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Keywords:

  • cats;
  • mast cell tumours;
  • kit fifth IgD;
  • mutation;
  • tyrosine kinase inhibition

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

The purpose of the current study was to investigate the mutation status of KIT in feline mast cell tumours (MCTs) and to examine the effects of tyrosine kinase inhibition on the phosphorylation of mutant kit in vitro and in clinical cases of cats. Sequence analysis of KIT identified mutations in 42/62 MCTs (67·7%). The vast majority of the mutations were distributed in exons 8 and 9, both of which encode the fifth immunoglobulin-like domain (IgD) of kit. All five types of kit with a mutation in the fifth IgD were then expressed in 293 cells and examined for phosphorylation status. The mutant kit proteins showed ligand-independent phosphorylation. The tyrosine kinase inhibitor imatinib mesylate suppressed the phosphorylation of these mutant kit proteins in transfectant cells. In a clinical study of 10 cats with MCTs, beneficial response to imatinib mesylate was observed in 7/8 cats that had a mutation in the fifth IgD of kit in tumour cells. Mutations in the fifth IgD of kit thus appear to be common and potentially sensitive to imatinib mesylate in feline MCTs. These data provide an in vivo model for paediatric mastocytosis where mutations in the fifth IgD of kit also occur.

The neoplastic proliferation of mast cells in cats, referred to as a mast cell tumour (MCT), is one of the common tumours in this species, in contrast to a similar disease in humans, mastocytosis, which is rare. MCTs occur in middle-aged to elderly cats and represent the second most commonly encountered cutaneous tumour in cats, accounting for 21% of cutaneous tumours (Miller et al, 1991). Cutaneous MCTs tend to be swollen, inflamed, ulcerated and itchy and sometimes cause gastrointestinal ulceration due to the release of mediators, such as histamine and heparin, from the neoplastic mast cells. MCTs also occur in visceral organs, particularly the spleen (Spangler & Culbertson, 1992), but they occur less frequently than cutaneous MCTs. Cutaneous MCTs are usually benign with good outcomes following surgical excision; however, they occasionally result in systemic involvement and fatal outcomes (Litster & Sorenmo, 2006; Wilcock et al, 1986). With regard to splenic MCTs, widespread dissemination with gastrointestinal ulceration is much more common.

Kit, a receptor tyrosine kinase encoded by KIT, plays a crucial role in cell growth by binding its ligand stem cell factor to various cells, including mast cells (Roskoski, 2005). Kit consists of five immunoglobulin-like domains (IgD) in the extracellular domain, a transmembrane domain and intracellular domains that include a juxtamembrane domain and a kinase domain, which is split by a kinase insert sequence into an adenosine triphosphate-binding site and an activation loop (Yarden & Ullrich, 1988). The gain-of-function mutations of kit, such as constitutive ligand-independent kinase activation, have been demonstrated to be closely related to the pathogenesis of several specific types of human tumours (Longley et al, 2001; Orfao et al, 2007), including acute myeloid leukaemia (AML), gastrointestinal stromal tumour (GIST) and mastocytosis.

The kinase inhibitor imatinib mesylate is a drug that effectively targets the Bcr-Abl kinase in chronic myeloid leukaemia (CML) (Druker et al, 2006) and certain subsets of acute lymphoblastic leukaemia (ALL) (Vignetti et al, 2007) in humans. Imatinib mesylate is also active in the treatment of GIST (Demetri et al, 2002) and mastocytosis (Akin et al, 2004; Frost et al, 2002; Heinrich et al, 2008; Pardanani et al, 2003; Zhang et al, 2006) by targeting mutant kit. However, its beneficial effects in mastocytosis are limited (Vega-Ruiz et al, 2009), because the vast majority (>90%) of adult patients possess a KIT mutation in exon 17, known as D816V, in the activation loop (Garcia-Montero et al, 2006) that is resistant to imatinib mesylate (Pardanani et al, 2003; Akin et al, 2003). The current Spanish Network on Mastocytosis (REMA) recommendations regarding the use of imatinib therapy in mastocytosis only include (i) those exceptional cases of aggressive systemic mastocytosis and mast cell leukaemia who are negative for the D816V KIT mutation; (ii) systemic mastocytosis patients carrying juxtamembrane KIT mutations (e.g. K509I and F522C) associated with an aggressive course of the disease; and (iii) aggressive cases of systemic mastocytosis with associated clonal haematological non-mast cell lineage disease associated with FIP1L1/PDGFRA gene rearrangements (systemic mastocytosis associated with chronic eosinophilic leukaemia) (Orfao et al, 2007). Of note, in these latter cases, imatinib therapy should be prescribed to bring eosinophil counts, and thus the chronic eosinophilic leukaemia-component, under control, whereas the systemic mastocytosis associated with chronic eosinophilic leukaemia typically behaves as an indolent disease that does not require imatinib or any other targeted or cytoreductive therapy (Orfao et al, 2007). Only few c-kit mutations have been reported to clinically respond to imatinib therapy (e.g. p.Asp816Thr, p.Lys509Ile, p.Phe522Cys, p.Val560Gly, p.Asp419del) (Hoffmann et al, 2008). In contrast to adult mastocytosis, studies have suggested that there are differences in the status of mutations in paediatric mastocytosis (Büttner et al, 1998; Longley et al, 1999). Regarding this point, Lanternier et al (2008) found mutations in KIT exon 8 and exon 9 that encode the fifth IgD of kit in three of 12 patients (25%) with paediatric mastocytosis. In addition, Hoffmann et al (2008) reported successful treatment with imatinib mesylate in the case of a child with progressive cutaneous mastocytosis who had a KIT mutation in exon 8 and they suggested the potential utility of imatinib mesylate in the treatment of paediatric mastocytosis. Similar to the report of Hoffmann et al (2008), we previously found that a cat that spontaneously developed MCTs possessed a mutation in KIT exon 8 and the tumours responded well to treatment with imatinib mesylate (Isotani et al, 2006), presuming a similarity in the underlying KIT mutation in the pathogenesis between human paediatric mastocytosis and feline MCT. Regarding surveillance of the KIT mutation in feline MCTs, only one report has been published (Dank et al, 2002). In that report, KIT exons 11, 12 and 17 that correspond to part of the juxtamembrane domain, ATP-binding site, and activation loop respectively, have been screened for mutation in 10 cats; however, no mutation was identified. These findings led to the hypothesis that mutation in the fifth IgD could be related to the pathogenesis of neoplastic proliferation of mast cells in cats.

In the present study, we therefore surveyed mutations in KIT genomic exons corresponding to the fifth IgD (exons 8 and 9) in feline MCTs; in addition, the nucleotide sequences of exons 11, 13 and 17, which are mutated in human mastocytosis, AML, or GIST, were also examined. In some cases for which tumour mRNAs were available, the entire coding sequence of kit was examined for mutation screening. On the basis of the screening, the phosphorylation status of mutant kit identified in feline MCTs was examined. Furthermore, we evaluated the effect of tyrosine kinase inhibition by imatinib mesylate on the phosphorylation of mutant kit in vitro and in clinical cases of feline MCTs.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

Animals and sample collections

Sixty-two cats that had cutaneous, splenic, or widespread MCTs were enrolled in this study. Ages of the cats ranged from 2 to 19 years with a median age of 11 years. Tumour samples were collected by surgical excision or fine needle aspiration biopsy of the tumour mass. Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy cats (6–10 years old) by density gradient centrifugation.

Analysis of KIT genomic nucleotide sequences

Genomic DNAs were extracted from frozen or formalin-fixed and paraffin-embedded tumour tissues using a DNeasy tissue kit (QIAGEN, Valencia, CA, USA). These DNA samples (100 ng) were subjected to polymerase chain reaction (PCR) amplification using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) with intronic primer sets to amplify feline KIT exon 8, 9, 11, 13 or 17 (Table I). After PCR amplification for 35 cycles, the products (10-μl aliquots) were size-fractionated on a 1·2% agarose gel and visualized with ethidium bromide staining. Amplified products were extracted from the gel and directly sequenced.

Table I.   Nucleotide sequences of primers specific to feline KIT used in PCR assays.
RegionForward primerReverse primer
Exon 85′-TGAAAGCAAGGGAGGGAGGAAGTC-3′5′-CTGCGGGCTAGAAATTGCGTGATA-3′
Exon 95′-CCCGCTGTTGGTCGTAAAAGGAA-3′5′-TTGGTGGAATGGACTGAAAATC-3′
Exon 115′-CTCCCCTAATAAGCGCTGTAATGA-3′5′-CAGGTGCAACAGAACAAAGGAAGT-3′
Exon 135′-GGGGCTTTCCGTGTAACCTC-3′5′-TCCCACCCATGACGATAAAACT-3′
Exon 175′-GTGTGACAGAAGCAGCATC-3′5′-GAGACTAACATCCTTCATTGG-3′
Entire coding sequence5′-CAGGAACGTGGAACGGACCTC-3′5′-GATCGTTCTCGCTGGGGAGAC-3′

Analysis of KIT cDNA whole nucleotide sequences

Total RNAs were extracted from freshly collected tumour samples of affected cats and PBMCs of normal cats using RNA-STAT 60 (Tel-TestB, Friendswood, TX, USA) as described previously (Bonkobara et al, 2001). After RNAs were reverse-transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen), an aliquot of cDNAs was subjected for PCR amplification to amplify the entire coding nucleotide sequence of KIT cDNA using Platinum Taq DNA polymerase and a primer set (Table I). After PCR amplification for 35 cycles, the resulting PCR products were cloned into the plasmid vector pCR2.1 using the TOPO TA cloning kit (Invitrogen) and the nucleotide sequences were determined by at least three independent clones.

Construction of haemagglutinin-tagged kit expression vectors

To produce feline mutant kit tagged with a C-terminal haemagglutinin (HA) epitope (kit-HA), the entire coding sequence of kit was excised from the plasmid vector containing mutant KIT gene by PCR amplification with the feline-specific KIT forward primer containing a KpnI restriction site and a reverse primer containing a NotI site linked to a sequence encoding for a HA epitope at the 5′ ends. Using these restriction sites, the PCR products were inserted into a mammalian expression vector pcDNA3.1 (Invitrogen). Wild-type kit-HA expression vector was constructed in a similar manner using a plasmid vector containing the feline KIT gene isolated from PBMCs, in which the nucleotide sequence was identical among three normal cats. Expression vector producing canine mutant kit-HA that contained the imatinib mesylate-sensitive mutation c.1725_1783+10dup (Isotani et al, 2008) was also prepared in the same manner.

Analysis of kit tyrosine phosphorylation

The 293 cells were plated in a six-well plate (3 × 105 cells per well) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum and antibiotics for 12 h. Feline wild-type and mutant kit-HA and canine mutant kit-HA expression vector were then transiently transfected using FuGene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN, USA). After transfection, the 293 cells were cultured for another 12 h and then serum starved for 12 h in DMEM. The 293 cells were then cultured further with or without 100 ng/ml of feline recombinant stem cell factor (SCF) (R&D Systems, Minneapolis, MN, USA) for 10 min. For the study of tyrosine kinase inhibition, 293 cells expressing wild-type and mutant kit-HA were cultured in DMEM containing various concentrations (0, 0·01, 0·1, 1 and 10 μmol/l) of imatinib mesylate (Glivec; Novartis, Basel, Switzerland) for 90 min after 12 h of serum starvation. The 293 cells expressing wild-type kit-HA were cultured further with the addition of 100 ng/ml of SCF for 10 min. After culturing, cells were washed twice with phosphate-buffered saline and lysed using cell lysis buffer containing 20 mmol/l Tris (pH7·5), 150 mmol/l sodium chloride, 1 mmol/l ethylenediaminetetraacetic acid, 1 mmol/l ethylene glycol tetraacetic acid, 1% Triton X-100, 2·5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l sodium orthovanadate, 1 mg/ml leupeptin and 1 mmol/l phenylmethylsulfonyl fluoride. The extracts were precipitated with Protein G-agarose beads (Invitrogen) bound with 0·4 μg of mouse anti-HA monoclonal antibody (Sigma-Aldrich, St Louis, MO, USA). The immunoprecipitated proteins were equally divided into two batches and each batch was separately applied to a 6% SDS-PAGE-gel and transferred onto a polyvinylidene fluoride membrane (Bio-Rad, Richmond, CA, USA). The membrane prepared from one batch was blotted with polyclonal goat anti-human kit antibody (Invitrogen) followed by biotin-conjugated rabbit anti-goat immunoglobulin (Invitrogen), and the membrane prepared from the other batch was blotted with monoclonal mouse anti-phosphotyrosine antibody (Calbiochem, San Diego, CA, USA) followed by biotin-conjugated rabbit anti-mouse IgG (Sigma-Aldrich). After incubation of the membranes with peroxidase-conjugated streptavidin, immunoreactive bands were visualized with an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK) and a VearsaDoc 5000 detector (Bio-Rad), and signal levels were semi-quantified by Quantity One analysis software (Bio-Rad).

Treatment of clinical cases of cats with mast cell tumour by imatinib mesylate

Ten client-owned cats with MCTs were included in the clinical study. The cats had tumours in the skin (six cats), spleen and liver (two cats), or the skin, spleen and liver (two cats). Five of the 10 cats had mastocytemia. The 10 cats underwent treatment with imatinib mesylate at an oral dose of 10 mg/kg daily for 2–32 weeks. Seven of these 10 cats had previously received treatment, including splenectomy, administration of a steroid (prednisolone or prednisone), vinblastine, lomustine, cyclophosphamide, nitrogen mustard, or chlorambucil, or combinations of these therapies. The remaining three cats received imatinib mesylate as an initial treatment. Responses of MCT to imatinib mesylate were assessed by changes in cutaneous tumour mass size based on the following criteria: complete remission (CR), the disappearance of all target lesions; partial remission (PR), at least a 30% decrease in the sum of the longest diameters of target lesions; progressive disease (PD), at least a 20% increase in the sum of the longest diameters of target lesions or the appearance of one or more new lesions; stable disease (SD), a decrease in tumour size of <30% or an increase of <20%. Tumour response of cats that did not have a measureable lesion was evaluated by ultrasonographic imaging following fine needle aspiration biopsy of the affected organs and monitoring of the mast cell number in peripheral blood smear slides.

Statistical analysis

Statistical analysis was performed with an unpaired two-tailed Student t-test. Significance was accepted at < 0·05.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

KIT mutations

Genomic samples from 62 MCTs were screened for mutation in KIT exons 8, 9, 11, 13 and 17. Samples suitable for RNA isolation were available in 17 of 62 MCTs and the entire nucleotide sequence of KIT cDNA was analysed using these 17 samples. Mutations were found in KIT exon 6 (c.957_966delinsT), exon 8 (c.1244_1255dup, c.1256_1264delinsTCA and c.1256_1262delinsT), exon 9 (c.1430G>T and c.1517_1518delinsTT) and exon 11 (c.1661_1663delinsGCAAGTGCACCC) (Fig 1). All of the mutations were in-frame mutations that alter the amino acid composition of kit (Fig 1). The locations of these mutations are illustrated in Fig 2. Mutations in exon 6, exons 8 and 9 and exon 11 altered kit amino acid composition in the fourth IgD, fifth IgD and juxtamembrane domain, respectively.

image

Figure 1.  In-frame mutations of the KIT gene in feline mast cell tumours. Nucleotide mutations and alteration of amino acids by the nucleotide mutations are shown. Bold underlined nucleotides indicate inserted, duplicated, or substituted nucleotides and deleted nucleotides are indicated in the brackets. Bold dotted amino acids indicate amino acids altered by the nucleotide mutations.

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image

Figure 2.  Location of mutations. Positions of mutations in KIT exons 6, 8, 9 and 11 (boxed) are indicated by arrows. The regions of kit corresponding to these exons are indicated in the domain structure of feline kit (top) and the locations of altered amino acids are indicated by dots on the domain structure. D1–D5, immunoglobulin-like domain 1–5; TM, transmembrane domain; JM, juxtamembrane domain; TK1, tyrosine kinase domain 1 (adenosine triphosphate-binding site); TK2, tyrosine kinase domain 2 (activation loop).

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The frequencies of the mutations are shown in Table II. Overall, KIT mutations were found in 42 of 62 MCTs (67·7%). Mutation was most frequent in exon 8 (28/62 cats, 45·2%), including c.1244_1255dup (25 cats), c.1256_1264delinsTCA (one cat) and c.1256_1262delinsT (two cats), followed by exon 9 (15/62 cats, 24·2%), including c.1430G>T (12 cats) and c.1517_1518delinsTT (three cats). Two cats had mutations in exon 8 (c.1244_1255dup) and exon 9 (c.1430G>T) and one cat had mutations in exon 8 (c.1244_1255dup) and exon 9 (c.1517_1518delinsTT) simultaneously. Mutations in exon 6 (1/17 cats, 5·9%) and exon 11 (1/62 cats, 1·6%) were found infrequently.

Table II.   Frequency of mutations in feline mast cell tumours.
MutationNo. of cats examinedNo. of positive cats (%)
  1. *Three cats possessed mutations in exon 8 (c.1244_1255dup) and exon 9 (c.1430G>T, two cats; c.1517_1518delinsTT, one cat) simultaneously.

Exon 6171 (5·9)
 c.957_966delinsT1
Exon 86228* (45·2)
 c.1244_1255dup25
 c.1256_1264delinsTCA1
 c.1256_1262delinsT2
Exon 96215* (24·2)
 c.1430G>T12
 c.1517_1518delinsTT3
Exon 11621 (1·6)
 c.1661_1663delinsGCAAGTGCACCC1
Exon 13620 (0)
Exon 17620 (0)
Other exons170 (0)

Ligand-independent phosphorylation of mutant kit

Because the fifth IgD of kit (exons 8 and 9) was the region in which mutations frequently occurred, we focused on the mutations found in this region for further analysis. To investigate the effect of KIT mutation on the phosphorylation of kit, expression vectors encoding feline wild-type kit-HA and mutant kit-HA that contain mutations of c.1244_1255dup, c.1256_1264delinsTCA, c.1256_1262delinsT, c.1430G>T, or c.1517_1518delinsTT were transfected to 293 cells and the phosphorylation status of immunoprecipitated kit-HA was compared by Western blotting. As shown in Fig 3A, signals of anti-phosphotyrosine in the wild-type kit-HA was weak in the absence of SCF and was markedly increased in the presence of SCF, indicating that wild-type kit-HA was phosphorylated in response to SCF. Conversely, all of the mutant kit-HA proteins demonstrated strong signals for anti-phosphotyrosine in the presence as well as absence of SCF. Semi-quantified signal levels of anti-phosphotyrosine normalized by that of anti-kit are shown in Fig 3B. Regardless of the presence or absence of SCF stimulation, signal levels for anti-phosphotyrosine in mutant kit-HA proteins were significantly higher than that in the SCF-non-stimulated wild-type kit-HA (< 0·05) and reached almost half of that in the SCF-stimulated wild-type kit-HA.

image

Figure 3.  Ligand-independent phosphorylation of mutant kit. (A) Tyrosine phosphorylation of wild-type and mutant kit proteins tagged with C-terminal haemagglutinin epitope (kit-HA) expressed in 293 cells in the presence or absence of stem cell factor. Wild-type or mutant KIT genes indicated at the top were ligated with a gene encoding haemagglutinin and transfected to 293 cells to produce kit-HA. After stimulating the cells with (+) or without (−) 100 ng/ml of stem cell factor, the tyrosine phosphorylation of kit-HA was analysed by immunoprecipitation of kit-HA with mouse anti-haemagglutinin monoclonal antibody, followed by Western blotting with a mouse anti-phosphotyrosine antibody (αp-tyr) or goat anti-human kit antibody (αkit). Signals of the immunoreactive bands were detected by a VearsaDoc 5000 detector. (B) Signal levels of the bands in each lane in panel A were semi-quantified with Quantity One analysis software, and the sums of the signal levels of the upper 140-kDa and lower 125-kDa bands for anti-phosphotyrosine were normalized by that for anti-kit. The normalized signal level of anti-phosphotyrosine in SCF-stimulated (+) feline wild-type kit-HA (second column from the left: feline wt-KIT) was set at 1·0. Data are represented by mean (columns) and SD (bars) of three independent experiments. *< 0·05 vs. SCF-non-stimulated (−) wild-type kit-HA (far left column: feline wt-KIT).

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Suppression of SCF-independent tyrosine phosphorylation of mutant kit by the tyrosine kinase inhibitor imatinib mesylate

We next examined whether SCF-independent tyrosine phosphorylation of the mutant kit-HA was suppressed by imatinib mesylate. SCF-stimulated feline wild-type kit-HA and canine mutant kit-HA into which an imatinib mesylate-sensitive mutation was inserted were included in the assay as controls of the experiment. Similar to the SCF-stimulated feline wild-type kit-HA and canine mutant kit-HA, tyrosine phosphorylation of all mutant kit-HA proteins were progressively suppressed according to the increasing concentration of imatinib mesylate and almost completely inhibited at a concentration of 10 μmol/l (Fig 4).

image

Figure 4.  Effects of the tyrosine kinase inhibitor imatinib mesylate on tyrosine phosphorylation of wild-type and mutant kit. The 293 cells were transfected with feline wild-type or mutant KIT genes or the imatinib mesylate-sensitive canine mutant KIT gene ligated with a haemagglutinin gene to produce kit-HA. After treatment of the cells with imatinib mesylate, the tyrosine phosphorylation of kit-HA was analysed by immunoprecipitation of kit-HA with mouse anti-haemagglutinin monoclonal antibody, followed by Western blotting with a mouse anti-phosphotyrosine antibody (αp-tyr) or goat anti-human kit antibody (αkit). Signals of the immuno-reactive bands were detected by the VearsaDoc 5000 detector.

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Mutation status and responses to imatinib mesylate in cats with MCTs

The status of the KIT mutation and the response to imatinib mesylate in 10 cases of feline MCTs are shown in Table III. Among the 10 cats enrolled, MCTs of eight cats had a KIT mutation in exon 8 (five cats, Cases 1–5) or exon 9 (three cats, Cases 6–8). Mutations in exon 8 were all c.1244_1255dup and those in exon 9 were c.1430G>T (Cases 6 and 7) and c.1517_1518delinsTT (Case 8). The MCTs of two cats (Cases 9 and 10) had no detectable mutation. Of the eight cats that had mutation in KIT, objective responses were achieved in seven cats including six PR (Cases 1–3 and 6–8) and one CR (Case 4) after 2–3 weeks of treatment with imatinib mesylate; one cat (Case 5) failed to achieve an objective response during 13 weeks of the treatment. Between two cats that had no detectable KIT mutation, one cat (Case 9) achieved PR after 3 weeks of treatment with imatinib mesylate and the other cat (Case 10) did not present objective responses during 4 weeks of treatment. Of the eight cats that achieved objective responses, six cats (Cases 2, 3 and 6–9) continued treatment until 9–32 weeks. During these periods, PR was maintained in four cats (Cases 3, 7, 8 and 9) and disease progressed in two cats (Cases 2 and 6). Treatment periods with imatinib mesylate varied greatly among individual cases in this study. This variation was mainly due to the termination of treatment by clients for personal reasons, such as financial burden (Cases 1, 3, 4, 7 and 8). Cases 5 and 10 stopped receiving treatment at the clients’ request because these cats did not show objective responses to treatment. Three cats stopped receiving treatment due to progression of disease (Cases 2 and 6) or moderate elevation of serum aspartate aminotransferase and alanine aminotransferase levels (Case 9). In this experiment, four cats (Cases 3, 4, 6 and 9) were concomitantly treated with a steroid (prednisolone or predonisone) or a combination of the steroid with vinblastine or lomustine during treatment with imatinib mesylate. In Case 6, lomustine was started after the achievement of PR.

Table III.   Mutation status of KIT and clinical responses to imatinib mesylate in cats with mast cell tumours.
Case no. Affected region/ mastocytemiaRegion of KIT examinedMutationPrevious treatmentConcomitant treatment Best response/ time to response (weeks)Treatment period (weeks)/status at the end of treatment
  1. (S), solitary lesion; (M), multiple lesions; SPN, splenectomy; VBL, vinblastine; CCNU, lomustine; CLB, chlorambucil; CPM, cyclophosphamide; NM, nitrogen mustard; P, prednisolone/prednisone; CR, complete remission; PR, partial remission; SD, stable disease; PD, progressive disease.

  2. *Lomustine was concomitantly used with imatinib mesylate after the achievement of partial remission.

  3. †Prednisolone was concomitantly used with imatinib mesylate in the first 5 weeks by tapering the dose.

 1Skin (S)/−Exon 8, 9, 11, 13, 17Exon 8 (c.1244_1255dup)NoneNonePR/33/PR
 2Skin (M), spleen, liver/+Entire KITExon 8 (c.1244_1255dup)SPNNonePR/332/PD
 3Skin (M)/−Exon 8, 9, 11, 13, 17Exon 8 (c.1244_1255dup)VBL, CLB, PPPR/318/PR
 4Spleen, liver/+Exon 8, 9, 11, 13, 17Exon 8 (c.1244_1255dup)SPN, CPM, VBL, PVBL, PCR/22/CR
 5Spleen, liver/+Exon 8, 9, 11, 13, 17Exon 8 (c.1244_1255dup)SPN, CCNU, VBL, NMNoneSD13/SD
 6Skin (M)/−Exon 8, 9, 11, 13, 17Exon 9 (c.1430G>T)VBL, PCCNU*, PPR/211/PD
 7Skin (S)/−Exon 8, 9, 11, 13, 17Exon 9 (c.1430G>T)NoneNonePR/29/PR
 8Skin (S)/−Exon 8, 9, 11, 13, 17Exon 9 (c.1517_1518delinsTT)PNonePR/312/PR
 9Skin (M), spleen, liver/+Entire KITNonePP†PR/314/PR
10Skin (S)/+Exon 8, 9, 11, 13, 17NoneNoneNoneSD4/SD

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

In the current study, KIT mutations occurred in MCTs in approximately 70% of the feline cases. The vast majority of the mutations were located in the region corresponding to the fifth IgD, reaching a prevalence of this region of 65%. This prevalence of the mutation in the fifth IgD appears much higher compared to that in cases of paediatric mastocytosis (<5 years-old) (Lanternier et al, 2008). In comparisons with other mast cell neoplasms, the mutation status in feline MCTs was different from that in adult human mastocytosis (Garcia-Montero et al, 2006) as well as that in canine MCT, in which mutations most frequently occurred in the juxtamembrane domain (16·8%), followed by the fifth IgD, including in exon 8 (4·7%) and exon 9 (4·2%) (Letard et al, 2008). Other than mast cell neoplasms, a significant frequency of KIT mutations corresponding to the fifth IgD have been reported in human core binding factor acute myeloid leukaemia (CBF-AML) (26%) (Care et al, 2003) and GIST (18%) (Heinrich et al, 2003); however, the frequency of mutations in this region was much higher in feline MCTs. From our findings, structural abnormality of the fifth IgD of kit caused by mutations is suggested to be critically involved in the pathogenesis of MCTs in cats.

All three types of mutations found in KIT exon 8 (c.1244_1255dup, c.1256_1264delinsTCA and c.1256_1262delinsT) consistently altered the residue of His419, and a mutation in exon 9 (c.1517_1518delinsTT) substituted the residue of Asn506. In a recent report by Yuzawa et al (2007), Tyr418 and Asn505, which correspond to feline His419 and Asn506 respectively, were mapped to the fifth IgD–fifth IgD interface and were shown to play an important role in the homophilic dimerization of two neighbouring receptors after binding of SCF. Moreover, these two regions overlap with or are located in a neighbouring residue of mutations that have been reported in patients with paediatric mastocytosis (Lanternier et al, 2008; Hoffmann et al, 2008), familial mastocytosis (Hartmann et al, 2005), CBF-AML (Care et al, 2003) and GIST (Heinrich et al, 2003) as oncogenic mutations. Therefore, these mutations found in feline MCTs were considered to affect the affinity of homophilic dimerization of kit, resulting in the activation of the kinase domain without ligand binding.

The second most common activating mutation in feline MCT, c.1430G>T (exon 9), substituted Ser477 to Ile477 and caused ligand-independent activation of kit. Mutation around this residue (Ser476 in humans) has not been reported in human tumours; however, identical substitution was reported on the corresponding codon of Ser479 in canine MCT as an activating mutation (Letard et al, 2008). Although the role of this residue is not known, it is considered to be an important residue that critically affects the structure and function of kit by mutational alteration.

We further examined the effects of the kinase inhibitor imatinib mesylate on the phosphorylation of mutant kit proteins in vitro and in clinical cases of feline MCTs. Clinical cases of cats with MCTs harbouring mutations c.1256_1264delinsTCA and c.1256_1262delinsT were not included in this study. Ligand-independent phosphorylation of all mutant kit proteins was suppressed by imatinib mesylate in vitro. Reflecting these in vitro experiments, spontaneous feline MCTs with KIT mutation of c.1244_1255dup, c.1430G>T and c.1517_1518delinsTT responded well to imatinib mesylate, except for one cat that had a mutation of c.1244_1255dup. The reason for the ineffectiveness of imatinib mesylate in this cat is currently unknown. A possible explanation is that several chemotherapies prior to imatinib mesylate treatment may have contributed to the attenuation of the effect of imatinib mesylate via induction of the drug elimination system, such as with p-glycoprotein. Alternatively, the cat may have an imatinib mesylate resistance mutation in other KIT exons or in other protein-tyrosine kinases. Although MCTs of two cats (Cases 9 and 10) had no detectable KIT mutation, these cats still underwent treatment with imatinib mesylate. Treatment with the usual chemotherapeutic drugs was not selected for these cats because their owners declined it. In accordance with the REMA recommendation for the use of imatinib therapy in mastocytosis (Orfao et al, 2007), these two cats possessed neither a mutation corresponding to a D816V KIT mutation nor chronic eosinophilic leukaemia. Moreover, some cases of MCTs in dogs (Isotani et al, 2008) and of mastocytosis in humans (Pardanani et al, 2003) have been demonstrated to respond to imatinib mesylate without a KIT mutation. We thus considered that these cats might respond to imatinib mesylate treatment, which was then started after informed consent was obtained from the cats’ owners. Of these two cats, one showed a response to imatinib mesylate. Although the entire KIT nucleotide sequence of five independent cDNA clones were examined in this case, no mutation was identified, suggesting that aberrant phosphorylation in feline MCTs may not be solely attributable to mutations in KIT. In this case, targets in a presently undefined novel oncogenic kinase could underlie the response of MCTs or imatinib mesylate could suppress the growth-promoting role of wild-type kit on MCTs. In the clinical experiment, concomitant treatments with a steroid or a steroid with vinblastine may have an effect on the results of treatment with imatinib mesylate. However, administration of these agents had been initiated >3 weeks prior to the beginning of imatinib mesylate treatment and no favourable tumour response had been noted in these periods. Thus, it is unlikely that these concomitant treatments contributed to the objective response.

In the present study, mutations in the fifth IgD of kit were frequent in feline MCTs and suggested to be important in the neoplastic proliferation of mast cells via ligand-independent phosphorylation of kit. These mutations seem to relate to the clinical response to imatinib mesylate in cats with MCT. Although a hotspot for kit mutations and responsiveness to imatinib mesylate in feline MCTs are considered to be different from those in cases of adult mastocytosis in humans, they could be similar to those in cases of paediatric mastocytosis. Our findings suggest that feline MCTs may be an interesting spontaneous tumour model for the development of kinase-targeted therapy in the treatment of progressive paediatric mastocytosis as well as other human tumours that are attributed to mutations in the fifth IgD of kit.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

The authors would like to thank Mr Ryuta Uemura, Ms Maimi Hara and Ms Noriko Ishii for helping with the sample process, Dr Shigeru Aoki, Dr Yu Sasaki, Dr Ikumi Ishikawa, Dr Miki Tominaga, Dr Rina Kato, Dr Tetsuya Kobayashi, Dr Yuhei Shimada and Dr Kimimasa Takahashi for helping with sample collection, and Canine-Lab Inc. (Dr Yosuke Uematsu and Dr Tomohiro Yamaguchi) for helping with sequencing analysis. This research was supported partially by the Grant-in-Aid for Scientific Research (No. 11050) and the Strategic Research Base Development Program for Private Universities 2008 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

Conflict-of-interest disclosure

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References

The authors declare no competing financial interests.

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  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict-of-interest disclosure
  8. References
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