Proposed Diagnostic Criteria and Classification of Canine Mast Cell Neoplasms: A Consensus Proposal

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.

Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells

Mast cells are immune cells that store large amounts of mast cell-restricted proteases in their secretory granules, including tryptase, chymase and carboxypeptidase A3. In mouse mast cells, it has been shown that tryptase, in addition to its canonical location in secretory granules, can be found in the nuclear compartment where it can impact on core histones. Here we asked whether tryptase can execute core histone processing in human mast cell leukemia cells, and whether tryptase thereby can affect the epigenetic modification of core histones. Our findings reveal that triggering of cell death in HMC-1 mast cell leukemia cells is associated with extensive cleavage of core histone 3 (H3) and more restricted cleavage of H2B. Tryptase inhibition caused a complete blockade of such processing. Our data also show that HMC-1 cell death was associated with a major reduction of several epigenetic histone marks, including H3 lysine-4-mono-methylation (H3K4me1), H3K9me2, H3 serine-10-phosphorylation (H3S10p) and H2B lysine-16-acetylation (H2BK16ac), and that tryptase inhibition reverses the effect of cell death on these epigenetic marks. Further, we show that tryptase is present in the nucleus of both viable and dying mast cell leukemia cells. In line with a role for tryptase in regulating nuclear events, tryptase inhibition caused increased proliferation of the mast cell leukemia cells. Altogether, the present study emphasizes a novel principle for how epigenetic modification of core histones is regulated, and provides novel insight into the biological function of human mast cell tryptase.

Possible Early Clinical Management of Systemic Mastocytosis Patients with Recently FDA Approved Drug Avapritinib By Molecular Diagnosis Using a Highly Sensitive KIT D816 Mutation Assay

Recently in June 2021, the Food and Drug Administration approved avapritinib (Ayvakit™) for adult patients with advanced systemic mastocytosis (AdvSM), including patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL). Mastocytosis is a heterogeneous, neoplastic disorder characterized by infiltration of abnormal mast cells in one or more organs. Mastocytosis subtypes are defined by disease distribution and the clinical features include, cutaneous mastocytosis (CM), where mast cell infiltration is confined to the skin, and systemic mastocytosis (SM) in which at least one extracutaneous organ is involved, with or without evidence of skin lesions. CM is most common in pediatric patients whereas SM typically presents in adulthood. The presence of somatic, activating mutations in KIT codon 816, predominantly D816V (A2447T), can be identified in the mast cells of 95% or more of patients with systemic mastocytosis (SM) and represent clonal markers in the disease. KIT is located on chromosome 4q12 and encodes for the mast/stem cell growth factor receptor, a type III receptor tyrosine kinase. Detection of KIT D816 mutations serve as a World Health Organization (WHO) minor criterion for the diagnosis of systematic mastocytosis and appear to confer relative resistance to tyrosine kinase inhibitors. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has developed a highly sensitive clinical test for the detection of KIT D816 mutations and molecular diagnosis of systemic mastocytosis patients. The KIT D816 mutation assay is a unique laboratory developed test based on highly sensitive allele specific PCR and its performance characteristics were determined by our laboratory. The sensitivity of the KIT D816 assay is 0.25% allele proportion. The specificity for the D816V mutation is > 99%. Systemic Mastocytosis (SM) is classified as myeloid neoplasm by WHO, however SM may be missed in suspected myeloid neoplasm work up by conventional sequencing technology including NGS which might not be able to achieve higher sensitivity. It is important to have a highly sensitive KIT D816 mutation analysis assay to detect low levels of KIT D816 allele burden that may be present in blood or bone marrow especially at early stage disease. Our highly sensitive laboratory developed KIT D816 assay based on allele specific PCR with a limit of detection of 0.25% could help physicians identify SM patients and clinically manage the disease. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Recently FDA approved Avapritinib for adult patients with advanced systemic mastocytosis (AdvSM), including patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL).

Comprehensive Clinicopathologic and Molecular Analysis of Mast Cell Leukemia With Associated Hematologic Neoplasm: A Report and In-Depth Study of 5 Cases

Mast cell leukemia with associated hematologic neoplasm (MCL-AHN) is a rare and highly aggressive entity that remains understudied due to the paucity of cases. We present a case of a 45-year-old man who was concurrently diagnosed with mast cell leukemia and acute myeloid leukemia. We identified four additional patients who had MCL-AHN in our institution and performed whole-exome sequencing of all available tumors. Our series revealed a novel and identical NR2F6 variant shared among two of the patients. This case series and sequencing results demonstrate the importance of fully characterizing rare tumors that are resistant to treatment.

Capitalizing on paradoxical activation of the MAPK pathway for treatment of Imatinib-resistant mast cell leukemia and chronic myelogenous leukemia

Prevention of fatal side effects during the therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called “paradoxical activation”. By re-analyzing the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KITV560G,D816V-expressing human mast cell leukemia (MCL) cell line HMC-1.2, low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. This allowed then for the effective suppression of HMC-1.2 proliferation and metabolic activity, as well as induction of apoptotic cell death in the presence of nanomolar concentrations of a combination of Ponatinib and of the MEK inhibitor Trametinib. Effectiveness of this drug combination was recapitulated in the human MC line ROSA expressing KITD816V as well as in Imatinib-resistant Ba/F3-BCR-ABLT315I cells. In conclusion, Imatinib-resistance in cells expressing respective mutants of the tyrosine kinases KIT or BCR-ABL can be efficiently bypassed by a low-concentration combination of the TKI Ponatinib and the MEK inhibitor Trametinib, potentially reducing negative side-effects in the therapy of leukemia patients.