C), were analysed. Apart from 37 dogs with ML, four normal dogs as well as two dogs with mammary carcinomas, one dog with bladder carcinoma, and one dog with malignant fibrous histiocytoma were included. Staging of ML was based on the modified World Health Organization (WHO) staging system for canine ML. The diagnosis of all tumours was verified by histopathology. The TK activity (units per litre [U/L]) ranged from 1.0 to 607.9 in the TK-REA analysis and from 1.1 to 510 in the TK-ELISA (normal reference value <7U/L). The range for LDH was between 12 and 1194 U/L (normal reference value <228 U/L). There was a significant correlation between the TK-REA and the TK-ELISA. The correlation coefficient (CC) was 0.97 and the standard error of the estimate (SEE) was 3.7 U/L. There was no correlation between LDH and either the TK-REA or the TK-ELISA (CC=0.53 for both assays; SEE=26.7 and 12.7 U/L, respectively). Most of the variation in LDH was still within the normal reference range. The mean LDH in dogs with high-stage (stage IV+V) disease was 201.9 U/L. The corresponding values for the TK-REA and TK-ELISA were 109 and 109.9 U/L, respectively. The significant relation between the TK-REA and the TK-ELISA was confirmed by Bland-Altman analysis. The TK-ELISA assay, because of its relative simplicity, will permit measurement of TK in cases of ML in dogs to become a routine procedure.

Abstract: Multiple endocrine neoplasia (MEN) is defined as concurrent neoplasia or hyperplasia in more than one endocrine gland. MEN is well known in humans and has also been reported in small animals. We report on a dog family of a mixed breed with Alaskan malamute as a major influence, where three members developed thyroid carcinomas and another dog had clinical signs mimicking the other three but without a confirmed diagnosis. The age of onset of the tumour was between 96-109 months. Clinical, biochemical and immunohistochemical examinations revealed that the affected individuals typically demonstrated symptoms including calcitonin positive thyroid cancer, hypothyroidism and chronic dermatitis. In addition, elevated serum calcium and multinodular adrenocortical hyperplasia were demonstrated in a single member. The diagnosis observed is similar to the familial form of medullary thyroid carcinoma (FMTC) in human. This is the first report of FMTC in dog. Up to 95% of FMTC and MEN2 is known to be caused by activating mutations in the RET gene. The dog Ret gene was analysed as a candidate in this pedigree. The complete dog Ret genomic sequence was predicted in silico. The lack of demonstratable Ret mutation suggests the involvement of alternative predisposing mutation in this pedigree. The unique occurrence of familial MTC makes this potentially an important model in further defining the genetic basis of MTC.

Abstract: The main objective of this study was to describe the incidence of mammary tumors (MTs) and the survival after MTs, in female dogs between 3 and 10 years of age (insured for veterinary care and with life insurance in a Swedish animal-insurance company) from 1995 to 2002. Measures of incidence are presented crudely, by breed and across age categories and birth cohorts (1991-1998). The survivals until MT diagnosis and after a MT diagnosis were computed. The overall incidence for any MT claim was 111 dogs per 10,000 dog-years at risk (DYAR). The overall MT rate in the 1992 and 1993 birth cohorts was 154 dogs per 10,000 DYAR. The incidence for any MT claim increased with age and varied by breed, from 319 dogs per 10,000 DYAR in the English springer spaniel to 5 dogs per 10,000 DYAR in the rough-haired collie. At the ages 6, 8 and 10 years, 1%, 6% and 13% respectively, of all females had at least one MT claim. The MT mortality was 6 deaths per 10,000 DYAR and increased with age. The overall-case fatality was 6%.

Abstract: Heterogeneous gene expression in tumours and the degradation of RNA when sampling under non-RNAse-free conditions may limit the potential benefit of cDNA array studies. This study examines changes in the integrity of RNA by means of RNA gel electrophoresis at various post-operative intervals on canine mammary tumours (n=10) and malignant lymphoma (n=1). The tumours were cut into pieces (3-5 mm diameter, approximately 50 mg) and kept in tubes without RNAse-free buffer at room temperature. No special precautions were taken to avoid the influences of Rnase; rather, normal surgical procedures were used. We found that total RNA of the mammary tumours started to degrade within 30 min of the operation, and the rate of degradation increased up to 4 h, which was the last time point included in this study. RNA in the lymphoma tumours degraded more rapidly, and was completely degraded at 30 min post-operation. The degradation of mRNA in the mammary tumours, as studied by human cDNA arrays, was heterogeneous, i.e. some mRNA degraded completely, some only partially. This indicates that the mRNA degradation rate varied depending on the type of mRNA. However, since we found that gene expression differs depending on the part of the mammary tumour examined, one cannot exclude that the variation in the mRNA degradation rate may simply reflect heterogeneous gene expression within the tumour. We conclude that RNA integrity is unaffected immediately after sampling under non-RNAse-free conditions; however, the tumour sample should be preserved under RNAse-free conditions within 15 min to avoid RNA degradation. This is a much shorter time interval than previously reported in other similar studies; however, these studies generally treated normal tissue, under which 3-5 h non-RNAse-free conditions have been found not to affect RNA quality.

Abstract: BACKGROUND: Several authors have recently reported encouraging results from Electrochemical treatment (EChT) in malignant tumours. However, EChT is not established and mechanisms are not completely understood. In vivo studies were conducted to evaluate the toxic changes and effectiveness of EChT on an animal tumour model. METHODS: Tumours were induced by injecting cells from the R3230AC rat mammary tumour cell line clone D subcutaneously, in 28 female Fischer 344 rats. EChT was conducted by inserting a platinum electrode into the tumours. The positive and negative control groups were subjected to the same conditions but without current. The rats were kept for 0, 7 or 14 days post-treatment. Three hours prior to euthanasia an i.p. injection of Bromodioxyuridine (BrdU) was given. The rats were euthanized, the lesions extirpated and samples were collected for histopathological, and immunohistochemical examination. RESULTS: Significant changes in cell proliferation rate were seen both in the cathode and anode regions. Apoptosis were induced in the anodic treated area outside the primary necrosis, detected with the TUNEL method. DISCUSSION: The results suggest that secondary cell destruction was caused by necrosis with cathodic EChT and apoptosis or necrosis with anodic EChT.

Abstract: Serum thymidine kinase (sTK) activity was evaluated as a tumor marker for canine malignant lymphoma (ML). The objective was to investigate if sTK, as in humans, could be used as a prognostic marker for survival time in dogs with ML and if sTK could identify early signs of progression of disease in treated dogs. Serum samples from 52 dogs with ML were tested for initial TK activity. Samples from 21 normal dogs and 25 dogs with nonhematologic neoplasms were used for comparison. Forty-four dogs with ML were treated. Serum TK activity was measured in treated dogs before each treatment and every 4 weeks thereafter until relapse. Dogs with ML had 2-180 times higher TK activity (TK 5-900 U/L) than normal dogs (TK <7 U/L) based on the mean + 2 standard deviations. In the group of other neoplasms, only 2 dogs had a moderate increase (6.4 and 7.5 U/L) compared with the controls. Mean sTK activities in the dogs with ML that had gone into complete remission (CR) were not significantly different from activities in healthy controls (P = .68). Mean sTK at least 3 weeks before and at the time of relapse was significantly higher than activity measured at CR (P < .0001). Dogs with ML that initially had sTK >30 U/L had significantly shorter survival times (P < .0001). Furthermore, sTK activity reflected the clinical staging of ML. Measuring sTK can be used as a powerful objective tumor marker for prognosis and for predicting relapse before recurrence of clinically detectable disease in dogs with ML undergoing chemotherapy.

Abstract: INTRODUCTION: Electrochemical treatment (EChT) has been taken under serious consideration as being one of several techniques for local treatment of malignancies. The advantage of EChT is the minimal invasive approach and the absence of serious side effects. Macroscopic, histopathological and ultra-structural findings in liver following a four-electrode configuration (dog) and a two-electrode EChT design (dog and rat) were studied. MATERIALS AND METHODS: 30 female Sprague-Dawley rats and four female beagle dogs were studied with EChT using Platinum:Iridium electrodes and the delivered dose was 5, 10 or 90 C (As). After EChT, the animals were euthanized. RESULTS: The distribution of the lesions was predictable, irrespective of dose and electrode configuration. Destruction volumes were found to fit into a logarithmic curve (dose-response). Histopathological examination confirmed a spherical (rat) and cylindrical/ellipsoidal (dog) lesion. The type of necrosis differed due to electrode polarity. Ultra-structural analysis showed distinct features of cell damage depending on the distance from the electrode. Histopathological and ultra-structural examination demonstrated that the liver tissue close to the border of the lesion displayed a normal morphology. CONCLUSIONS: The in vivo dose-planning model is reliable, even in species with larger tissue mass such as dogs. A multi-electrode EChT-design could obtain predictable lesions. The cellular toxicity following EChT is clearly identified and varies with the distance from the electrode and polarity. The distinct border between the lesion and normal tissue suggests that EChT in a clinical setting for the treatment of liver tumours can give a reliable destruction margin.

Abstract: INTRODUCTION: The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event. METHODS: Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2'-deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis. RESULTS: In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9- and 10-treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min. CONCLUSIONS: pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic.

Abstract: BACKGROUND: A reinvented technique for tumour therapy, electrochemical treatment (EChT), is attracting increasing attention. This study compared results from treatment of liver and mammary tissue focusing on destruction and pH changes in the tissue close to the treatment electrodes. Subsequently, data were compared with a dose-planning model. METHODS: Mammary or liver tissue in 50 adult female Sprague Dawley rats was given EChT with a constant, direct current. The electrodes used were Pt/Ir (9:1) with spherical tips. In situ pH measurements were taken with a micro-combination glass electrode. RESULTS: Spherical lesions were produced in both liver and mammary tissue. No significant difference was detected when comparing the size of the lesions in the two kinds of tissue. Similar pH profiles were obtained in tissue surrounding the electrodes, with pH values changing rapidly from unphysiological to neutral status within the space of a few millimetres. The pH at the border of the macroscopic destruction zone, regardless of tissue type or coulomb dosage, correlated well with specific values (4.5-5.5 at the anode and between 9 and 10 at the cathode). CONCLUSION: The analogous destruction patterns in mammary and liver tissue support the hypothesis that EChT has similar results in at least these two different types of tissue. This implies that the destructive pattern caused by the treatment may be the same also in tumours.

Abstract: The electrochemical treatment (EChT) of tumours implies that tumour tissue is treated with a continuous direct current through two or more electrodes placed in or near the tumour. The treatment offers considerable promise of a safe, simple and relatively noninvasive anti-tumour therapy for treatment of localised malignant as well as benign tumours. Although more than 10,000 patients have been treated in China during the past 10 years, EChT has not yet been universally accepted. The reason for this is the lack of essential preclinical studies and controlled clinical trials. Uncertainties regarding the destruction mechanism of EChT also hinder the development of an optimised and reliable dose-planning methodology. This article reviews the collected Chinese and occidental experiences of the electrochemical treatment of tumours, alone and in combination with other therapies. The current knowledge of the destruction mechanism underlying EChT is presented along with different approaches towards a dose planning methodology. In addition, we discuss our view of different important parameters that have to be accounted for, if clinical trials are to be initiated outside of China.