Chapter 6.1
Advances in the management of skin cancer
Skin cancer is one of the most commonly diagnosed cancers in the world today in both humans and our pet population. Advances in molecular techniques are now affording us an opportunity to develop therapeutics targeted at specific cancer-related cellular pathways. However, despite progress in conventional treatments, such as chemotherapy and radiation, and the new targeted therapies, some cancers, such as melanoma and cutaneous lymphoma, continue to cause significant mortality and morbidity.
This short synopsis is not complete but is aimed at providing an insight into current advanced treatments and horizon therapies for cutaneous malignancies in dogs and cats with comparative aspects.
Introduction
In human medicine, the term ‘skin cancer’ is often restricted to either cutaneous malignant melanoma or nonmelanoma ‘skin cancers’, which are a group largely made up of basal cell carcinoma and squamous cell carcinoma. Currently, between 2 and 3 million nonmelanoma skin cancers and 132,000 melanoma skin cancers occur globally each year. One in every three cancers diagnosed is a skin cancer.1 As the molecular mechanisms of cutaneous neoplasias are elucidated, the similarities between the human and animal diseases become more apparent. Animal models can provide rich foundations for translational research, both in vivo and in vitro, into some of the most aggressive and devastating cancers affecting both humans and animals. Molecular targeted therapies have the general advantages of a more targeted cellular approach to the management of cancer, with the aims of better outcome and fewer adverse effects. In this short synopsis, we focus on the most current and horizon therapies for malignant cutaneous neoplasms in both human and animal patients.
Receptor tyrosine kinase inhibitors
One of the most significant advances in the management of cutaneous neoplasms in animal patients in recent years has been the introduction of receptor tyrosine kinase (RTK) inhibitors for veterinary use. Receptor tyrosine kinases are a large family of transmembrane receptors that, when stimulated by their cognate ligands (growth factors), trigger cytoplasmic and nuclear signalling, resulting in cell proliferation and differentiation.2 These growth factor receptors are divided into 20 families according to their different extracellular ligand-binding domains. A vast array of cancer types in both humans and animals have demonstrated dysregulation of RTKs. Receptor tyrosine kinases have also been implicated in new blood vessel formation (angiogenesis) in tumours and the process of metastasis. The normal activity of RTKs – such as the platelet-derived growth factor receptor (PDGFR) or the vascular endothelial growth factor receptor (VEGFR) – may be utilized by the tumour stroma to maintain angiogenesis, promote metastasis and support the tumour niche environment.
The development of inhibitors of the RTK (RTKi) pathways has been a hotly contested arena in the pharmaceutical industry. The initial success of imatinib (Gleevec®; Novartis, Basel, Switzerland) in human leukaemia suggested that this approach to cancer therapy could be highly successful in a range of other cancers. Specific small-molecule tyrosine kinase inhibitors (RTKis) are able to block the activity of receptors by competitive inhibition of ATP binding (Figure 1). Recently, the following two RTKis were approved by the European Medicines Agency and the US Food and Drug Administration for use in mast cell tumours (MCTs) in dogs: (i) toceranib phosphate (Palladia®; Pfizer Inc., Manhattan, New York, NY, USA), approved for use in recurrent, nonresectable grade II/III MCTs;3 and (ii) masitinib (Masivet®; AB Sciences, Paris, France), approved for use in nonresectable grade II/III MCTs with established c-KIT mutation, which should be confirmed before treatment.4
Figure 1. Mechanism of action of receptor tyrosine kinase (RTK) inhibitors. In normal cellular physiology, ligand binding to RTK causes phosphorylation of serine residues through the activity of ATP. Phosphorylation activates downstream proteins, ultimately affecting cellular proliferation. The RTK inhibitors (RTKi) block the ATP binding site, thereby preventing phosphorylation.
Both drugs were developed to target RTKs, in particular c-KIT on canine MCTs.5,6 Toceranib also targets VEGFR2 and PDGFR, suggesting that the use of this drug would also target tumour stroma (through PDGFR) and neoangiogensis (through VEGFR2), perhaps having an additional effect on tumour metastasis. Both drugs have demonstrated efficacy as single agents in prospective clinical trials in dogs with mast cell tumours.7,8 The initial clinical trials for toceranib revealed biological activity in a variety of tumour types, including carcinomas, sarcomas, melanomas and lymphomas, with the highest response rates in dogs with MCTs.9 A recent retrospective study surveyed the biological activity of toceranib in other solid tumours, including apocrine gland anal sac adenocarcinoma, metastatic osteosarcoma, thyroid carcinoma, head and neck carcinoma and nasal carcinoma.10 A response achieving clinical benefit (complete remission, partial remission or stable disease) was seen in 74% of treated dogs, indicating that further studies are needed to evaluate the efficacy of this drug for targeting other tumour types.
Mutations in KIT have been found to occur in approximately 15% of mucosal and 23% of acral melanomas in human studies.11 Trials are currently underway to evaluate the RTKi imatinib in mucosal and acral melanomas with KIT mutations. A recent preliminary veterinary investigation found that buccal amelanotic melanomas showed strong positive staining for the c-KIT protein compared with melanotic melanomas.12 These findings may provide the framework for future studies into the role of KIT in melanoma, using a canine model for human melanoma. The overall prognosis for metastatic melanoma remains grave for both human and veterinary patients, with future translational research having the potential to benefit both species.
Squamous cell carcinoma has been shown to express the RTK epidermal growth factor receptor (EGFR), which can be exploited for directed molecular targeted therapy. In a recent meta-analysis of cutaneous human head and neck squamous cell carcinoma (SCC), overexpression of EGFR was found in 56% of primary tumours and 58% of regional metastases.13 Erlotinib (Tarceva®; OSI Pharmaceuticals, LLC, Farmingdale, New York, NY, USA) and gefitinib (Iressa®; AstraZeneca plc, London, UK) are EGFR inhibitors, previously approved to treat haematopoietic malignancies, which are now being tested in clinical trials of human patients with cutaneous SCC.14 Overexpression of EGFR is also a feature of feline SCC, which generally has a poor to grave prognosis because most cases are presented in the advanced stages.15 Epidermal growth factor receptor is not a target for either of the veterinary approved RTKis, toceranib or masitinib.5 In our laboratory, we recently investigated the effects of the RTKi gefitinib on a feline SCC cell line.16 Treatment with gefitinib resulted in decreased cell proliferation but ultimately the development of drug resistance. Resistance could be overcome with RNAi targeted to EGFR, and suggests that ultimately multimodal therapy may become important.
Veterinary studies combining RTKs with conventional chemotherapy drugs are currently lacking. However, Robat et al.17 recently conducted a phase 1 clinical study to evaluate the safety of the combination of vinblastine and toceranib phosphate (Palladia®; Pfizer) in dogs with MCTs. In that study, the dose-limiting toxicity for the simultaneous combination was neutropenia, and the maximally tolerated combined doses were 1.6 mg/m2 every other week (vinblastine) and 3.25 mg/kg orally, every other day (toceranib). This represents greater than a 50% reduction in dose intensity for vinblastine (compared with single-agent use) and, as such, does not support this combination based on current chemotherapy combination paradigms. While a strict adherence to dose paradigms speaks against the combination, evidence of significant activity (71% objective response) and enhanced myelosuppression suggest additive or synergistic activity. A prospective randomized evaluation comparing this combination with standard single-agent treatments needs to be conducted to interrogate this possibility.
The use of RTKis (inhibitors) with radiotherapy, another proven treatment modality, has recently been investigated. A muticentre prospective trial was conducted to establish the tolerability of using toceranib phosphate in combination with palliative radiotherapy in nonresectable MCTs.18 In that study, toceranib was administered for 1 week before initiation of radiotherapy consisting of a total of 24 Gy delivered in three or four fractions. The overall response rate was 76.4%, with 58.8% of dogs achieving a complete response and 17.6% a partial response. The median time to best response was 32 days, and the median progression-free interval was 316 days. The overall median survival time was not reached with a median follow-up of 374 days. These response rates and durations were higher than those reported for toceranib as a single-agent treatment for MCT, but the study did include the addition of prednisolone. Predictably, the most common toxicities were related to the gastrointestinal tract and the liver.
Subpopulations of T lymphocytes are often dysregulated in cancer, with decreases in numbers of effector T cells and increases in regulatory T cells. In murine models, T cells have been shown to have a direct suppressive effect on the immune response to tumours.19 Additionally, regulatory T-cell numbers have been correlated with clinical outcome in both human and veterinary cancer patients.20,21 A recent study conducted by Mitchell et al.22 combined toceranib phosphate and metronomic dosing of cyclophosphamide in an attempt to improve tumour control by suppression of regulatory T cells and restoration of T-cell-mediated immune responses. In that study, 15 client-owned dogs with advanced tumours were entered into a prospective clinical trial. Results demonstrated that toceranib significantly decreased the number and percentage of regulatory T cells in the peripheral blood of dogs with cancer. Dogs receiving toceranib and cyclophosphamide demonstrated a significant increase in serum concentrations of interferon-γ, which was inversely correlated with regulatory T-cell numbers after 6 weeks of combination treatment. These data support an immunomodulatory function of RTKis in cancer.
Small-molecule tyrosine kinase inhibitors are a new and exciting class of drugs that give the veterinary practitioner a further readily accessible modality in treating MCTs. All the potential indications for this class of drugs are not yet known, but the number of cancers proving responsive to these agents is on the rise as our knowledge of cellular signalling pathways is exploited. The oral formulations would suggest that this class of drugs is easy to administer and monitor in clinical patients. It is essential, however, that the same precautions and considerations be given to using these drugs as we would for conventional cytotoxic therapy, because adverse drug-specific reactions have been described with both medications.
Retinoids
Retinoids, both naturally occurring compounds and synthetically derived substances, are derivatives of vitamin A. These are biological response modifiers, which have antineoplastic activities.23 Retinoids act via activation of the nuclear retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each of which has three isoforms (α, β and γ). Antineoplastic effects of retinoids occur via suppression of cellular proliferation, induction of terminal differentiation and, ultimately, apoptosis.24,25
Retinoids have been used topically in human cutaneous T cell lymphoma (CETL) for more than 20 years 26,27 and, more recently, in canine and feline CETL with mixed results (40-50% response rate of 5-13 months).28,29 Isotretinoin (Accutane®; Roche Pharmaceuticals, Basel, Switzerland) and etretinate (Tegison®; Roche Pharmaceuticals) have been removed from the North American human market due mutagenic and teratogenic effects, which persist even long after discontinuation of therapy. Acitretin (Soriatane®; Stiefel Laboratories, Inc., Research Triangle Park, NC, USA or Neotigason®; Actavis UK Ltd, Barnstaple, UK), a RAR agonist, is still commercially available; however, objective studies on efficacy are seriously lacking. Latterly, bexarotene (Targretin®; Eisai Co., Ltd, Tokyo, Japan), a synthetic RXR agonist, has been efficacious as a single agent in patients with CETL.29 It has biological activity taken orally as well as applied topically and has shown efficacy in human patients with mycosis fungoides and Sézary syndrome.30 The expression of retinoid receptors in normal canine lymphoid tissue and canine nodal lymphoma has been recently investigated.31 Normal lymphoid tissue did not express retinoid receptors, whereas canine nodal lymphomas exhibited strong expression. Cutaneous T-cell lymphomas were shown to express both RARs and RXRs in 29 of 30 dogs with CETL, which suggests that retinoid therapy may be beneficial.32 To date, however, there are no large-scale studies that have tested this hypothesis.
Toll-like receptor agonists
Toll-like receptors are a subset of pattern recognition receptors within the innate immune system that function to recognize pathogens.33 Their expression results in production of immunoregulatory cytokines, such as tumour necrosis factor-α, interferon-α and interferon-γ, and increases in immune surveillance. Toll-like receptor agonists that mediate these effects in the immune system have been used clinically for several years to treat basal cell carcinoma and cutaneous lymphoma in humans.34,35 Dendritic cells present in the dermis are key antigen-presenting cells for induction of adaptive antitumour immune responses. Imiquimod and resiquimod are Toll-like receptor agonists with promise as targeted therapies for CETL.36,37 Imiquimod has shown limited potential as a sole agent due to variable bioavailability and may be more useful as an adjunct therapy. Resiquimod has markedly greater bioavailability with topical application and, consequently, the potential to induce systemic immune activation, resulting in targeting of tumour cells. These compounds have shown initial success both in terms of enhancing immune responses and in eliciting antitumour activity in human CETL patients.38 Recently, Kim et al.39 used in situ vaccination of CETL lesions with the Toll-like receptor 9 agonist CpG oligonucleotides in conjunction with local radiation, which led to regression of distant CETL lesions. These findings highlight the potential pivotal role that stimulation of the innate immune system with immunomodulatory agents can play in treatment of cancer and the need to explore more means of multimodal immunotherapy in both human and veterinary patients to provide enhanced disease control.
Histone deacetylase inhibitors
Dissection of the tumour kinome and exploration of key signalling pathways has identified new targets. Histones are small nuclear proteins responsible for packaging of DNA within nucleosomes. Transcription of DNA occurs in the acetylated state, whereas the hypoacetylated state results in compaction of DNA and repression of transcription. Abberant hypoacetylation is characteristic of some cancers suppressing cellular differentiation. Histone deacetylase (HDAC) inhibitors can activate both the death receptor and intrinsic apoptotic pathways and result in the reactivation of tumour suppressor gene transcription, leading to cytotoxicity and apoptosis.40 In the human field, the HDAC inhibitors vorinostat (Zolinza®; Merck, Whitehouse Station, NJ, USA) and romidepsin (Istodax®; Celgene, Summit, NJ, USA) have been approved for use in CETL.41,42 These drugs are generally very well tolerated and have little to no effects on normal cells. The activity of HDAC inhibitors appears to be multifactorial, and pathways include myc, JAK-STAT, B-Tumour Growth Factor and Bcl-6.43,44 The HDAC inhibitors OSU-HDAC42 and SAHA have recently been investigated in canine tumour cell lines (including T-cell lymphosarcoma and MCT) and found to have potent histone acetylation activity at micromolar concentrations.45 This represents an exciting potential therapeutic target for many canine solid tumours and a role for new drug development.
BRAF inhibitors
The Cancer Genome Project46 brought to light an unanticipated pathway for induction of melanoma.47 Mutations were identified in the BRAF oncogene, which is the serine/threonine kinase downstream of melanocyte-stimulating hormone. The high frequency of BRAF mutation in melanoma (50%) may be due to melanocyte biology. Ultraviolet B light causes upregulation of melanocyte-stimulating hormone, which in turn binds to its receptor, upregulating cyclic AMP and causing increased proliferation of melanocytes. This melanocyte-specific signalling pathway elucidates why there are higher numbers of BRAF mutations in melanoma compared with other cancers.
The BRAF inhibitor vemurafenib (Figure 2) was recently approved (2011) by the US Federal Drug Administration and European Medicines Agency for treatment of human metastatic melanoma.48,49 This drug has a response rate of up to 78% in patients expressing the BRAF-V600E mutation, with no response in patients who lack this mutation. Despite excellent results, almost all patients develop progressive disease due to vemurafenib resistance. A variety of mechanisms of resistance have been described, precluding development of definitive subsequent treatment strategies.
Figure 2. BRAF is a major protein intermediate in RAF/MEK.ERK signalling in normal cells but causes excessive proliferation in melanoma cells with BRAF mutations. The BRAF inhibitors cause cell death by apoptosis.
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