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Keywords:

  • c-kit;
  • Dog;
  • Gleevec;
  • Mast cell tumor;
  • Mutation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Background: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT.

Hypothesis: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit.

Animals: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0–25.3 cm) before treatment.

Methods: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1–9 weeks.

Results: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission).

Conclusions and Clinical Importance: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Imatinib mesylate (Gleeveca) is a small-molecule tyrosine kinase inhibitor. Imatinib mesylate competes with adenosine triphosphate (ATP) for the ATP binding site of protein-tyrosine kinase and prevents downstream signaling.1 It has potent therapeutic activity against human tumors driven by constitutively phosphorylated protein-tyrosine kinases such as Bcr-Abl in Philadelphia-positive chronic myeloid leukemia2 and mutated KIT, which is caused by mutation of the corresponding gene c-kit, in gastrointestinal stromal tumor (GIST).3

Mutations consisting of internal tandem duplication (ITD) within c-kit exon 11, which correspond to the juxtamembrane region of KIT, have been frequently found in high grade mast cell tumor (MCT) in dogs.4–7 The mutations have been shown to induce constitutive phosphorylation of KIT,4,8,9 suggesting the importance of such mutations for the development or progression of MCT in dogs. It has been reported that canine MCT cell lines with a mutation in the juxtamembrane region regressed after treatment with imatinib mesylate in xenografted SCID mice.10 Moreover, SU11654, an indolinone tyrosine kinase inhibitor with different chemical structure from imatinib mesylate, induced objective tumor responses in 11 dogs, including 9 ITD-positive and 2 ITD-negative cases, among 22 dogs with advanced MCT.11 This report indicates the importance of targeting dysregulated protein-tyrosine kinase in the therapeutic intervention of MCT in dogs, including ITD-positive and possibly ITD-negative cases. Imatinib mesylate is a commercially available drug targeting protein-tyrosine kinase. Although some hepatotoxicity from imatinib mesylate has been reported in preclinical experiments using healthy dogs,12,13 the drug is worth evaluating for treatment of canine MCT. In the present study, we examined the therapeutic potential of imatinib mesylate in canine MCT.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Dogs

Twenty-one dogs (8 mixed, 3 Labrador Retriever, 2 Golden Retriever, 2 Shiba, 1 Welsh Corgi, 1 Maltese, 1 French Bulldog, 1 Cairn Terrier, 1 Beagle, 1 Italian Greyhound) with a median age of 10 years (range 3–15 years) and a median body weight of 12 kg (range 3–31 kg) were enrolled in this study. Nine dogs were male and 12 were female. All of the dogs had obvious MCT in skin. Tumor size was determined according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines,14 in which the size was measured as the sum of the longest diameters of the lesions.

Diagnosis of MCT was made from histopathology of surgically excised tissue or cytology of fine needle aspiration of the mass. The histologic grade of tumor was determined according to the Patnaik histologic grading system.15 Regional lymph node assessment with fine needle aspiration and/or buffy coat analysis was performed on some dogs.

Eleven of these 21 dogs had received treatment, including surgery, administration of prednisolone, vinblastine, or lomustine, or combinations of these therapies. The remaining 10 dogs received imatinib mesylate as an initial treatment.

Detection of ITD Mutation

Genomic DNAs were extracted from tumor cells collected by fine needle aspiration or from surgically excised tumor tissues with a DNeasy tissue kit.b These DNA samples (20 ng) were subjected to polymerase chain reaction (PCR) amplification with platinum Taq DNA polymerasec with the following primer set to amplify canine c-kit exon 1116: forward primer, 5′-CCCATGTATGAAGTACAGTGGAAG-3′ and reverse primer, 5′-GTTCCCTAAAGTCATTGTTACACG-3′. After PCR amplification for 35 cycles, the products (10-μL aliquots) were size-fractionated on a 2% agarose gel and visualized with ethidium bromide staining. A band of PCR products larger than the estimated size (190 bp) of wild-type c-kit amplification product was extracted from the gel and the nucleotide sequences were determined.

Analysis of c-kit Whole Nucleotide Sequence

Total RNAs were extracted from tumor cells collected by fine needle aspiration with RNA-STAT 60d as described previously.17 After RNAs were reverse transcribed into cDNA with SuperScript III reverse transcriptase,e an aliquot of cDNA was subjected for PCR amplification to amplify the entire coding nucleotide sequence of c-kit with platinum Taq DNA polymerase and the following primer set: forward primer, 5′-TTGGGCGCGAGCAGGAAC-3′ and reverse primer, 5′-AGCCGAAGGAGGACAGAATAACCA-3′. After PCR amplification for 30 cycles, the resulting PCR products were cloned into the plasmid vector pCR2.1 with the TOPO TA cloning kitf and the nucleotide sequences were determined.

Treatment and Assessment of Response

Dogs were treated with imatinib mesylate at an oral dose of 10 mg/kg daily for 1–9 weeks. Imatinib mesylate was formerly available as a commercially prepared 100-mg capsule, but is currently available only as a 100-mg tablet. Reformulated capsules were prepared if the commercial size was not suitable. During the treatment, 6 of the 21 dogs were given prednisoloneg at doses of 0.25–2 mg/kg.

Responses of MCT to imatinib mesylate were assessed based on the RECIST guidelines. The following criteria was used: 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 tumor size of <30% or an increase of <20%.

To evaluate toxicity, all of the dogs underwent physical and hematologic examinations at days 7, 14, and periodically throughout their treatment if it persisted past 14 days.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

In the 21 dogs enrolled in the present study, the tumor ranged in size from 1.0 to 25.3 cm, with a median size of 7.2 cm. Tumor locations included the head, neck, trunk, inguinal region, and extremities. Of the 21 dogs, 8 had solitary tumors and 13 had multiple tumors. The histologic grade of MCT was determined in 10 dogs; grade II was found in 7 and grade III was found in 3. Cytologic diagnosis was made in the remaining 11 dogs in which histologic grade could not be determined. Large numbers of mast cells were found in regional lymph nodes in 14 of 17 dogs examined, suggesting metastasis. Mast cells were identified by buffy coat analysis in 6 of 18 dogs examined.

PCR analysis for c-kit exon 11 was performed and a larger than expected PCR product was detected in tumor samples from 5 dogs, in addition to the expected wild-type PCR product (Fig 1A). In contrast, no band other than the product band of wild-type c-kit was observed in tumor samples from the rest of the 16 dogs (Cases 6–21). The large bands in Cases 1–5 possessed an ITD mutation within their nucleotide sequences (Fig 1B). The duplications were located near the 3′ end of exon 11 and in 2 cases (Cases 1 and 4) were extending into the neighboring intron. All of the duplications were in frame and ranged in size from 27 to 69 bp.

image

Figure 1.  Detection of internal tandem duplication mutations in c-kit exon 11. (A) Agarose gel electrophoresis of polymerase chain reaction (PCR) amplification products of c-kit exon 11 using genomic DNA extracted from tumors. The PCR product of wild type c-kit is 190 bp. A large PCR product was detected in Cases 1–5. (B) Genomic nucleotide sequence of c-kit exon 11 (large capitals) and the neighboring intron (small capitals). Bars under the nucleotide sequence indicate the regions of duplication.

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Among 21 dogs enrolled in this study, 10 dogs, including all 5 dogs with ITD mutation, experienced a CR or PR within 14 days of treatment. By contrast, PD or SD was noted on days 7 and/or 14 of treatment in the remaining 11 dogs. Treatment with imatinib mesylate was terminated on day 7 or 14 in 15 dogs at the owner's request because of financial burden. The remaining 6 dogs continued treatment with imatinib mesylate for a total duration of 21–63 days. The tumor volumes in these dogs were gradually reduced; however, none of these dogs achieved CR.

Four of the 5 cases without ITD mutation that achieved PR were examined for the entire coding nucleotide sequence of c-kit with mRNA extracted from MCT. From the analysis, no mutations such as ITD, deletion, insertion, and point mutation were noted within the nucleotide sequence.

Among 10 dogs that responded to imatinib mesylate, 5 dogs were administered predonisolone in the period of evaluation because they had vomiting, inappetance, or both. With the exception of 1 dog, the administration of prednisolone was initiated more than 3 weeks before beginning imatinib mesylate and no clinical response had been recorded. In 1 dog, predonisolone was initially given to the dog simultaneously with imatinib mesylate.

During the periods of treatment with imatinib mesylate, no abnormalities in hematologic test results, including CBC and serum biochemistry, and physical examination were noted in all dogs. One owner reported that 1 dog vomited, which was resolved with an antiemetic drug, during treatment with imatinib mesylate.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

In the present study, there is evidence that imatinib mesylate is active in the treatment of some dogs with MCT, achieving CR or PR within short periods. Among the responsive tumors, half possessed an ITD mutation. An ITD mutation reportedly causes aberrant constitutive phosphorylation of KIT, and it is believed to be the primary stimulus for uncontrolled growth in certain tumors.6 This could explain why all of the ITD-positive MCT cases exhibited clinical response to imatinib mesylate in this study.

In addition to the ITD-positive MCT cases, we found that some dogs with ITD-negative MCT responded to imatinib mesylate at the same rate as did ITD-positive MCT dogs. Regression of ITD-negative MCT occurs in dogs, in addition to ITD-positive MCT dogs with the tyrosine kinase inhibitor SU11654.11 Our results also indicate that aberrant phosphorylation in MCT of dogs might not be solely attributable to an ITD mutation in exon 11 of c-kit. Other than ITD mutation within c-kit exon 11, various mutations in c-kit exons 9, 11, 13, and 17 have been shown to be responsible for aberrant phosphorylation of KIT in human GIST, and these mutation-possessing GIST are known to be sensitive to imatinib mesylate.18 We thus examined mutations within entire nucleotide sequence of c-kit in ITD-negative MCT cases that responded to imatinib mesylate; however, no mutation was detected within the nucleotide sequences. It was thus considered that other mechanisms underlie the aberrantly regulated protein-tyrosine kinase; for example, the presence of mutation in PDGFR-α or -β, both of which were found in human patients with imatinib mesylate-sensitive mastocytosis,19,20 and gain-of-function mutations in other receptor tyrosine kinases could be involved in these cases.

In contrast to the immediate tumor response in 10 dogs, no objective tumor response to the treatment with imatinib mesylate was observed in 11 dogs within 7 or 14 days. Two dogs with MCT achieved PR after 6 and 9 weeks of treatment with SU11654, suggesting the potential of gradual tumor response to tyrosine kinase inhibitor.11 Although continued treatment of these 11 dogs with imatinib mesylate might have had therapeutic benefits, we were unable to pursue it in this study.

Regarding the toxicity of imatinib mesylate on dogs, hepatic toxicosis has been reported in 2 papers; one indicated elevated serum liver enzyme activity with hepatocellular and bile duct necrosis in normal dogs at a dose corresponding to a clinical dose for humans12 and the other indicated progressive liver toxicosis in normal dogs at a dose of 100 mg/kg but not at 3 mg/kg.13 In the present study, such abnormality in the liver panel of serum biochemistry, including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, was not noted in all dogs during treatment with imatinib mesylate. Although an owner reported that 1 dog vomited, the dosage used in the present study is unlikely to induce serious toxicosis in dogs.

This study has several limitations. First, information from which to evaluate clinical stage was insufficient. Not all dogs underwent regional lymph node assessment with cytology, buffy coat analysis, and abdominal ultrasound examination. No dog underwent bone marrow aspiration and spleen/liver aspiration at the beginning of the treatment. Second, samples from only half of the dogs in this study underwent histopathology examination and the review of the histopathologic samples was not performed by a single pathologist. Third, 6 of 21 dogs enrolled in this study received prednisolone with imatinib mesylate. Five of the 10 dogs that responded to treatment were also on prednisolone; in contrast, 1 of the 11 dogs that exhibited no response to imatinib mesylate was on prednisolone. Although 4 of these 5 dogs were on prednisolone before initiation of imatinib mesylate without any known response, this still remains a significant confounder. Therefore, we cannot exclude the possibility that prednisolone contributed to the observed response. Furthermore, the observed tumor reduction in 1 dog in which the dog was given prednisolone and imatinib mesylate concurrently could have been secondary to prednisolone. Finally, only 6 of 21 dogs received imatinib mesylate for longer than 14 days and follow-up examination is lacking in this study. It is thus possible that longer duration of treatment would have resulted in the development of hepatotoxicity in a subset of dogs.

In conclusion, this study indicates that imatinib mesylate might have biologic activity in some dogs with MCT. For prediction of the tumor response to imatinib mesylate, detection of a mutation in c-kit exon 11 is likely to be valuable; however, there are dogs with MCT in which other aberrantly regulated protein-tyrosine kinases may account for the clinical response to imatinib mesylate.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

aGleevec, Novartis Pharma AG, Basel, Switzerland

bDNeasy tissue kit, QIAGEN, Valencia, CA

cPlatinum Taq DNA polymerase High Fidelity, Invitrogen, Carlsbad, CA

dRNA STAT-60, Tel-TestB, Friendswood, TX

eSuperScript III reverse transcriptase, Invitrogen

fTOPO TA cloning kit, Invitrogen

gPredonine, Shionogi, Osaka, Japan

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

This research was supported partially by a Grant-in-Aid for Scientific Research (No. 18580323) and “Academic Frontier” Project for Private Universities: matching fund subsidy (2005–2009) from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References