New treatments and techniques in veterinary oncology (Proceedings)

Tyrosine kinase inhibitors

Tyrosine kinases are proteins that phosphorylate other proteins on tyrosine residues thereby regulating cell growth and differentiation. They bind ATP and use this to add phosphate groups to key residues on themselves (termed autophosphorylation) and on other molecules, resulting in intracellular signaling and alterations in gene transcription that affect cell proliferation and survival. Those tyrosine kinases expressed on the cell surface (receptor tyrosine kinases, RTKs) bind growth factors that regulate their activation. Examples include Kit, Met, EGFR, all of which are known to be dysregulated in particular forms of cancer VEGFR, PDGFR, and FGFR are also important in promoting the growth of tumor blood vessels known as tumor angiogenesis. The most well described example of tyrosine kinase dysregulation is the Bcr-Abl fusion protein found in human patients with chronic myelogenous leukemia (CML) that results in chronic activation of the cytoplasmic kinase Abl. An RTK frequently altered in cancer is Kit, normally expressed on hematopoietic stem cells, melanocytes, and on mast cells. Dysregulation of Kit occurs in several human cancers including systemic mastocytosis acute myleogenous leukemia (AML) and gastrointestinal stromal tumors (GISTs) Tyrosine kinase dysfunction has been far less characterized in veterinary oncology. Several authors have identified the presence of Kit mutations in dog mast cell tumors (MCTs) and these also result in uncontrolled signaling Up to 30% of all dog MCTs may carry Kit mutations and these have been shown to be significantly associated with tumor grade, local recurrence and decreased survival. Mutations in Kit have also been found in canine GISTs and are virtually identical to those seen in the human disease.

The most well studied TKI in humans is Gleevec (imatinib mesylate, Novartis), an orally administered drug that blocks the activity of the cytoplasmic kinase Abl. This drug was designed specifically to target the constitutively active Bcr-Abl fusion protein found in human patients with Chronic Myelogenous Leukemia (CML) For those individuals in the chronic phase of CML, Gleevec induces a remission rate close to 95% and most patients remain in remission for longer than 1 year Gleevec also binds to the ATP binding pocket of the RTK Kit. Gleevec induces responses in 50-70% of patients with GISTs, far better than the 5% response rate treatment with a variety of chemotherapeutics. Based on the high response rate, Gleevec has become standard of care therapy for patients with GISTs. Gleevec has recently been used to treat cancers in dogs and cats. Gleevec (imatinib mesylate) Gleevec has been used in dogs to treat MCTs. However, it has idiosyncratic hepatotoxicity in proportion of dogs necessitating discontinuation of therapy. A recent study demonstrated response to therapy in 10/21 dogs treated with Gleevec; the RR was 100% in dogs whose MCTs possessed a Kit ITD (n=5) (39). No dogs exhibited hepatotoxicity, although the duration of treatment was relatively short in most patients. Another study reported partial responses to therapy in 3 dogs with systemic mast cell disease treated with Gleevec. A phase I clinical trial evaluating the toxicity of Gleevec was performed in 9 cats with a variety of tumors Doses of 10-15 mg/kg were well tolerated with no evidence of hematologic/hepatic toxicity and only mildgastrointestinal toxicity. Recently, a cat with systemic mastocytosis was treated with Gleevec and experienced a complete remission.

Palladia (SU11654, Pfizer), exhibits activity against members of the split-kinase RTK family (VEGFR, PDGFR and Kit) In 57 dogs with a variety of cancers (carcinomas, sarcomas, MCTs, melanomas, and lymphomas) objective responses occurred in 16 dogs (RR=28%) with stable disease in an additional 15 dogs for an overall biological activity of 54%, including sarcomas, carcinomas, melanomas, myeloma, and MCTs. The highest RR was in MCTs, with 10/11 dogs with Kit mutations exhibiting responses (n=9) or stable disease (n=1). A placebo controlled randomized study of Palladia was then performed in dogs with non-resectable Grade II and III MCTs During the blinded phase, the RR in Palladia treated (n=86) dogs was 37.2% (7 CR, 25 PR) versus 7.9% (5 PR) in placebo-treated (n=63) dogs. Of 58 dogs that received Palladia following placebo-escape, 41.4% (8 CR, 16 PR) experienced an objective response. The overall RR for all 145 dogs receiving Palladia was 42.8% (21 CR, 41 PR). Dogs with Kit mutations were much more likely to respond (SD>10 weeks, CR, or PR) to Palladia than those without (82% vs 55%) and dogs without lymph node metastasis had a higher response rate than those with involvement (67% vs. 46%).

Kinavet (AB Science) is another TKI that primarily targets Kit. In a phase II study of a Kinavet in 13 dogs with grade II and III MCTs there were 2 CRs, 2 PRs, and stable disease in an additional 2 dogs (Axiak et al, VCS 2006). A randomized double blind placebo controlled phase III clinical trial of Kinavet was then performed in over 200 dogs with non-metastatic Grade II or III MCTs(38). While the overall RR was not significantly different between placebo and Kinavet treated dogs (15% vs. 16%), there was a significant difference in time to progression between the two groups (75 vs 118 days), suggesting that Kinavet has biologic activity in MCTs. Although dogs with MCTs possessing Kit mutations did not experience a significantly greater response to therapy when treated with Kinavet (20%) compared to placebo (10%), they did experience a significantly longer time to progression in dogs with Kit mutations.

Both Palladia and Kinavet also have the capacity to cause unique side effects. Palladia can induce a mild, non-life threatening neutropenia in a subset of treated patients. This does not seem to predispose dogs to bacterial infection, and often resolves with dose reduction. Also a poorly understood lameness can be seen as weel Kinavet can induce a protein losing nephropathy and asyndrome of hemolytic anemia in a small subset of patients, the origins of which are not clear.

Polymerase Chain Reaction (PCR) for Antigen Receptor Gene Rearrangements (PARR) Assay and Flow Cytometry

DNA is extracted from a sample of either a lymph node or circulating lymphocytes (in an animal with lymphocytosis). Amplification of immunoglobulin and T-cell receptor sequences is performed. If the proliferation is due to a neoplastic cause, then the population of lymphocytes would be expected to have derived from a single "clone" of cells, and there should be a large accumulation of the specific receptor (immunoglobulin for B-cell) seen as a band on gel electrophoresis. If the lymphocytosis or nodal enlargement is due to a heterogeneous population of lymphocytes (as would be expected in a patient with a reactive, inflammatory, infectious cause), there is no distinct band. This is a powerful tool in dogs with a slowly progressive lymphoid disease, where it is important prognostically, and to decide whether to institute therapy or to look for another cause. Flow cytometry can identify and quantify lymphocyte subsets using cluster of differentiation (CD) markers. The full prognostic significance of panels of these markers has yet to be determined in cats and dogs with lymphoma, but it could be a powerful tool in directing therapy and making a prognosis for our patients. However, the use of flow cytometry to detect the presence of CD-34 in leukemic blood samples has enormous prognostic significance. CD-34 is a putative stem cell marker and the finding of tumor cells with CD-34 in a patient with lymphocytosis implies that patient has acute lymphoid leukemia (ALL), rather than chronic lymphoid leukemia (CLL). This can be of value in patients where the lymphocytosis is neoplastic (see PARR above), but the lymphoid cells are "reactive" or "medium sized" and a true distinction between CLL and ALL cannot be made. In one study, dogs that were CD-34 positive had a median survival of 16 days; if CD-34 negative, regardless of other markers dogs with all other subtypes lived considerably longer (between a median of 289 and 474 days).

Half-body radiotherapy

Several studies have come out in the last few years looking at the role of ½ body radiation therapy as a meanes to improve survival of dogs with lymphoma. The NCSU approach was to deliver the radiation therapy in 2 consecutive daily 4 Gy fractions (instead of a single dose) following a 11 week CHOP protocol. Dogs in remission from lymphoma following chemotherapy appeared to benefit from the addition of radiation and toxicity was mild and self-limiting. 52 dogs received radiation therapy and median first remission was 10 months; 31 relapsed and 20 went on to receive further chemotherapy. The second remission rate was 85%. The overall median remission was 16 months. A more recent report by Lurie of 13 dogs used 6 or 8 Gy fractions but the dose rate was lower than previous studies. Dogs received 1 cycle of CHOP and radiation therapy but no more chemotherapy. One dog died from treatment complications an hematological toxicity was more common and more severe after 8 Gy treatment. GI toxicity was more likely following postcaudal half-body irradiation with 8 Gy. The protocol was well tolerated and treatment intensification using a 2-week inter-radiation interval was possible in all dogs treated with 6 Gy. Preliminary survival data for these dogs were very encouraging, In dogs that were in CR prior to irradiation, the 1-year and 3-year progression-free survival rates were 89% and 64% respectively, indicating that the low dose rate may play a significant role in improving outcome although more studies need to be done.

Merial melanoma vaccine

Since melanoma is thought to be an immunogenic tumor, immunotherapy treatments have been investigated. Tyrosinase is a normal enzyme found in melanocytes and is essential in melanin synthesis. Tyrosinase is also found in melanoma cells. Xenogeneic vaccines use DNA coding for the same protein, but from a different species, to try to overcome the immune system's reluctance to recognize self. The tyrosinase gene is inserted into a double stranded DNA ring. The DNA is then injected through a transdermal device, which deposits the vaccine into the muscle. The DNA is transcribed and translated and the resulting protein stimulates both antibody and T cell responses. The tyrosinase from a different species helps to stimulate the immune response yet the response is targeted against melanoma cells. Collaboration between the Animal Medical Center (AMC) and Memorial Sloan-Kettering Cancer Center (MSKCC) has resulted in several clinical trials investigating potential xenogeneic vaccines. The USDA conditionally licensed Merial melanoma vaccine, which contains plasmid DNA expressing human tyrosinase. It is a therapeutic cancer vaccine indicated for use in Stage II and Stage III oral melanomas with local disease control. The first four vaccines are given every two weeks, followed by booster vaccines given every six months. Each vaccine is administered in the muscle of the medial thigh. It is administered with the Canine Transdermal Device, a high-pressure spring instrument that ensures intramuscular injection. Few side effects have been reported with the vaccine. Mild local injection reactions, such as mild erythema, have been noted and ther were also two dogs that developed autoimmune depigmentation). One dog developed a hematoma at the site that resolved. A transient low-grade fever may also be noted. Survival data is encouraging with median survival as follows: Stage I > 939 days with 92.8% survival, Stage II > 908 days with 79% living 1 year and 63% living 2 years, Stage III > 1646 days with 77% living 1 year, 65% living 2 years and 57% living 3 years.

Chemoembolization

Chemoembolization involves intra-arterial chemotherapy administration in conjunction with subsequent particle embolization. This technique has been demonstrated to result in a 10- to 50-fold increase in intratumoral drug concentrations. The subsequent particle embolization results in tumor cell necrosis and paralyzes tumor cell excretion of chemotherapy resulting in minimized systemic toxicity. This procedure is most commonly used in the treatment of diffuse hepatocellular carcinoma in humans but has also been used to treat other tumors of the liver and elsewhere in the body including the nasal cavity recently in dogs and cats. Most hepatic tumors rely on hepatic arterial blood supply (up to 95%) for growth in contrast to the normal liver parenchyma, which receives most of its blood supply by the portal vein (approximately 80%). Chemotherapy is often mixed with Lipiodol, (Melville Laboratories, Savage, New Jersey), an oily substance that supplies radiographic contrast to the chemotherapy and acts as a tumor localizer. More recently, drug-eluting beads that bind to various chemotherapeutics have been evaluated to enhance the concentration and extend the duration of tumor-chemotherapy exposure.

Recent references

TKIs

London CA, Malpas PB, et al. Multi-center, placebo-controlled, double-blind, randomized study of oral toceranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, for the treatment of dogs with recurrent (either local or distant) mast cell tumor following surgical excision. Clin Cancer Res. 2009 Jun 1;15(11):3856-65.

Hahn KA, Ogilvie G, et al. Masitinib is safe and effective for the treatment of canine mast cell tumors. J Vet Intern Med. 2008 Nov-Dec;22(6):1301-9

PARR

Lana SE, Jackson TL, et al. Utility of polymerase chain reaction for analysis of antigen receptor rearrangement in staging and predicting prognosis in dogs with lymphoma. J Vet Intern Med. 2006 Mar-Apr;20(2):329-34.

Williams MJ, Avery AC, et al. Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis. J Vet Intern Med. 2008 May-Jun;22(3):596-601.

Keller RL, Avery AC, et al. Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement. Vet Clin Pathol. 2004;33(3):145-9.

½ Body Radiotherapy

Gustafson NR, Lana SE et al. A preliminary assessment of whole-body radiotherapy interposed within a chemotherapy protocol for caninelymphoma. Vet Comp Oncol. 2004 Sep;2(3):125-31.

Lurie DM, Kent MS, et al. A toxicity study of low-dose rate half-body irradiation and chemotherapy in dogs with lymphoma. Vet Comp Oncol. 2008 Dec;6(4):257-67.

Rassnick KM, McEntee MC, et al. Comparison of 3 protocols for treatment after induction of remission in dogs with lymphoma. J Vet Intern Med. 2007 Nov-Dec;21(6):1364-73.

Williams LE, Johnson JL et al. Chemotherapy followed by half-body radiation therapy for canine lymphoma. J Vet Intern Med. 2004 Sep-Oct;18(5):703-9.

Melanoma Vaccine

Bergman PJ, Camps-Palau MA, et al. Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center. Vaccine. 2006 May 22;24(21):4582-5. Epub

2005 Aug 24. PubMed PMID: 16188351.

Bergman PJ, McKnight J, et al. Long-term survival of dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human tyrosinase: a phase I trial. Clin Cancer Res. 2003 Apr;9(4):1284-90. PubMed PMID: 12684396.

Chemoembolization

Weisse C. Hepatic chemoembolization: a novel regional therapy. Vet Clin North Am Small Anim Pract. 2009 May;39(3):627-30.

Weisse CW, Berent AC, Todd KL, Solomon JA. Potential applications of interventional radiology in veterinary medicine. J Am Vet Med Assoc. 2008 Nov 15;233(10):1564-74.

Marioni-Henry K, Schwarz T, Weisse C, Muravnick KB. Cystic nasal adenocarcinoma in a cat treated with piroxicam and chemoembolization. J Am Anim Hosp Assoc. 2007 Nov-Dec;43(6):347-51.