These results provide evidence that ITDs in c-kit occur frequently in MCTs of dogs. The high prevalence of c-kit activating mutations in MCTs of dogs combined with the relative abundance of mast cell disease in dogs provide an ideal naturally developing tumor in which to test the safety and efficacy of novel small-molecule kinase inhibitors such as imatinib mesylate.

IntroductionThe proto-oncogene c-kit was first identified as the oncogenic component of the acutely transforming Hardy-Zuckerman 4-feline sarcoma virus. 1 It encodes Kit, a type 3 receptor tyrosine kinase (RTK) that binds the ligand stem cell factor (SCF). 2 Structurally, Kit is related to the receptors for platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, and FLT-3 ligand (reviewed in Ashman 3 ). All contain an extracellular ligand-binding domain composed of 3 to 7 immunoglobulinlike regions, a transmembrane domain, a negative regulatory juxtamembrane (JM) domain, and 2 kinase domains in which resides a kinase insert. SCF-Kit interactions promote the development of mast cells from hematopoietic progenitors; mice deficient in either SCF or Kit (Sl or W mutant mice) exhibit a near-complete absence of mast cells, anemia, and thrombocytopenia. 2,4-6 These mice are also sterile and lack skin pigmentation because of the absence of melanocytes. With regard to mast cells, SCF-Kit interactions are intimately involved in several critical processes. Most important, Kit signaling is required for the differentiation and maturation of mast cells in vivo. 2,[7][8][9][10] Other activities attributable to SCF-Kit binding include chemotaxis and haptotaxis of mast cells and the promotion of cell survival and proliferation. 2,7,8,11,12 Several years ago, investigators began to study the potential role of Kit dysfunction in malignant mast cells. Point mutations in c-kit that lead to constitutive activation of Kit in the absence of ligand binding were identified in 3 malignant mast cell lines (human HMC-1, rat RBL, and mouse P815), providing an indication that dysregulation of Kit may promote uncontrolled growth or survival of mast cells. [13][14][15] Following this discovery, similarly activating mutations were identified in the CD34 ϩ hematopoietic precursors from patients with mastocytosis and in cells from patients with urticaria pigmentosa, thus providing the first evidence that c-kit may play a role in spontaneous mast cell disease. 16,17 Recent studies have consistently demonstrated the presence of activating point mutations in the catalytic domain of c-kit in human patients with aggressive mastocytosis, mast cell leukemia, and hematologic disorders with associated mastocytosis. [17][18][19][20][21][22][23] In contrast to the disease in humans, mast cell neoplasms are one of the most common malignant tumors of the dog, representing between 7% and 21% of all canine tumors. 24,25 Given the link between mast cell disorders and dysfunctional Kit, we investigated whether similar mutations in c-kit were present in canine mast cell tumors (MCTs). Although c-kit derived from the canine MCTs did not contain the previously described activating mutations, 30% to 50% of the tumors examined had novel mutations consisting of tandem duplications in exons 11 and 12 of the gene. 26 As is the case with tandem duplications in exons 11 and 12 of Flt-3, these mutations also result in constitutive phosph...

To determine whether live-attenuated feline immunodeficiency virus (FIV) proviral DNA will induce protective immunity, a plasmid clone constructed with a FIV provirus containing a deletion in the viral accessory gene vif (FIV-pPPR-Deltavif) was inoculated as proviral DNA into four cats by the intramuscular route. After 43 weeks, these cats were boosted with the same proviral plasmid. Analysis of peripheral blood mononuclear cells at several time points after the primary and booster inoculations revealed no detectable virus or proviral DNA. At 6 weeks after the booster, immunized cats and additional naive control cats were challenged with a cell-free preparation of the infectious biological isolate FIV-PPR by the intraperitoneal route. Virus was detected after challenge in unvaccinated control cats but not in any of the FIV-pPPR-Deltavif-immunized cats. Both FIV Gag- and Env-specific cytotoxic T lymphocyte (CTL) activities were detected in peripheral blood cells of control cats after challenge infection, whereas only one of four cats immunized with FIV-pPPR-Deltavif DNA exhibited a measurable CTL response to Env following challenge. Although anti-Gag antibodies were not detected after both proviral DNA inoculation and challenge, anti-Env antibodies were found in FIV-pPPR-Deltavif-immunized cats after vaccination as well as after challenge. These findings indicate that inoculation with FIV-pPPR-Deltavif proviral DNA induced resistance to challenge with infectious FIV and that a vif deletion mutant may provide a relatively safe attenuated lentiviral vaccine.

An activating mutation in codon 599 of BRAF has been identified in approximately 60% of human cutaneous nevi and melanomas, but not melanomas of mucosal origin. The purpose of this study was to determine if BRAF mutations occur in canine oral malignant melanomas. The canine BRAF gene was first cloned from normal canine testicular cDNA, and a novel previously unreported splice variant involving exon 5 was identified during this process. To screen canine melanoma samples for BRAF mutation in codon 599, cDNA and genomic DNA were isolated from canine malignant melanoma cell lines and primary tumor samples respectively, all from cases seen at the Veterinary Medical Teaching Hospital at the University of California, Davis. Polymerase chain reaction (PCR) was performed for exon 15 using primers based at the 5' end of exon 15 and the 5' end of intron 15 and the resultant products were directly sequenced. No mutations in codon 599 or exon 15 were identified in any of the 17 samples evaluated. However, all of the melanoma cell lines expressed BRAF and demonstrated high levels of basal ERK phosphorylation suggesting that dysregulation of this pathway is present. Therefore, similar to the case with human mucosal melanomas, canine oral malignant melanomas do not possess codon 599 BRAF mutations commonly identified in human cutaneous melanomas. This finding supports the notion that melanomas arising from non-sun-exposed sites exhibit distinct mechanisms of molecular transformation.

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