2015 ACVIM Forum Research Abstract Program

CHRONIC LYMPHOCYTIC LEUKEMIA. Emily Rout 1 , Robert Burnett 1 , Courtney Abbott 1 , Stacey George 1 , Anne Avery 1 . 1 Colorado State University, Fort Collins, CO, USA Canine B cell chronic lymphocytic leukemia (CLL) is common in dogs and shares many features with human CLL. In human CLL, immunoglobulin (Ig) gene usage and mutation status are important markers for disease behavior, but these factors have not been assessed in canine B-CLL. We sequenced the immunoglobulin heavy chain variable region (VH) genes from neoplastic peripheral blood B cells in 45 dogs with B-CLL. Thirteen VH genes were used. Excluding the Boxer breed, the most commonly used genes were VH1-44, VH1-62, and VH1-35. In non-neoplastic B cells, VH1-44 and VH1-62 are the most commonly used genes. 59% of all genes used in the B-CLL patients had greater than 2% mutations compared to germline sequence and were classified as mutated, while 41% were unmutated. Of the cases using VH1-44, one third used an unmutated gene. All the cases using VH1-62 and VH1-35 used mutated genes. VH1-41 is overrepresented in Boxers with B-CLL, but is not preferentially used in Boxers with other B-cell neoplasms. Of 8 Boxer dogs with B-CLL that were sequenced, 6 used an unmutated VH1-41 gene. These findings suggest that antigen selection may play a role in the pathogenesis of B-CLL in Boxers. These data lay the foundation to correlate VH gene usage and mutation status with outcome in dogs, further establish canine patients as good models of human CLL, and identify groups, like the Boxers, that may be useful in studying the pathogenesis of CLL. Imatinib has potent anti-tumor activity against canine mast cell tumor (MCT) carrying c-kit mutation. Although the tumors initially respond well to imatinib, they eventually develop resistance. Here, molecular mechanisms that confer imatinib resistance to neoplastic mast cells were investigated using an imatinib-sensitive canine neoplastic mast cell line VI-MC carrying a KIT c.1523A>T activating mutation. Two imatinib-resistant sublines were established by culturing VI-MC cells in increasing concentrations of imatinib (1 M-resistant, rVI-MC1; 10 M-resistant, rVI-MC10). Both sublines had a second KIT mutation c.2443G>C. Recombinant KIT with the second mutation was insensitive to 1 M but sensitive to 10 M imatinib. The effect of imatinib on the phosphorylation of KIT and its downstream signalling proteins was then examined using these sublines. KIT and ERK were constitutively phosphorylated in both sublines and their phosphorylation was suppressed by 10 M imatinib in rVI-MC1 cells. However, KIT but not ERK phosphorylation was supressed in rVI-MC10 cells. The phosphorylation of ERK in rVI-MC10 cells was also not diminished by the Src family kinase (SFK) inhibitor dasatinib. This second mutation in KIT may play an important role for imatinib resistance in neoplastic mast cells. Furthermore, KIT/SFK-independent activation of ERK would be involved in imatinib resistance when the neoplastic cells are exposed to higher concentrations of imatinib. Gemcitabine is an antimetabolite chemotherapy agent with clinical activity against a number of human malignancies. Gemcitabine is activated to its cytotoxic triphosphate form (dFdCTP) and in a concentration-dependent manner, competes for incorporation into the DNA and induces apoptosis. Whereas intracellular accumulation of dFdCTP is schedule dependent and relies on achieving and maintaining optimal extracellular pl