The use of COLD‐<scp>PCR</scp>, <scp>DHPLC</scp> and GeneScanning for the highly sensitive detection of <i>c‐<scp>KIT</scp></i> somatic mutations in canine mast cell tumours

Gentilini, Fabio; Mantovani, Vilma; Turba, María Elena

doi:10.1111/vco.12039

Cited by 4 publications

(6 citation statements)

References 34 publications

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“…In a more recent study, using vinblastine and toceranib, no positive correlation between the presence of the mutation and the response to treatment was observed, that is, the effectiveness of the drug was not altered by the mutation. However, the methodologies employed in this study were not sufficiently sensitive ( 72 ) and did not include prevalent mutations beyond exons 8 and 11. In addition, they have a conflict of interest with the pharmaceutical industry, which is responsible for producing toceranib, which could contribute to the unjustifiable use of TKIs ( 57 ).…”

Section: Discussionmentioning

confidence: 99%

“…Despite these results indicated by the extracted data, it is important to note that all studies have performed conventional polymerase chain reaction (PCR)/sequencing methods for the detection of somatic mutations in canine mast cell samples. This can be considered a limitation since data indicate that this methodology has limited sensitivity ( 72 , 74 ). In addition, all mutated tumors were based only on internal tandem duplications (ITD), and not on single-point mutations, and most performed sequencing only of exons 8 and 11, not providing information about other exons that may be constitutively activating, such as exon 9, 14, and 17 ( 12 , 13 , 75 ).…”

Section: Discussionmentioning

confidence: 99%

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Tyrosine kinase inhibitors as an alternative treatment in canine mast cell tumor

et al. 2023

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The current gold standard treatment for canine mast cell tumors (MCT) uses vinblastine sulfate (VBL) as chemotherapy, although tyrosine kinase inhibitors (TKI) have recently been shown to be worthy candidates for treatment. This systematic review aimed to analyze the overall survival (OS), progression-free survival (PFS), overall response rate (ORR), and complete (CR) or partial response (PR) in dogs with MCT treated with TKI compared to standard VBL treatment. The systematic review was registered in the Open Science Framework (OSF) database under the identifier 10.17605/OSF.IO/WYPN4 (https://osf.io/). An electronic search was performed in nine databases. References from eligible studies were also selected to find more registers. A total of 28 studies met the eligibility criteria, and one more was recovered from the references of eligible studies, totaling 29 selected studies. The overall response rate, complete response, and partial response were higher in dogs treated with tyrosine kinase inhibitors than in dogs treated with vinblastine. The overall survival and progression-free survival of vinblastine-treated dogs were higher compared to tyrosine kinase inhibitors-treated dogs. Dogs with mutated KIT treated with tyrosine kinase inhibitors have longer overall survival and progression-free survival compared to those treated with vinblastine. It is important to consider the limitation of the study which should temper the interpretation of the results, videlicet, the extracted data lacked sample standardization and included variables such as animal characteristics, mutation detection methods, tumor characteristics, and treatment types which may have influenced the outcome of the study.Systematic review registrationhttps://osf.io/, identifier: 10.17605/OSF.IO/WYPN4.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Tyrosine kinase inhibitors as an alternative treatment in canine mast cell tumor

et al. 2023

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“…C‐kit somatic mutation screening was performed in a commercial laboratory (Genefast) on the cytologic samples of the spleen according to previously published methods 1–3 . The analysis included the following mutation tests: Ex 11, internal tandem duplication (ITD), insertions and deletions; Ex 8, point mutation Q430R, insertions and deletions; Ex 9, point mutation S479I, point mutation N508I; and Ex 17, point mutation c.2443 G > C. Point mutation S479I in exon 9 of the canine c‐kit gene was detected (Figure 5).…”

Section: Case Presentationmentioning

confidence: 99%

“…C-kit somatic mutation screening was performed in a commercial laboratory (Genefast) on the cytologic samples of the spleen according to previously published methods. [1][2][3] The analysis included the…”

Section: A S E Pr E S E Ntati O Nmentioning

confidence: 99%

Case of mastocytemia and systemic mastocytosis in a dog: ProCyte Dx and Sysmex XT‐2000i scattergram findings, morphologic features, and c‐kit somatic mutation analysis

Palić

Heier

Acke

et al. 2023

Veterinary Clinical Pathol

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A 10‐year‐old female Golden Retriever was presented for a recheck after the complete removal of low‐grade complex mammary carcinoma. The in‐house ProCyte Dx automated counts revealed moderate regenerative anemia and moderate eosinophilia. The ProCyte Dx WBC scattergram showed a cloud in an unusual place parallel and to the right of the monocyte dot plot location. Cells were classified as either monocytes or neutrophils with no clear separation. Complete blood count analysis performed in the laboratory on a Sysmex XT‐2000iV analyzer showed moderate regenerative anemia and WBC count within RI; a differential count was not provided by the instrument. On the Sysmex XT‐2000iV DIFF scattergram, neutrophil and eosinophil dot plots were present at the respective locations and appeared separated, but the instrument did not provide numerical results. In addition to the normal lymphocyte dot plot location, the second cloud of cells classified as lymphocytes was displayed to the right of the monocyte dot plot area. On the WBC/BASO scattergram, the second population of cells was present above and to the right of the leukocyte cluster. Morphologic assessment of the blood smear detected mastocytemia with 16% poorly granulated and degranulated mast cells. FNAs from the liver and spleen contained large aggregates of poorly granulated mast cells. C‐kit somatic mutation screening detected the presence of point mutation S479I in exon 9 of the canine c‐KIT gene. This is the first description of abnormal scattergrams from ProCyte Dx and Sysmex XT‐2000iV analyzers in a dog with concurrent mastocytemia and systemic mastocytosis, and where cytologic assessments of a blood smear, liver, and spleen, and c‐kit somatic mutation analysis were performed.

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“…Elutions were carried out with a mixture of Buffer A (0.1 mol/L triethylammonium acetate-TEAA) and Buffer B (0.1 mol/L TEAA, 25% acetonitrile). The amplicons were run at two different partiallydenaturing temperatures of 56.3°C and 57.8°C used for the DHPLC screening of the entire exon 9, including the p.Asp508Ile mutation as previously reported [25]. The data analysis was carried out using Navigator software, (ADS Biotec Limited).…”

Section: Dhplc and Sequencingmentioning

confidence: 99%

The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs

Gentilini

Turba²,

Dally

et al. 2020

BMC Vet Res

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Background: Gain-of-function mutations in KIT are driver events of oncogenesis in mast cell tumours (MCTs) affecting companion animals. Somatic mutations of KIT determine the constitutive activation of the tyrosine kinase receptor leading to a worse prognosis and a shorter survival time than MCTs harbouring wild-type KIT. However, canine MCTs carrying KIT somatic mutations generally respond well to tyrosine kinase inhibitors; hence their presence represents a predictor of treatment effectiveness, and its detection allows implementing a stratified medical approach. Despite this, veterinary oncologists experience treatment failures, even with targeted therapies whose cause cannot be elucidated. The first case of an MCT-affected dog caused by a secondary mutation in the tyrosine kinase domain responsible for resistance has recently been reported. The knowledge of this and all the other mutations responsible for resistance would allow the effective bedside implementation of a deeply stratified and more effective medical approach. Case presentation: The second case of a canine MCT carrying a different resistance mutation is herein described. The case was characterised by aggressive behaviour and early metastasis unresponsive to both vinblastine-and masitinib-based treatments. Molecular profiling of the tumoural masses revealed two different mutations; other than the already known activating mutation p.Asn508Ile in KIT exon 9, which is tyrosine kinase inhibitor-sensitive, a nearly adjacent secondary missense mutation, p.Ala510Val, which had never before been described, was detected. In vitro transfection experiments showed that the secondary mutation did not cause the constitutive activation by itself but played a role in conferring resistance to masitinib. Conclusions: This study highlighted the importance of the accurate molecular profiling of an MCT in order to improve understanding of the molecular mechanism underlying tumourigenesis and reveal chemoresistance in MCTs for more effective therapies. The detection of the somatic mutations responsible for resistance should be included in the molecular screening of MCTs, and a systematic analysis of all the cases characterised by unexpected refractoriness to therapies should be investigated in depth at both the genetic and the phenotypic level.

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The use of COLD‐PCR, DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

Cited by 4 publications

References 34 publications

Tyrosine kinase inhibitors as an alternative treatment in canine mast cell tumor

Tyrosine kinase inhibitors as an alternative treatment in canine mast cell tumor

Case of mastocytemia and systemic mastocytosis in a dog: ProCyte Dx and Sysmex XT‐2000i scattergram findings, morphologic features, and c‐kit somatic mutation analysis

The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs

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