Canine mast cell tumours part I: Clinical and survival outcomes.

BACKGROUND: Dogs have a species-specific susceptibility for developing mast cell tumours (MCTs). Mutations in the KIT proto-oncogene (KIT) are known to contribute to the neoplastic biology of mast cells. In dogs, the most common KIT mutation is an internal tandem duplication (ITD) in exon 11 which has been considered a useful prognostic supplement to traditional histopathological tumour grading. OBJECTIVE: The aim of this retrospective study was to explore the importance of KIT exon 11 ITD mutation status and known clinical and pathological indices in predicting prognosis in a cohort of Australian dogs diagnosed with MCT. METHODS: Clinical parameters, survival data, and KIT mutation status were collected and assessed for 220 dogs with cutaneous or subcutaneous MCT (n = 189 and n = 31, respectively). RESULTS: In at least one of the multivariable models, tumour grade (cutaneous Kiupel low or high grade) or tumour subcutaneous location, multiple concurrent MCTs, metastasis at the time of surgery, and senior age were statistically significant in predicting the outcome (MCT-related death and/or second MCT diagnosis) at 6- or 12-month post-tumour excision. KIT exon 11 ITD mutation status was not a significant predictor in any of the final multivariable models and was strongly correlated with high histological grade (p < 0.001). CONCLUSION: In this sample of dogs, tumour histological grading remained the single most powerful prognostic indicator for MCT outcome. However, concurrent evaluation of multiple prognostically significant parameters provides information of potential value to inform therapeutic management for each patient.

MAST CELL TUMORS IN CHEETAH (ACINONYX JUBATUS): A CASE SERIES.

Mast cell tumors in nondomestic felids are rarely reported and their biological characteristics are not well described. A retrospective review of the pathology records of 52 zoo-housed cheetahs (Acinonyx jubatus) identified five cases of mast cell tumor, involving four closely related individuals. The age at initial presentation varied from 14 mo to 6 yr. Four cases presented as solitary or multiple cutaneous masses that were mostly slow growing, up to 20 mm diameter, and predominantly nonulcerated. The diagnosis was made by fine needle aspiration cytology of a lesion in one case and by excisional biopsy in the others. Histopathologically, the lesions resembled low- to intermediate-grade canine mast cell tumors, with variations in the degree of anisocytosis and anisokaryosis. Surgical excision was incomplete for 80% of the cutaneous lesions, but local recurrence was not observed in any case. One animal with cutaneous lesions subsequently developed fatal visceral mastocytosis involving the spleen, liver, and adrenal gland. There was no evidence of lymph node invasion or paraneoplastic gastrointestinal signs in any of the cases.

KIT mutations in mast cell tumours from cheetahs (Acinonyx jubatus) and domestic cats (Felis catus).

Mast cell tumours (MCT) have been documented in numerous species and mutations within the KIT proto-oncogene are implicated in the neoplastic biology of mast cells in humans, dogs and cats. This study determined high KIT gene nucleotide and Kit amino acid sequence homology between several species known to suffer mast cell neoplasia and especially high sequence conservation between the cheetah (Acinonyx jubatus) and domestic cat (Felis catus) KIT sequences. As a result, we hypothesised that KIT mutations would exist in the neoplastic DNA of four cheetahs diagnosed with MCT from a recent case series. PCR and Sanger sequencing identified conservative exon 6 KIT mutations in two of the four cheetahs. The mutations were different between the two cheetahs. Only wild-type DNA in exons 6, 8, 9 and 11 of KIT was observed in the MCTs of the remaining two cheetahs. Twenty cutaneous MCTs from domestic cats were collected for KIT mutation comparison. Twelve tumours possessed a mutation within KIT exons 6, 8 or 9 (60%, 95% CI 38.5%-81.5%). No mutations were detected in exon 11. There was no significant association between domestic feline MCT KIT mutation status and tumour histological grade (traditional schematic, P = .934; Sabattini 2-tier schematic, P = .762) or mitotic index (P = .750). KIT mRNA and Kit protein sequences are conserved across species but the role of KIT in feline MCT pathogenesis is not completely understood.

Comparative aspects of mast cell neoplasia in animals and the role of KIT in prognosis and treatment.

Mast cell neoplasia clinical presentation and biological behaviour vary considerably across mammalian species, ranging from a solitary benign mass to an aggressive systemic malignancy. Mutations in the KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) gene are common molecular abnormalities involved in mast cell tumorigenesis. KIT mutations often occur in dog, cat and human neoplastic mast cells and result in altered Kit protein structure and function. In dogs, certain KIT mutations are associated with more malignant and lethal disease. In contrast, KIT mutations in feline and human mast cell neoplasms are not correlated with prognosis, but are of value in diagnosis and treatment planning in humans. KIT genetic abnormalities have not been well investigated in other species, although aberrant cytoplasmic Kit protein staining detected in neoplasms of the ferret, horse and cow resembles aberrant Kit staining patterns detected in neoplastic mast cells of dogs, cats and humans. Mutations within KIT are classified as either regulatory-type or enzymatic pocket-type mutations according to their location within the KIT Proto-Oncogene. Mutations within the enzymatic pocket domain confer tumour resistance to tyrosine kinase inhibitors (TKIs). Hence, knowledge of tumour KIT mutation status adds valuable information for optimizing patient treatment strategies. The use of TKIs in combination with conventional chemotherapeutics has opened a new treatment avenue for patients unresponsive to existing drugs. This review highlights the similarities and differences of mast cell neoplasia in mammals with a special focus on the involvement of KIT in the canine and feline forms in comparison to human mast cell neoplasia.

DNA purification increases PCR-amplifiable DNA extracted from formalin-fixed, paraffin-embedded canine mast cell tumors for routine KIT mutation detection.

DNA amplification by PCR detects KIT exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes (HPRT and KIT) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8-60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A260/A280 and A260/A230) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both HPRT and KIT primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using KIT gene primers.

Epigenetics and phenotypic variation in mammals.

What causes phenotypic variation? By now it is clear that phenotype is a result of the interaction between genotype and environment, in addition to variation not readily attributable to either. Epigenetic phenomena associated with phenotypic variation at the biochemical, cellular, tissue, and organism level are now well recognized and are likely to contribute to the "intangible variation" alluded to. While it is clear that epigenetic modifications are mitotically heritable, the fidelity of this process is not well understood. Inheritance through more than one generation of meioses is even less well studied. So it remains unclear to what extent epigenetic changes contribute to phenotypic variation in natural populations. How might such evidence be obtained? What are the features of phenotypes that might suggest an epigenetic component? How much of the epigenetic component is truly independent of genetic changes? The answers to such questions must come from studies designed specifically to detect subtle, stochastically determined phenotypic variation in suitable animal models.

Mechanisms of embryonal tumor initiation: distinct roles for MycN expression and MYCN amplification.

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.