Deregulation of miR-27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer-associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2-derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha-smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR-27a and let-7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR-27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR-27a in canine fibroblasts and involved in their activation. Importantly, miR-27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin-6 (IL-6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR-27a may play an important role in the tumour-microenvironment crosstalk.

A synonymous germline variant in a gene encoding a cell adhesion molecule is associated with cutaneous mast cell tumour development in Labrador and Golden Retrievers.

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.

Identification of molecular genetic contributants to canine cutaneous mast cell tumour metastasis by global gene expression analysis.

Cutaneous mast cell tumours are one of the most common canine cancers. Approximately 25% of the tumours metastasise. Activating c-kit mutations are present in about 20% of tumours, but metastases occur in the absence of mutations. Tumour metastasis is associated with significantly diminished survival in spite of adjuvant chemotherapy. Available prognostic tests do not reliably predict whether a tumour will metastasise. In this study we compared the global expression profiles of 20 primary cutaneous mast cell tumours that metastasised with those of 20 primary tumours that did not metastasise. The objective was to identify genes associated with mast cell tumour metastatic progression that may represent targets for therapeutic intervention and biomarkers for prediction of tumour metastasis. Canine Gene 1.1 ST Arrays were employed for genome-wide expression analysis of formalin-fixed, paraffin-embedded biopsies of mast cell tumours borne by dogs that either died due to confirmed mast cell tumour metastasis, or were still alive more than 1000 days post-surgery. Decreased gene expression in the metastasising tumours appears to be associated with a loss of cell polarity, reduced cell-cell and cell-ECM adhesion, and increased cell deformability and motility. Dysregulated gene expression may also promote extracellular matrix and base membrane degradation, suppression of cell cycle arrest and apoptosis, and angiogenesis. Down-regulation of gene expression in the metastasising tumours may be achieved at least in part by small nucleolar RNA-derived RNA and microRNA-effected gene silencing. Employing cross-validation, a linear discriminant analysis-based classifier featuring 19 genes that displayed two-fold differences in expression between metastasising and non-metastasising tumours was estimated to classify metastasising and non-metastasising tumours with accuracies of 90-100% and 70-100%, respectively. The differential expression of 9 of the discriminator genes was confirmed by quantitative reverse transcription-PCR.

Paediatric mastocytosis: long-term follow-up of 53 patients with whole sequencing of KIT. A prospective study.

BACKGROUND: Mastocytosis is a heterogeneous group of clinical disorders characterized by the abnormal accumulation of mast cells. The adult and paediatric forms differ in their clinical and genetic features and outcomes. OBJECTIVES: To describe the clinical evolution of a well-characterized cohort of paediatric mastocytosis (PM), and to analyse the relationship between KIT mutation and the clinical course. METHODS: This was a prospective cohort study performed at the National Clinical Reference Center for Mastocytosis. Diagnosis was confirmed by identification of KIT mutation on lesional skin biopsy. Mastocytosis subtype, mast cell mediator-related symptoms (MC MRS) and clinical course were recorded. Fifty-three patients with PM and > 4 years of disease course were enrolled. The mean ± SD age at the final evaluation was 13·2 ± 4·8 years. The main outcome was the type of KIT mutation as a predictor of evolution and clinical characteristics. RESULTS: Patients presented with maculopapular cutaneous mastocytosis (n = 44), diffuse cutaneous mastocytosis (n = 6) or mastocytoma (n = 3). The mean duration of disease was 12·1 years. Substantial or partial cutaneous regression (18 of 53 and 16 of 53), stabilization or aggravation (16 of 53) and complete cutaneous regression (three of 53) were noted. MC MRS mainly regressed (21 of 53). For 22 patients, evolution of MC MRS and evolution of cutaneous lesions were different. No significant association between evolution and KIT mutation or between evolution and type of cutaneous mastocytosis was found. A late onset of the disease (after 2 years) is associated with worse evolution. CONCLUSIONS: PM is not systematically self-regressive. MC MRS manifestations and cutaneous lesions can persist or increase overtime. KIT mutation is not a predictor of evolution.

Transcriptomic analysis identified up-regulation of a solute carrier transporter and UDP glucuronosyltransferases in dogs with aggressive cutaneous mast cell tumours.

Gene expression analyses have been recently used in cancer research to identify genes associated with tumorigenesis and potential prognostic markers or therapeutic targets. In the present study, the transcriptome of dogs that had died because of mast cell tumours (MCTs) was characterised to identify a fingerprint having significant influence on prognosis determination and treatment selection. A dataset (GSE50433) obtained using a commercial canine DNA microarray platform was used. The transcriptome of seven biopsies obtained from dogs with histologically confirmed, surgically removed MCTs, treated with chemotherapy, and dead for MCT-related causes, was compared with the transcriptional portrait of 40 samples obtained from dogs with histologically confirmed, surgically removed MCTs and that were still alive at the end of the follow-up period. Among the differentially expressed genes (DEGs), eight transcripts were validated by quantitative real time PCR and their mRNA levels were measured in a cohort of 22 additional MCTs. Statistical analysis identified 375 DEGs (fold change 2, false discovery rate 5%). The functional annotation analysis indicated that the DEGs were associated with drug metabolism and cell cycle pathways. Particularly, members of solute carrier transporter (SLC) and UDP glucuronosyltransferase (UGT) gene families were identified as dysregulated. Principal component analysis (PCA) of the 22 additional MCTs identified the separate cluster dogs dead for MCT-related causes. SLCs and UGTs have been recently recognised in human cancer as important key factors in tumour progression and chemo-resistance. An in-depth analysis of their roles in aggressive canine MCT is warranted in future studies.

Adult-onset mastocytosis in the skin is highly suggestive of systemic mastocytosis.

Adult-onset urticaria pigmentosa/mastocytosis in the skin almost always persists throughout life. The prevalence of systemic mastocytosis in such patients is not precisely known. Bone marrow biopsies from 59 patients with mastocytosis in the skin and all available skin biopsies (n=27) were subjected to a meticulous cytological, histological, immunohistochemical, and molecular analysis for the presence of WHO-defined diagnostic criteria for systemic mastocytosis: compact mast cell infiltrates (major criterion); atypical mast cell morphology, KIT D816V, abnormal expression of CD25 by mast cells, and serum tryptase levels >20 ng/ml (minor criteria). Systemic mastocytosis is diagnosed when the major diagnostic criterion plus one minor criterion or at least three minor criteria are fulfilled. Systemic mastocytosis was confirmed in 57 patients (97%) by the diagnosis of compact mast cell infiltrates plus at least one minor diagnostic criterion (n=42, 71%) or at least three minor diagnostic criteria (n=15, 25%). In two patients, only two minor diagnostic criteria were detectable, insufficient for the diagnosis of systemic mastocytosis. By the use of highly sensitive molecular methods, including the analysis of microdissected mast cells, KIT D816V was found in all 58 bone marrow biopsies investigated for it but only in 74% (20/27) of the skin biopsies. It is important to state that even in cases with insufficient diagnostic criteria for systemic mastocytosis, KIT D816V-positive mast cells were detected in the bone marrow. This study demonstrates, for the first time, that almost all patients with adult-onset mastocytosis in the skin, in fact, have systemic mastocytosis with cutaneous involvement.

Identification of KIT activating mutations in paediatric solitary mastocytoma.

AIMS: Mastocytosis is an abnormal mast cell proliferation involving one or more organs, in particular the skin and bone marrow. In children, disease is usually limited to the skin, with three distinct clinical presentations: urticaria pigmentosa, diffuse cutaneous mastocytosis and solitary mastocytoma. Although the KIT D816V mutation is typically found in adult-onset mastocytosis, it is less commonly seen in childhood-onset mastocytosis, and the frequency of KIT mutations in paediatric solitary mastocytoma is poorly documented. METHODS AND RESULTS: In this study we analysed KIT exons 8, 9, 11, 13 and 17 in nine cases of paediatric solitary mastocytoma using a laboratory-developed Sanger sequencing assay. A KIT mutation was identified in six cases (67%), including three with the D816V mutation typical of adult-onset disease, and another three with an internal tandem duplication (p.A502_Y503dup) in exon 9, previously described in gastrointestinal stromal tumour. CONCLUSIONS: Paediatric solitary mastocytoma is frequently associated with KIT activating mutations, in keeping with a clonal process. KIT mutational status appears insufficient to explain the divergent biology of childhood and adult-onset disease.

Imatinib elicited a favorable response in a dog with a mast cell tumor carrying a c-kit c.1523A>T mutation via suppression of constitutive KIT activation.

Target therapy using the tyrosine kinase inhibitor imatinib is one of the new therapeutic approaches for canine mast cell tumors (MCTs). In the present report, we demonstrate a clinical response to imatinib in a dog with MCT carrying a c-kit c.1523A>T mutation. Moreover, the effect of this mutation on the phosphorylation status of KIT and the inhibitory potency of imatinib on the phosphorylation of the mutant KIT were examined in vitro. A dog with a MCT tumor mass on the right forelimb sole with lymph node metastasis and mastocytemia was treated with imatinib. The MCT mass markedly shrank and mastocytemia became undetectable with 2 weeks of treatment. The lymph node enlarged by metastasis became normal in size with 5 weeks of treatment. From the sequencing analysis of c-kit in tumor cells, a substitution mutation c.1523A>T that alters the amino acid composition (p.Asn508Ile) within the extracellular domain of KIT was identified. The mutant KIT expressed on 293 cells showed ligand-independent phosphorylation and imatinib suppressed this phosphorylation in a dose-dependent manner. From these findings, imatinib was considered to elicit a clinical response in a canine case of MCT via inhibition of the constitutively activated KIT caused by a c-kit c.1523A>T mutation.