Histiocytic sarcoma (HS) is an aggressive malignant neoplasm of dendritic cell origin that is common in certain breeds of dogs. High prevalence of fatal, disseminated HS has been described in Bernese Mountain Dogs (BMDs). Support for genetic predisposition to develop HS has been presented in several studies, but to date, causative genetic events have not been reported. In addition, no driver mutations have been identified in tumours. Recently, E76K gain-of-function mutation in SHP2 encoded by the PTPN11 gene has been described in human histiocytic malignancies. In our study, we identified the PTPN11 in HS of BMDs. Amplification of exon 3 of the PTPN11 gene followed by Sanger sequencing was used to detect the mutation and estimate the prevalence in HS from 30 BMDs, 13 Golden Retrievers and 10 other dog breeds. The overall prevalence of PTPN11 in HS of BMDs was 36.67% compared with 8.69% in other breeds. No mutation was identified in normal tissues from 10 BMDs with HS that carried the mutation and 12 control dogs with no neoplastic disease, including 6 BMDs. Increased immunoreactivity for AKT, phosphorylated ERK1/2 and phosphorylated AKT in a small subset of BMDs with PTPN11 suggests that a gain-of-function might be mediated by the ERK and AKT pathways. These data suggest PTPN11 as an important driver mutation of HS in BMDs. This information may not only aid in unravelling the tumourigenic events associated with HS in BMDs, but also help in identifying more promising therapeutic strategies.

Histiocytic sarcoma in humans is an aggressive orphan disease with a poor prognosis as treatment options are limited. Dogs are the only species that spontaneously develops histiocytic sarcoma with an appreciable frequency, and may have value as a translational model system. In the current study, high-throughput drug screening utilizing histiocytic sarcoma cells isolated from canine neoplasms identified these cells as particularly sensitive to a MEK inhibitor, trametinib. One of the canine cell lines carries a mutation in PTPN11 (E76K), and another one in KRAS (Q61H), which are associated with the activation of oncogenic MAPK signaling. Both mutations were previously reported in human histiocytic sarcoma. Trametinib inhibited sensitive cell lines by promoting cell apoptosis, indicated by a significant increase in caspase 3/7. Furthermore, findings were successfully recapitulated in an intrasplenic orthotopic xenograft mouse model, which represents a disseminated aggressive form of histiocytic sarcoma. Mice with histiocytic sarcoma xenograft neoplasms that were treated with trametinib had significantly longer survival times. Target engagement was validated as activity of ERK, downstream of MEK, was significantly downregulated in neoplasms of treated mice. Additionally, trametinib was found in plasma and neoplastic tissues within projected therapeutic levels. These findings demonstrate that in dogs, histiocytic sarcoma may be associated with a dysfunctional MAPK pathway, at least in some cases, and may be effectively targeted through MEK inhibition. Clinical trials to test safety and efficacy of trametinib in dogs with histiocytic sarcoma are warranted, and may provide valuable translational information to similar diseases in humans..

While the genetic contributions to the predisposition of Bernese mountain dogs (BMDs) to histiocytic sarcoma (HS) remains unclear, some insights into key genetic drivers have been gained. Our group recently reported a mutation in the PTPN11 gene (E76K). We have now identified a second missense mutation in PTPN11 (G503V), and a mutation in KRAS (Q61H) present in HS cell lines. These mutations are associated with malignancies in humans, and known to be gain-of-function mutations that result in activation of the mitogen-activated protein kinase (MAPK) pathway. The goal of the present study was to evaluate the prevalence of these mutations in a large sample of HS cases from BMDs and golden retrievers, and in lymphoma cases, from a cohort of BMDs. Mutations in PTPN11 were present in HS in 41/96 (43%) BMDs, and in 3/13 (23%) golden retrievers. PTPN11 mutations E76K and G503V did not coexist in the same neoplasm. The KRAS mutation was much less frequent, with a prevalence of 3.1% (3/96). We did not identify either PTPN11 nor KRAS mutations in any of the lymphoma samples. These results point out the potential relevance of PTPN11 and KRAS mutations as activators of the oncogenic MAPK pathway for canine HS, particularly in BMDs.

BackgroundHistiocytic sarcoma is a rare disorder in humans, however it is seen with appreciable frequency in certain breeds of dogs, such as Bernese mountain dog. The purpose of this study was to fully characterize a novel canine histiocytic sarcoma cell line, and utilize it as a tool to screen for potential therapeutic drugs.MethodsThe histiocytic sarcoma cell line was characterized by expression of cellular markers as determined by immunohistochemistry and flow cytometry techniques. The neoplastic cells were also evaluated for their capability of phagocytizing beads particles, and their potential to grow as xenograft in an immunodeficient mouse. We investigated the in vitro cytotoxic activity of a panel of thirteen compounds using the MTS proliferation assay. Inhibitory effects of different drugs were compared using one-way ANOVA, and multiple means were compared using Tukey’s test.ResultsNeoplastic cells expressed CD11c, CD14, CD18, CD45, CD172a, CD204, MHC I, and vimentin. Expression of MHC II was upregulated after exposure to LPS. Furthermore, the established cell line clearly demonstrated phagocytic activity similar to positive controls of macrophage cell line. The xenograft mouse developed a palpable subcutaneous soft tissue mass after 29 days of inoculation, which histologically resembled the primary neoplasm. Dasatinib, a tyrosine kinase pan-inhibitor, significantly inhibited the growth of the cells in vitro within a clinically achievable and tolerable plasma concentration. The inhibitory response to dasatinib was augmented when combined with doxorubicin.ConclusionsIn the present study we demonstrated that a novel canine histiocytic sarcoma cell line presents a valuable tool to evaluate novel treatment approaches. The neoplastic cell line favorably responded to dasatinib, which represents a promising anticancer strategy for the treatment of this malignancy in dogs and similar disorders in humans.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4132-0) contains supplementary material, which is available to authorized users.

This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0525/-/DC1. M.P. is currently affiliated with the Royal (Dick) School of Veterinary Studies and the Roslin Institute, Roslin, Midlothian, U.K.