Stuart C. Helfand

Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit

Spontaneous mast cell tumors (MCT) are the most common malignant neoplasm in the dog, representing between 7% and 21% of all canine tumors, an incidence much higher than that found in humans. These tumors often behave in an aggressive manner, metastasizing to local lymph nodes, liver, spleen, and bone marrow. The proto-oncogene c-kit is known to play a critical role in the development and function of mast cells. Point mutations in the kinase domain of c-kit leading to tyrosine phosphorylation in the absence of ligand binding have been identified in three mastocytoma lines, (P815, RBL, and HMC-1), and some human patients with various forms of mastocytosis. We now demonstrate that although c-kit derived from canine MCT did not contain the previously described activating point mutations, 5 of the 11 tumors analyzed possessed novel mutations consisting of tandem duplications involving exons 11 and 12. We also show that one such duplication, detected in a canine mastocytoma cell line, was associated with constitutive phosphorylation of c-kit protein (KIT), suggesting that these mutations may contribute to the development or progression of canine MCT.

Expression of stem cell factor receptor (c-kit) by the malignant mast cells from spontaneous canine mast cell tumours

Stem cell factor receptor (SCFR, c-kit), normally expressed on haematopoietic and mast cells, plays a regulatory role in cellular growth and differentiation. Dysregulated expression of SCFR may contribute to neoplastic transformation. We investigated expression of SCFR on malignant canine mast cells obtained directly from spontaneous canine mast cell neoplasms, in an attempt to determine whether these undifferentiated cells maintained expression of this growth-promoting cytokine receptor. Malignant mast cells (histological grade 2) from skin tumours or lymph node metastases were collected from canine patients, and SCFRs were detected by flow cytometric analysis of these cells. All of the tumours bound mouse and canine recombinant stem cell factor (SCF), indicating that the cells not only expressed SCFRs, but that the receptors possessed the functional property of ligand binding. Immunoglobulin Fc receptors for canine IgE were identified on these cells by flow cytometry, a further indication that the cells analysed were mast cells and retained some differentiated features. Immunohistochemical analysis of formalin-fixed, paraffin wax-embedded mast cell tumour biopsies confirmed expression of SCFRs by malignant cells from each tumour. The relative binding of SCF to suspensions of tumour cells, as assessed by flow cytometry, correlated with the intensity of immunolabelling for SCFR in sections of the same tumours, suggesting variability in SCFR expression between tumours. Agarose gel electrophoresis of the products of SCFR reverse transcription-polymerase chain reaction derived from each tumour had the molecular weight predicted for canine SCFR cDNA on the basis of the mouse and human counterparts. This further confirmed SCFR expression by malignant canine mast cells. Taken together, these results show that a membrane receptor capable of triggering cell growth is expressed by malignant canine mast cells, suggesting a role for this receptor in the aetiology of canine mast cell cancer. This relatively common malignancy of the dog would seem to present an opportunity for the investigation of the potential role of the SCF/SCFR pathway in the development of spontaneous malignancies of mast cells.

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (αvβ3 integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that ‘nuclear factor of activated T cells’ activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.

Mutations of Phosphatase and Tensin Homolog Deleted from Chromosome 10 in Canine Hemangiosarcoma

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain.

Inactivation of the p16 cyclin-dependent kinase inhibitor in high-grade canine non-Hodgkin's T-cell lymphoma

The significance of p16/Rb tumor suppressor pathway inactivation in T-cell non-Hodgkins lymphoma (NHL) remains incompletely understood. We used naturally occurring canine NHL to test the hypothesis that p16 inactivation has specific pathologic correlates. Forty-eight samples (22 T-cell NHL and 26 B-cell NHL) were included. As applicable, metaphase- or array-based comparative genomic hybridization, Southern blotting, promoter methylation, and Rb phosphorylation were used to determine the presence, expression, and activity of p16. Fishers exact test was used to test for significance. Deletion of p16 (or loss of dog chromosome 11) was restricted to high-grade T-cell NHL (lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified). These were characterized by a concomitant increase of tumor cells with Rb phosphorylation at canonical CDK4 sites. Rb phosphorylation also was seen in high-grade B-cell NHL (diffuse large B-cell lymphoma and Burkitt-type lymphoma), but in those cases, it appeared to be associated with c-Myc overexpression. The data show that p16 deletion or inactivation occurs almost exclusively in high-grade T-cell NHL; however, alternative pathways can generate functional phenotypes of Rb deficiency in low-grade T-cell NHL and in high-grade B-cell NHL. Both morphologic classification according to World Health Organization criteria and assessment of Rb phosphorylation are prognostically valuable parameters for canine NHL.

Aberrant p53 expression in feline vaccine-associated sarcomas and correlation with prognosis.

Eighty spontaneously occurring feline vaccine-associated sarcomas (VAS) were evaluated to determine the immunohistochemical expression of the tumor suppressor gene p53. Sixty-five of 80 VAS (81%) exhibited positive immunoreactivity with Mab240, a murine monoclonal antibody that specifically recognizes mutated p53. Only 44 of 81 tumors (55%) were positive with rabbit polyclonal antibody CM-1. CM-1 often yielded nonspecific staining of nonneoplastic tissues. Nonspecific staining was greatly reduced or absent with Mab240. Cytoplasmic staining for p53 was a consistent pattern of VAS, occurring in 44% of tumors evaluated. Cats with tumors that exhibited cytoplasmic p53 had significantly shorter time to tumor recurrence compared to those cats with tumors that exhibited nuclear p53 staining (P = 0.0284), but no significant difference in survival outcome was observed. Immunohistochemical detection of p53 offers a prognostic tool for VAS, and, because abnormal p53 expression appears to be a common feature of feline VAS, molecular targeting of mutant p53, may offer a promising new therapeutic opportunity for this cancer.

Enhancing antimelanoma immune responses through apoptosis

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas+ melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

Current Studies of Liposome Muramyl Tripeptide (CGP 19835A Lipid) Therapy for Metastasis in Spontaneous Tumors: A Progress Review*

Targeted delivery of macrophage activating agents is an attractive approach to treat micrometastatic disease. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) is a potent activator of monocytes/macrophages in humans, mice, and dogs. We have conducted clinical trials in dogs with malignant and highly metastatic spontaneous tumors. Presented are results of our trials evaluating L-MTP-PE in combination with surgery and chemotherapy in dogs with spontaneous osteosarcoma and hemangiosarcoma, particularly relevant malignancies having having many similarities to human cancer. Osteosarcoma dogs received chemotherapy following surgery (cisplatin q 28 days x 4). At completion of chemotherapy, dogs were randomized to receive L-MTP-PE or placebo. The L-MTP-PE group had a significantly longer median survival time compared to the placebo group (p < 0.021). Dogs with splenic hemangiosarcoma received combination chemotherapy following surgery (doxorubicin and cyclophosphamide q 21 days x 4). At the first chemotherapy, dogs were randomized to receive L-MTP-PE or placebo. The L-MTP-PE group had a significantly longer median survival time compared to the placebo group (p < 0.03). These studies show that L-MTP-PE is an effective agent for treatment of metastasis and can be safely administered in combination with chemotherapy.

Immunophysiological studies of interleukin-2 and canine lymphocytes

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dogs PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.