Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

Vinblastine as a second rescue for the treatment of canine multicentric lymphoma in 39 cases (2005 to 2014).

OBJECTIVES: The objective of this study was to retrospectively evaluate response and outcome of dogs with multicentric lymphoma treated with single-agent vinblastine as a second rescue. METHODS: Medical records from 39 client-owned dogs receiving vinblastine rescue treatment (having relapsed on or following completion of UW-Madison and CCNU/L-asparaginase protocols), between 2005 and 2014, were reviewed for information regarding clinical presentation, diagnostic testing, drug dosage, number of treatments, side effects, response and outcome. RESULTS: The median starting dose of vinblastine was 2·6 mg/m(2) (1·7 to 2·8 mg/m(2) ), administered weekly until disease progression. Of the 39 dogs treated, 3 dogs (7·7%) achieved a complete remission, 7 dogs (17·9%) achieved a partial response, 18 dogs (46·2%) maintained stable disease and 11 (28·2%) had progressive disease. Ten dogs (25·6%) developed a grade III or IV neutropenia, and 4 dogs (10·3%) developed grade III or IV thrombocytopenia (one dog in both categories). After starting vinblastine, the median progression-free survival was 29·5 days (0 to 77 days) and overall median survival time was 46 days (4 to 250 days). Duration of first remission was identified as a positive predictor of outcome. CLINICAL SIGNIFICANCE: Single-agent vinblastine is well tolerated in dogs with relapsed or refractory lymphoma. Responses were incomplete and short-lasting.

Effects of Toceranib Phosphate on Systolic Blood Pressure and Proteinuria in Dogs.

BACKGROUND: Systemic hypertension and proteinuria are established adverse effects of tyrosine kinase inhibitor treatment in people. OBJECTIVE: The objective of this study was to investigate changes in systolic blood pressure and the incidence of proteinuria secondary to treatment with toceranib phosphate in dogs with cancer. ANIMALS: Twenty-six control dogs and 30 dogs with cancer were evaluated for the first part of the study (baseline characteristics). For the second part (effect of toceranib phosphate treatment), 48 client-owned dogs were evaluated, including 20 control dogs and 28 dogs with various types of neoplasia. METHODS: Prospective cohort study. Client-owned healthy control dogs and dogs with cancer were enrolled. Blood pressure and urine protein:creatinine ratios were measured before treatment and 2 weeks after initiation of toceranib phosphate treatment. RESULTS: Systolic blood pressure was significantly (P = 0.0013) higher in previously normotensive treatment dogs after initiation of treatment with toceranib phosphate (152 mmHg ± 19) compared to baseline (136 mmHg ± 14). 37% of treated dogs developed SBP ≥ 160 mmHg. The prevalence of systemic hypertension (37%) and proteinuria (21%) at baseline in treatment dogs did not differ from that of age-matched healthy controls (15% [P = 0.13] and 0% [P = 0.069], respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Toceranib phosphate treatment might result in increased systolic blood pressures in dogs. Systemic hypertension should be considered a potential adverse effect of this drug in dogs. Systemic hypertension and proteinuria were detected at clinically relevant frequencies in the dogs with cancer before antineoplastic therapies suggesting that monitoring of these variables might be warranted in this population.

Osteosarcoma tissues and cell lines from patients with differing serum alkaline phosphatase concentrations display minimal differences in gene expression patterns.

Serum alkaline phosphatase (ALP) concentration is a prognostic factor for osteosarcoma in multiple studies, although its biological significance remains incompletely understood. To determine whether gene expression patterns differed in osteosarcoma from patients with differing serum ALP concentrations, microarray analysis was performed on 18 primary osteosarcoma samples and six osteosarcoma cell lines from dogs with normal and increased serum ALP concentration. No differences in gene expression patterns were noted between tumours or cell lines with differing serum ALP concentration using a gene-specific two-sample t-test. Using a more sensitive empirical Bayes procedure, defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was increased in both the tissue and cell lines of the normal ALP group. Using quantitative PCR (qPCR), differences in DCUN1D1 expression between the two groups failed to reach significance. The homogeneity of gene expression patterns of osteosarcoma associated differing serum ALP concentrations are consistent with previous studies suggesting serum ALP concentration is not associated with intrinsic differences of osteosarcoma cells.

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro.

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.

Activation of the canonical Wnt/ß-catenin signalling pathway is rare in canine malignant melanoma tissue and cell lines.

Canine malignant melanoma is a highly aggressive tumour associated with a poor overall survival rate due to both local disease recurrence and its highly metastatic nature. Similar to advanced melanoma in man, canine oral melanoma is poorly responsive to conventional anti-cancer therapies. The lack of sustainable disease control warrants investigation of novel therapies, preferably targeting features specific to the tumour and different from normal cells. The Wnt signalling pathway is known to contribute to melanocytic lineage development in vertebrates and perturbation of the Wnt/ß-catenin pathway has been implicated in numerous cancer types. Alterations of the Wnt/ß-catenin pathway are suggested to occur in a subset of human melanomas, although the precise role of the Wnt/ß-catenin pathway in melanoma is yet to be defined. This study investigates the activation status of the canonical Wnt/ß-catenin pathway in canine malignant melanoma and its potential as a therapeutic target for treating this disease. The data indicate that canonical Wnt/ß-catenin pathway activation is a rare event in canine oral malignant melanoma tissue and canine malignant melanoma cell lines.

Characterization of ß-catenin expression in canine osteosarcoma.

Osteosarcoma (OSA) is the most frequently occurring malignant primary bone tumour in dogs and children and arises from cells of the osteoblast lineage. Inappropriate Wnt signalling activity has been implicated in human OSA. Altered expression of ß-catenin, an integral member of the Wnt signalling pathway, has been associated with numerous human cancers, including OSA. In this study, 30 of the 37 primary canine OSA tissues and 2 of the 3 metastatic OSAs were positive for ß-catenin expression as determined by immunohistochemistry, whereas 2 normal bones stained negative for ß-catenin. No mutations were identified in exon 3 of ß-catenin in the three OSA cases in which DNA sequencing was performed. Finally, there was no relationship between ß-catenin expression and overall survival time or disease-free interval. Our results indicate ß-catenin is frequently expressed within the cytoplasm of neoplastic cells in canine OSA but contains no detectable mutations in exon 3, similar to human OSA.

Doxorubicin and cyclophosphamide for the treatment of canine lymphoma: a randomized, placebo-controlled study.

Median survival times (STs) for doxorubicin-treated canine lymphoma range from 5.7 to 9 months. Because dogs treated with multi-agent protocols have longer STs, we sought to evaluate whether adding cyclophosphamide would improve outcome in canine lymphoma patients while maintaining an acceptable level of toxicity. Thirty-two dogs with stage III-V multicentric lymphoma were treated with doxorubicin every 3 weeks for five total cycles and prednisone at a tapering dose for the first 4 weeks. Dogs were randomized to receive either cyclophosphamide or placebo concurrently. Seventeen dogs received doxorubicin and placebo, while 15 dogs received doxorubicin and cyclophosphamide. Response, toxicity, progression-free interval (PFI) and ST were evaluated. The combination of doxorubicin and cyclophosphamide was well tolerated, causing no increase in adverse events over doxorubicin alone. Despite a numeric improvement in outcome in cyclophosphamide treated dogs, the addition of cyclophosphamide did not result in statistically improved response rate, PFI or ST.

Pararadicular cementum deposition as a criterion for age estimation in human beings.

The purpose of this article is to ascertain if pararadicular cementum can be used as a reliable criterion for age estimation in human beings. Fifty-two nonrestorable teeth were extracted from 42 patients at the Veterans Administration Medical Center at Ann Arbor, Mich. The specimens were prepared to a thickness of 500 microns with a Buehler Isomet bone saw (Bronwell Scientific, Inc., Rochester, N.Y.). Longitudinal sections were cleaned of artifacts in an ultrasonic cleaner and stained with 1% alizarin red. Photomicrographs were taken of each prepared section. The cementum was composed of multiple light and dark bands that were counted on the photograph and added to the average eruption time of the individual tooth. There was an overall Pearson's product-moment correlation coefficient of r = 0.93 between the patient's predicted age with the use of cementum annulations as compared with the actual chronologic age of the person. Predicted age counts showed greater divergence from actual age in persons older than 55 years. A formula is presented to adjust for this discrepancy. The data indicate that quantitation of cementum annuli is a moderately reliable means for age estimation in humans.

Radiographic "working length" revisited.

The desired "working length" for the biomechanical preparation and resultant obturation of the root canal system is one of the most important phases of endodontics. Traditionally, radiographs are used to confirm working length of the root length and to evaluate the subsequent obturation of the root canal system. This study attempts to determine if radiographs are an accurate method of root length determination in a period when electronic apex locators are proposed as their replacement. Eight-seven vital and 24 nonvital teeth were accessed and files placed to and the radiographic working length before the file and teeth were extracted for sectioning. Results showed that the radiographic distance of the file from the apical vertex was 0.7 mm shorter than the actual file position. This discrepancy can lead the clinician to try to get closer to the radiographic apex, when in reality the end of the file is closer to the vertex than is suspected. The average distance short of the vertex as established by Kuttler should be increased to lie between 1.5 and 2.0 mm from the apical vertex to prevent overfilling the root canal.

Nonionizing method of locating the apical constriction (minor foramen) in root canals.

The electronic method was evaluated as to its accuracy when used to determine the position of the apical constriction (minor foramen) in root canals. There were 39 vital and 8 nonvital teeth for a total of 47 specimens from 22 patients. The specimens were prepared with a Buehler Isomet bone saw to a thickness of 500 microns. Distances were measured and recorded with the use of a Bioquant II image analysis system. The electronic method appeared to measure a mean value of 0.2 mm coronal to the cemetodentinal junction in 47 canals where the apex locator was set at a reference setting of 40. When a frequency curve was plotted, it showed that the probability of being within 0.76 mm (1 SD) of the cementodentinal junction was 68%. This appears to correlate to where Kuttler indicated the minor constriction to be located. These devices seem to offer a unique method to locate the apical constriction and thus to ensure proper working length while reducing ionizing radiation.

Influence of the major and minor foramen diameters on apical electronic probe measurements.

There have been conflicting reports on the accuracy of electronic devices used for determining working length. The influence of the major and minor diameters on electronic probe measurements were evaluated to ascertain whether anatomical features of the apical portion of the canal might be responsible for these discrepancies. Forty-seven nonrestorable teeth selected from 22 patients were studied. Conventional access was made. A Kerr file was placed to a position 0.5 mm from the major foramen as registered by the Neosono-D apex locator. The electronic probe length was then measured. After the tooth was extracted, the file was fixed with autopolymerizing composite in the canal. A Buehler Isomet lapadary saw was used to prepare specimens to a thickness of 500 microns. A Bioquant II Image Analysis System was used to measure and record distances. It was found that as the width of the major foramen increased, the discrepancy between the electronic probe tip length induction and the actual position of the major foramen increased.

Anatomy of the root apex and its histologic changes with age.

There were 87 vital and 24 nonvital teeth for a total of 111 specimens from 47 patients. The specimens were prepared to a thickness of 500 microns with a Buehler Isomet Bone Saw. Distances were measured and recorded with a Bioquant II Image Analysis System. The mean age of the patients was 48.9 years. The median age was 52 years. The range was 51 years, with a maximum of 77 years and a minimum of 26 years. There is a positive correlation, which could not have occurred by chance, that as age increases the deviation and the width of the foramen opening both increase. This increase appears to be a result of apical cemental thickening that occurs as the patient ages. The tissue in the apical area seems to be capable of generating additional cementum as the tooth ages. The cementodentinal junction width, or most apical extent of the dentin, remains constant.

Some ethical considerations of short-term workshops in the principles and methods of behavior modification.

Workshops and seminars to expose different sectors of the professional community to the principles and applications of behavior modification are briefly discussed. The possible misapplication of procedures by conference participants, whose only exposure to behavioral methods has been at these workshops is viewed as a potentially serious ethical issue. It is suggested that the goals of such seminars and workshops must be clarified, and methods of evaluation of the participants' skills devised, lest we contribute to the misapplication of procedures and to the criticism that behavioral methods are unethical approaches to treatment.