Mast Cell Leukemia Research Articles

Whole Genome Sequencing Identifies Non-KIT Mutations and Cytogenetic Aberrations in Systemic Mastocytosis but Has Limited Sensitivity for Detection of KIT D816V

▪Background: Systemic mastocytosis (SM) is a hematologic neoplasm characterized by the infiltration of clonal mast cells in the bone marrow or other extra-cutaneous organs. The clinical course varies between advanced and non-advanced (indolent and smoldering SM) forms of SM. The vast majority of patients harbor the activating D816V mutation in the KIT tyrosine kinase. Additional somatic mutations in other genes have been recognized as risk factors in SM. Cytogenetic aberrations are rarely found in SM but have been associated with advanced disease. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) have been described as an alternative to cytogenetics and targeted molecular genetic analysis in myeloid cancers.Aim: To assess the ability of WGS/WTS to detect cytogenetic aberrations and recurrent somatic mutations in SM.Methods: 120 patients (51 female, 69 male) diagnosed with SM were analyzed with WGS/WTS and results were compared with orthogonal data of KIT D816V PCR, targeted sequencing, and cytogenetics. 47 patients (39%) were diagnosed with advanced SM (1 mast cell leukemia, 3 aggressive SM, 43 SM with associated hematologic neoplasm). For WGS, 2x151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). BaseSpace's Tumor/Normal app v3 was used to call variants with Strelka Somatic Variant Caller v2.4.7 and structural variants (aberrations with >50bp in size) with Manta (v0.28.0). Genomic DNA from a mixture of multiple anonymous donors (Promega, Fitchburg, WI, USA) was used as normal. For WTS, 2x101 bp paired-end reads were produced with a median of 50 mio. reads per sample, aligned with STAR v2.5.0, and variants were called using Isaac Variant Caller v2.3.13.Results: WGS/WTS detected cytogenetic aberrations in 21% of patients: 2 patients displayed a complex aberrant karyotype, 3 balanced structural aberrations, 16 copy number alterations, and 6 copy number neutral losses of heterozygosity. Aberrations detected by chromosome banding analysis were also found by WGS in all but three patients (small clones with aberrations present in ≤20% of metaphases and <10% of interphase nuclei as determined by FISH). In contrast, WGS/WTS detected additional aberrations in 16 patients. The frequency of chromosomal aberrations detected by WGS/WTS was higher in advanced compared to non-advanced SM (34% vs. 12%, p<0.05). KIT D816V was detected by PCR in 98%, by WGS in 21% and by WTS in 46% of patients. The detection rate by WGS was significantly higher in advanced (36%) compared to non-advanced SM (12%, p<0.05) while no difference was observed for WTS (45% vs. 47%). Somatic mutations outside of KIT were analyzed within a subset of 121 genes recurrently mutated in hematologic neoplasms. 46% of patients showed non-KIT mutations with a median of 2 mutations per patient. Both frequency of non-KIT mutations as well as the median number of mutations per patient was higher in advanced (83%; n=3) compared to non-advanced SM (22%, n=1, p<0.05). Finally, we analyzed the impact of genetic aberrations on survival in our SM cohort. Patients were grouped according to the presence of chromosomal aberrations and gene mutations (non-KIT) as assessed by WGS/WTS. SM patients with both types of aberrations (n=16), one type of aberration (n=47; gene mutations only n=38; chromosomal aberrations only n=8), or no aberration but KIT D816V (n=57) showed significant differences in overall survival (p<0.05, Figure 1).Conclusions: WGS/WTS has limited sensitivity for detection of KIT D816V in SM. This finding can be explained by the low KIT D816V mutation burden typically found in bone marrow aspirates of SM patients. In line, we observed a slightly higher detection rate in advanced SM and in RNA-based WTS analysis. As WGS/WTS will be applied for the diagnostic workup of myeloid malignancies in the future and SM associated with other hematologic neoplasms may be overlooked if not specifically investigated, additional PCR-based techniques are still mandatory to rule out KIT D816V as a diagnostic criterion for SM. In contrast, WGS/WTS detects both chromosomal aberrations and additional gene mutations in patients with SM and can be used as an alternative to cytogenetics and targeted sequencing for risk assessment. In particular, the absence of genetic aberrations in WGS/WTS identifies SM patients with indolent course of the disease and favorable prognosis. [Display omitted] DisclosuresHoermann: Novartis: Honoraria. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Cladribine therapy for advanced and indolent systemic mastocytosis: Mayo Clinic experience in 42 consecutive cases.

We describe our single institution experience with cladribine therapy in 42 patients with systemic mastocytosis (SM): 22 advanced (adv-SM; median age 65 years, 68% males) and 20 indolent/smouldering SM (ISM/SSM; median age 56 years, 45% males); subcategories included eight aggressive, 13 associated with another haematological neoplasm, one mast cell leukaemia, 17 ISM and three SSM. Overall/major response rates were 77%/45% for adv-SM and 70%/60% for ISM/SSM, and median (range) duration of response 10 (4-75) and 46 (4-140) months respectively. A >50% reduction in bone marrow mast cell burden and serum tryptase level was documented in 63% and 67% of patients with adv-SM and 50% and 46% with ISM/SSM respectively. The presence of KIT proto-oncogene, receptor tyrosine kinase (KIT)D816V predicted response in adv-SM: 17 (90%) of 19 with and none of three without the mutation responded (P < 0·01). Treatment-emergent adverse events were mostly limited to transient cytopenias: Grade 3/4 neutropenia, thrombocytopenia, or lymphopenia occurred in 27%, 27% and 27% of patients with adv-SM, and 5%, 5% and 30% with ISM/SSM respectively. The present study provides practical information that might be considered when making treatment choices between cladribine and newer KIT-targeted therapies and identifies the absence of KITD816V as a potential marker of cladribine resistance in advanced SM; the latter observation needs confirmation in a larger study.

Precision Medicine in Systemic Mastocytosis.

Mastocytosis is a rare hematological neoplasm characterized by the proliferation of abnormal clonal mast cells (MCs) in different cutaneous and extracutaneous organs. Its diagnosis is based on well-defined major and minor criteria, including the pathognomonic dense infiltrate of MCs detected in bone marrow (BM), elevated serum tryptase level, abnormal MCs CD25 expression, and the identification of KIT D816V mutation. The World Health Organization (WHO) classification subdivides mastocytosis into a cutaneous form (CM) and five systemic variants (SM), namely indolent/smoldering (ISM/SSM) and advanced SM (AdvSM) including aggressive SM (ASM), SM associated to hematological neoplasms (SM-AHN), and mast cell leukemia (MCL). More than 80% of patients with SM carry a somatic point mutation of KIT at codon 816, which may be targeted by kinase inhibitors. The presence of additional somatic mutations detected by next generation sequencing analysis may impact prognosis and drive treatment strategy, which ranges from symptomatic drugs in indolent forms to kinase-inhibitors active on KIT. Allogeneic stem cell transplant (SCT) may be considered in selected SM cases. Here, we review the clinical, diagnostic, and therapeutic issues of SM, with special emphasis on the translational implications of SM genetics for a precision medicine approach in clinical practice.

A MAST KNOWN CAUSE OF SHOCK

TOPIC: Critical Care TYPE: Fellow Case Reports INTRODUCTION: The main types of shock include cardiogenic, hypovolemic, distributive, and neurogenic shock. Management of shock requires identifying and correcting the underlying cause, and supporting end-organ perfusion with fluid resuscitation and vasoactive medications. Here we present a rare cause of distributive shock. CASE PRESENTATION: A 65-year-old man presented with 2-month history of progressive weakness. Laboratory studies reviewed new thrombocytopenia to 25 x10(9)/L and acute renal failure with creatinine of 3.24 mg/dL. His bone marrow biopsy was concerning for acute mast cell leukemia, and he was started on treatment with 1 gram of methylprednisolone. About 1 hour after receiving this infusion, he developed chest pain, tachycardia and tachypnea. He was urgently transferred to the ICU. In the ICU, his condition progressed into ventricular fibrillation requiring CPR, defibrillation, and intubation. He was in refractory circulatory shock requiring high doses of norepinephrine, epinephrine, vasopressin, and stress dose steroids. Given his underlying mast cell leukemia, empirical treatment for mast cell activation syndrome was started. He received scheduled diphenhydramine, famotidine, montelukast and cromolyn. Patient was quickly weaned off vasoactive medications in the next 24 hours. Tryptase obtained at time of his hypotensive event was markedly elevated at greater than 2000 ng/mL. He had worsening renal failure requiring renal replacement therapy, and given his underlying diagnosis of mast cell leukemia, a rare hematological malignancy with limited treatment options and high mortality, the focus of his care was transitioned to concentrate solely on his comfort. DISCUSSION: Mast cell disorders include cutaneous and systemic mastocytosis, allergic diseases, and idiopathic disorders. Acute mast cell leukemia is a rare form of systemic mastocytosis characterized by at least 20% immature mast cells in bone marrow. Sudden mast cell activation leads to degranulation of mast cell and release of pre-formed mediators including histamine. It also stimulates de novo synthesis of other cytokines and chemokines. Typical symptoms include flushing, lightheadedness and abdominal cramping, but can progress to syncope and distributive shock, as seen in our patient. Treatment includes identification and avoidance of triggers, and supportive care with antihistamines (both H1 and H2 blockers) and cromolyn (mast cell stabilizer). CONCLUSIONS: It is essential for critical care clinicians to recognize mast cell activation syndrome as a potential cause of refractory shock, especially in at risk patients with systemic mastocytosis. Prompt treatment with antihistamines and cromolyn can facilitate resolution of shock. REFERENCE #1: Akin C, Valent P, Metcalfe DD. Mast cell activation syndrome: Proposed diagnostic criteria. J Allergy Clin Immunol. 2010;126(6):1099-104.e4. doi:10.1016/j.jaci.2010.08.035 REFERENCE #2: Molderings GJ, Haenisch B, Brettner S, et al. Pharmacological treatment options for mast cell activation disease. Naunyn Schmiedebergs Arch Pharmacol. 2016;389(7):671-694. doi:10.1007/s00210-016-1247-1 DISCLOSURES: No relevant relationships by Alice Gallo De Moraes, source=Web Response No relevant relationships by Zhenmei Zhang, source=Web Response

The Role of Avapritinib for the Treatment of Systemic Mastocytosis.

Systemic mastocytosis is a rare hematologic disorder characterized by the clonal proliferation of mast cells in extra-cutaneous organs. This disease can be further subdivided into five different phenotypes: indolent systemic mastocytosis (ISM), smoldering systemic mastocytosis (SSM), aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated hematological neoplasm (SM-AHN) and mast cell leukemia (MCL). The tyrosine kinase inhibitor (and also potent KIT D816V inhibitor) avapritinib, initially approved for the treatment of gastrointestinal stromal tumors (GISTs) bearing a PDGFRA exon 18 mutation, also showed great promise in patients with systemic mastocytosis, a disease known to be driven by a mutation in KIT (D816V). We present an overview of this rare disorder, including a review of the current understanding of the genetic mechanisms which lead to the disease state, the action of the tyrosine kinase inhibitors, as well as the latest clinical trial data which led to the current recommendations for the use of avapritinib.

Nuclear αvβ3 integrin expression, post translational modifications and regulation in hematological malignancies.

αvβ3 integrin, a plasma membrane protein, is amply expressed on an array of tumors. We identified nuclear αvβ3 pool in ovarian cancer cells and were interested to explore this phenomenon in two rare and aggressive types of leukemia, T-cell acute lymphoblastic leukemia (T-ALL) and Mast cell leukemia (MCL) using Jurkat and HMC-1 cell lines, respectively. Moreover, we collected primary cells from patients with chronic lymphocytic leukemia (CLL, n=11), the most common chronic adult leukemia and used human lymphoblastoid cell lines (LCL) generated from normal B cells. Nuclear αvβ3 integrin was assessed by Western blots, confocal microscopy, and the ImageStream technology which combines flow-cytometry with microscopy. We further examined post translational modifications (phosphorylation/glycosylation), nuclear trafficking regulation using inhibitors for MAPK (U0126) and PI3K (LY294002), as well as nuclear interactions by performing Co-immunoprecipitation (Co-IP). αvβ3 integrin was identified in all cell models within the nucleus and is N-glycosylated. In primary CLL cells the β3 integrin monomer is tyrosine Y759 phosphorylated, suggesting an active receptor conformation. MAPK and PI3K inhibition in Jurkat and CLL cells led to αvβ3 enhancement in the nucleus and a reduction in the membrane. The nuclear αvβ3 integrin interacts with ERK, Histone H3 and Lamin B1 in Jurkat, Histone H3 in CLL cells, but not in control LCL cells. To conclude, this observational study provides the identification of nuclear αvβ3 in hematological malignancies and lays the basis for novel cancer-relevant actions, which may be independent from the membrane functions.

National clinical recommendations for the diagnosis and treatment of mastocytosis

Introduction. Recommendations cover the current state of diagnosis and treatment of mastocytosis.Aim — а consolidation of the Russian experts’ opinion on treatment for adult mastocytosis.Main findings. The recommendations have been developed taking into account foreign literature, national experience and world clinical evidence on therapy for systemic and cutaneous mastocytoses, mast cell leukaemia and other mastocytosis forms. The significance of bone marrow and peripheral blood molecular genetic testing for the presence of KITD816V gene variants is demonstrated. The treatment regimens described are based on midostaurin, imatinib, cladribine, hydroxycarbamide, interferon alfa and haematopoietic stem cell transplantation. Prognosis in different forms of mastocytosis is provided.

MPN-180: A Novel Mechanism of Action of Midostaurin in Systemic Mastocytosis: Beyond KIT Inhibition

Context: Constitutively active KIT tyrosine kinase harboring the D816V mutation is the major driver of systemic mastocytosis (SM) pathogenesis. We have previously reported that the loss of function of SETD2 histone methyltransferase due to reduced protein stability is a common cooperating event in advanced SM (advSM). Objective: To dissect the mechanisms of action of midostaurin in advSM and to explore the potential of SETD2 as a marker of response to therapy. Materials: The mast cell (MC) leukemia cell line HMC-1 (negative for SETD2) and primary neoplastic MCs from 10 advSM patients, collected at regular time points during midostaurin treatment. Results: This study started from our preliminary observations indicating that aurora kinase A (AKA) is overexpressed and hyperactivated in advSM and is implicated in SETD2 proteasomal degradation. The new gold standard of therapy in advSM is midostaurin, which targets KIT andseveral other kinases, including AKA. We, thus, investigated whether midostaurin treatment may result in efficient AKA inhibition and subsequent SETD2 rescue. To this purpose, the HMC-1 cell line was treated with 5 µM midostaurin for 24 h, and phospho-AKA (T288), SETD2, and H3K36me3 (surrogate marker of SETD2 activity) were evaluated by WB. Midostaurin reduced AKA phosphorylation by 60%, partially restoring SETD2 expression and activity. We next wondered whether the same happens in patients treated with midostaurin. We, thus, collected and performed WB analysis on neoplastic mast cells from advSM patients before and during midostaurin treatment. We found that midostaurin may target AKA and rescue SETD2 expression and function in vivo and that SETD2/H3K36me3 rescue correlates with the clinical response to midostaurin and the reduction of KIT D816V allele burden, as assessed by digital PCR at 3, 6, and 12 months. In 8 patients who responded to midostaurin, SETD2/H3K36me3 rescue was detected by WB as early as 3 months after treatment onset, whereas 2 refractory patients did not show any increase in SETD2/H3K36me3 and maintained an unaltered KIT allele burden. Conclusions: Midostaurin treatment induces AKA partial dephosphorylation and consequent rescue of SETD2 expression and function in vitro and in vivo SETD2/H3K36me3 may serve as a biomarker of response to midostaurin. Supported by AIRC project 23001

Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms

Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition ofMC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that invivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.

Avapritinib for Systemic Mastocytosis

ABSTRACT Introduction Systemic mastocytosis (SM) is a rare myeloid neoplasm driven in ≈95% of cases by activating KIT mutations, usually D816V. SM can be indolent (ISM), smoldering (SSM) and advanced (AdvSM), the latter characterized by organ damage resulting from infiltrating neoplastic mast cells. The vast majority of cases are indolent, with near-normal life expectancy, although symptoms can be severe. AdvSM, comprising aggressive SM, SM with an associated hematologic neoplasm and mast cell leukemia, however, carries a poor prognosis. Avapritinib is a highly potent and selective inhibitor of mutant KIT. Areas Covered We provide an overview of SM, including the current therapeutic landscape, and discuss avapritinib in detail: its chemistry and discovery, pharmacodynamic and pharmacokinetic data, current approval status and safety and efficacy profiles in both advanced and non-advanced SM. Expert Opinion With a response rate of 75% amongst evaluable patients with AdvSM and marked reductions observed in measures of mast cell and disease burden, avapritinib stands out as a highly effective targeted therapy for this mutant KIT-driven disease. Cognitive impairment may occur, and intracranial hemorrhage has been reported, particularly in association with severe thrombocytopenia. Early results in patients with ISM/SSM are encouraging. Avapritinib is now approved in the US for AdvSM.

SETD2 Non-Genomic Loss of Function and p53 Functional Inactivation in Advanced Systemic Mastocytosis (SM): Pathogenetic and Therapeutic Implications

KIT constitutive activation is thought to be necessary but not sufficient for the development of aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). The identification and dissection of cooperating pathogenetic pathways may set the basis for the development of novel therapeutic strategies.We have recently found that the HMC-1.1 and -1.2 MCL cell lines and ASM/MCL patients (pts) display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non-genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity, homologous recombination (HR), mismatch repair (MMR). Inhibition of proteasome-mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3. Reversing the detrimental effects of SETD2 and H36K36Me3 deficiency may be a promising therapeutic strategy.In this study, we aimed to investigate the effects of SETD2 loss of function in ASM and MCL and to assess the in vitro efficacy of second-generation proteasome inhibitors as therapeutic agents. To investigate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting and immunofluorescence to assess phosphorylated histone 2A.X (γH2AX), which marks ongoing DNA damage signaling and DNA double strand breaks and Rad51, which is a surrogate for active HR. Compared to cells from healthy controls, cells from SETD2- and H3K36Me3-deficient MCL pts had significantly higher levels of γH2AX and Rad51. To assess whether the increased DNA damage may translate into a greater number of submicroscopic genomic lesions, and whether the degree of SETD2 down-modulation may be inversely correlated with the degree of genomic complexity, SNP-array data from 13 pts were examined. Structural complexity score (SCS) and genome instability index (GII) were not significantly different (SCS, 387 vs 376; GII, 0.293 vs 0.337) between pts with > or <50% residual SETD2 and H3K36Me3 levels, suggesting that the activity of HR repair may counteract DNA damage events. Moreover, SETD2 down-modulation was not associated with reduced MMR proficiency, as assessed by lack of microsatellite instability. On the other hand, RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras and intron retention not observed in healthy donors.In renal cell carcinoma cell lines, SETD2 has been shown to bind and activate p53. After proteasome inhibition, SETD2 was indeed found to immunoprecipitate with p53 in HMC-1.1 and -1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with SP-141, a new class of MDM2 inhibitors that promote MDM2 auto-ubiquitination and degradation, also rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses for 24-48-72-96 hours induced cytostatic but not cytotoxic effects as shown by cell growth curves. Cytostatic effects resulted from a G2/M block of cell cycle induced by Gadd45a overexpression. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells (LD50=0.133µM and 0.050µM respectively).Finally, treatment with the second-generation proteasome inhibitors carfilzomib and ixazomib potently induced apoptosis and reduced colony growth in HMC-1.1 (LD50=0.10nM and 0.66nM respectively) and -1.2 (LD50=0.13nM and 0.72nM respectively) cells.In conclusion: MDM2-mediated ubiquitination contributes to SETD2 non-genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for TP53 gene mutations. Approaches aimed to inhibit MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies worth to be further explored.Supported by AIL and AIRC (16996) DisclosuresZanotti:Deciphera: Consultancy. Ciceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Valent:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Blueprint: Research Funding; Teva: Honoraria. Martinelli:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Roche: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy. Soverini:Incyte Biosciences: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

Clinical Activity in a Phase 1 Study of Blu-285, a Potent, Highly-Selective Inhibitor of KIT D816V in Advanced Systemic Mastocytosis (AdvSM)

Clinical Activity in a Phase 1 Study of Blu-285, a Potent, Highly-Selective Inhibitor of KIT D816V in Advanced Systemic Mastocytosis (AdvSM)

Systematic KIT Sequencing Identifies Unsuspected Systemic Mastocytosis with Associated Hematological Neoplasm

Background: Systemic mastocytosis (SM) can occur on its own or in association with non-mast cell disorders (SM with associated hematological neoplasm, SM-AHN). Because extensive bone marrow involvement by non-mast cell disease may obscure mast cells in routinely-stained biopsies, it is possible that SM-AHN is under-recognized. Given the high association between KIT D816V mutations and SM, we asked if systematic evaluation for somatic KIT mutations in patients with known or suspected hematologic malignancies could identify unsuspected cases of SM-AHN.Methods: We queried 5754 clinical next generation sequencing (NGS) samples from 4173 unique patients with known/suspected hematologic neoplasms evaluated at our institution between 1/1/15 and 6/30/17 using a custom, 95-gene, amplicon-based NGS panel (PMID 27339098). Exons 8-9, 11 and 17 of KIT were analyzed. In patients with a somatic KIT mutation but without known SM, marrows were assessed for SM with mast cell tryptase and CD25 immunohistochemistry (IHC).Results: We identified 37 somatic KIT mutations in 35 patients (Figure), including non-D816V (n=9) and D816V (n=26; 1 case with concurrent D816Y).Non-D816V KIT mutations occurred in 9 patients: 5 core-binding factor-rearranged acute myeloid leukemia (CBF-AML) [418_419insFF (n=1); 417_419TYDdelinsI (n=1); Y823C (n=1); D816Y (n=2; 1 with also V559D)], 1 AML with myelodysplasia-related changes (AML-MRC; 419delD), 1 acute promyelocytic leukemia (N822K), 1 chronic myelomonocytic leukemia (R634W), and 1 mast cell leukemia (D816Y). In addition to the patient with mast cell leukemia, 2 patients with CBF-AML had known SM [418_419insFF; D816Y and concurrent V559D]. IHC performed for this study on the remaining 6 cases did not reveal multifocal, dense aggregates of abnormal mast cells.Of the 26 cases with the D816V variant, 18 had an established diagnosis of SM [SM without AHN (n=6); SM with suspected AHN, based on marrow morphology and/or abnormal peripheral counts (n=5); SM-AHN where AHN = chronic myelomonocytic leukemia (n=6) or myelodysplastic syndrome (n=1)]. In the 3 remaining cases of CMML or MDS with KIT D816V (marked on Figure), SM was not clinically or pathologically suspected. IHC performed following identification of the KIT D816V mutation by NGS revealed clusters of spindled, CD25+ mast cells, consistent with SM-AHN. All 3 patients also had a serum tryptase >20 ng/mL and splenomegaly, although these parameters may be influenced by the AHN. In 1 patient, retrospective review of prior liver/lymph node biopsies revealed previously unrecognized aggregates of spindled, CD25+ mast cells accounting for unexplained fibrosis. The patient was started on Midostaurin following SM diagnosis with significant clinical response. The final 5 cases with the D816V variant were AML. This included 4 de novo or therapy-related AMLs in which the KIT mutation was not present at initial diagnosis. In 2 cases, the mutation appeared in peripheral blood at time of relapse after stem cell transplant (concurrent bone marrow biopsy not performed; pre-transplant marrows showed no evidence of SM by IHC). In the other 2 cases, there was no IHC evidence of SM in leukemic marrows bearing the KIT D816V mutation. The final AML was AML-MRC with known KIT mutation; increased, CD25+ mast cells were present at time of AML but were not spindled or clustered.None of the cases of isolated SM had additional driver mutations, whereas 15 of 16 cases of known or suspected SM-AHN exhibited driver mutations in genes that are recurrently mutated in myeloid neoplasms. In the SH-AHN cases, the KIT D816V variant allele fraction varied from subclonal to equivalent to levels of other driver mutations. A group of 30 cases of CMML (n=15) and MDS (n=15) without KIT mutations were used as controls. None of these cases showed IHC evidence of SM.Conclusion: We find that detection of the KIT D816V mutation in CMML or MDS is invariably associated with SM, even in cases in which SM is not clinically suspected. Our data strongly suggest that identification of the KIT D816V mutation in patients with a diagnosis of CMML or MDS should prompt additional testing for underlying SM given the potential prognostic and therapeutic implications. [Display omitted] DisclosuresAster:Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees. Steensma:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Incyte: Equity Ownership; Onconova: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Takeda: Consultancy; H3 Biosciences: Consultancy. Lindsley:Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; MedImmune: Research Funding. DeAngelo:Celgene: Research Funding; ARIAD: Consultancy, Research Funding; Glycomimetics: Research Funding; Blueprint Medicines: Honoraria, Research Funding; Immunogen: Honoraria, Research Funding; BMS: Consultancy; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding.

Phase 2 Study of Brentuximab Vedotin in Patients with Advanced Systemic Mastocytosis

Phase 2 Study of Brentuximab Vedotin in Patients with Advanced Systemic Mastocytosis

Selection and Efficacy of Cytoreductive Agents in Patients with Mastocytosis Included in the Registry of the European Competence Network on Mastocytosis (ECNM)

Selection and Efficacy of Cytoreductive Agents in Patients with Mastocytosis Included in the Registry of the European Competence Network on Mastocytosis (ECNM)

Digital Droplet PCR: Next Generation KIT D816V Testing As Advanced Tool for Diagnosis and Prognostication in Mastocytosis

Background: The somatic mutation KIT D816V is a diagnostic criterion for systemic mastocytosis (SM) and can be found in the majority of patients. Therefore, sensitive detection of KIT D816V in blood and bone marrow (BM) is important for diagnosis of SM. Additionally, precise quantification of the KIT D816V allele burden is relevant clinically as it helps to predict multilineage involvement and prognosis in advanced SM and might be useful as a biomarker for treatment response to midostaurin or other interventional drugs. However, despite the high relevance of reliable KIT D816V allele burden measurement, no single assay for the detection and quantification of the KIT mutation has been accepted as a global standard. In addition, differences in calibration material and data normalization hamper the standardization of KIT D816V quantification and complicate the comparison of results obtained in different study groups. Digital droplet PCR (ddPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations.Methods: We performed a validation study of the PrimePCR ddPCR mutation assay for KIT wild-type and the KIT D816V point mutation using the QX-200 droplet-reader on 302 DNA samples from 156 patients with mastocytosis (median age 56 years; 85 females, 71 males) in comparison to melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) (Sotlar et al., 2003, AJP 162, 737-74) and allele-specific quantitative real-time PCR (qPCR) (Kristensen et al., 2011, JMD 13, 180-188). According to WHO criteria, 16 patients were diagnosed with cutaneous mastocytosis, 5 with mastocytosis in the skin, 105 with indolent SM, 7 with smoldering SM, 1 with BM mastocytosis, 8 with aggressive SM, 12 with SM with associated hematologic neoplasm, and 2 with mast cell leukemia. Data were analyzed using the Bland-Altman plot, ordinary least squares regression and Passing-Bablok regression for method comparison, Kaplan-Meier survival plots, and log-rank test.Results: ddPCR was found to be highly sensitive, specific and precise in detecting KIT D816V with a limit of detection of 0.005% variant allele fraction and a coefficient of variation <20%. ddPCR identified the mutation in 90% of all mastocytosis patients compared to 89% with qPCR and 70% with clamp-PCR indicating a superior diagnostic yield of ddPCR over clamp-PCR (p<0.001). In addition, ddPCR was also found to be suitable to detect and quantify KIT D816V in DNA isolated from formalin-fixed paraffin-embedded (FFPE) BM sections. In peripheral blood and BM aspirates, ddPCR for KIT D816V showed a high concordance to qPCR (r=0.99). No systematic deviation for the conversion from qPCR to ddPCR was found (slope of 1.00 of log-transformed data; 95% confidence interval 0.98 to 1.02). In a next step, we confirmed the clinical value of KIT D816V allele burden measurements by ddPCR. In these studies, patients with advanced SM showed a significantly higher KIT D816V allele burden (median 2.43%) compared to patients with indolent SM (median 0.14%; p<0.001). Moreover, ddPCR confirmed the prognostic significance of a high KIT D816V allele burden regarding survival of KIT D816V+ SM patients (n=115) using a cutoff level of 2% mutant alleles. In the group with <2% mutant allele burden the median survival was not reached, whereas in patients with a KIT D816V allele burden of ≥2% the median survival was 7.7 years (p<0.001).Conclusion: We performed a comprehensive validation of ddPCR for KIT D816V in peripheral blood and BM in order to establish a new reliable, quantitative qPCR technique for patients with mastocytosis. ddPCR is highly sensitive and specific for detection and quantification of KIT D816V, gives highly reproducible results even at a very low variant allele fraction level, and confirms the clinical value of KIT D816V allele burden measurement in blood and BM aspirates. In addition, ddPCR for quantification of KIT D816V can be performed in FFPE BM section material. Finally, ddPCR-based quantification of KIT D816V is not limited by the necessity to use generally accepted calibration material for assay standardization. In this regard, we propose to assess the ddPCR for KIT D816V in multicentric studies to increase comparability of allele burden data from different study groups. These studies will be a next crucial step towards the implementation of KIT D816V allele burden measurement as a global standard in clinical practice. [Display omitted] DisclosuresSperr:Teva: Honoraria; Novartis: Other: Register; Meda: Research Funding; Phadia: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Valent:Blueprint: Research Funding; BMS: Honoraria; Incyte: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Hoermann:Gilead: Honoraria, Research Funding; Amgen: Honoraria; Ariad: Honoraria; Novartis: Honoraria.

Distinct Diagnostic and Clinical Features of Bone Marrow Mastocytosis: A Study of the Registry of the European Competence Network on Mastocytosis (ECNM)

Abstract Background. Bone marrow mastocytosis (BMM) is characterized by accumulation of neoplastic mast cells (MC) in the bone marrow (BM), absence of skin lesions and lack of overt infiltration of other extra-cutaneous organs by neoplastic MC. Although clinically relevant due to its association with life-threatening anaphylaxis, BMM is considered a provisional sub-entity of indolent systemic mastocytosis (ISM) in the 2016 WHO classification. Aims and methods. To define clinical and biological features of BMM we evaluated 1,343 adult patients (pts) with non-advanced systemic mastocytosis (SM) enrolled in the European Competence Network on Mastocytosis (ECNM) registry. The following characteristics were compared between pts with BMM (n=260), ISM (n=1,027) and smouldering SM (SSM, n=56): age, gender, diagnostic criteria, biochemical parameters at diagnosis [blood counts, albumin, alkaline phosphatase (AP), lactate dehydrogenase, tryptase], presence of mediator-related symptoms (diarrhea, flushing, headache, pruritus, hypotension, blistering, cramping, bone pain) and their severity (classified according to Valent P. et al. Eur J Clin Invest. 2007;37:435-53), constitutional symptoms, osteoporosis/osteopenia and allergy. The Student- t and the χ2 tests were used to assess the statistical significance of differences for continuous and categorical variables, respectively. Event-free survival (EFS) was calculated from diagnosis to progression into more severe SM (i.e. SSM, aggressive SM, SM associated with another hematological neoplasm or mast cell leukemia) or death. Results. Median age at diagnosis was 53 years (yrs) (range 19-83) for BMM vs 46 yrs (range 19-82) for ISM vs 52 yrs (range 25-79) for SSM (p 20 ng/mL at diagnosis, while 77% of ISM and 100% of SSM pts fulfilled this criterion. If performed, atypical MC morphology in BM smears, aberrant MC immunophenotype (i.e. CD25 and/or CD2 expression) and KIT D816V mutation were demonstrable in the majority of pts (CD25 and/or CD2-positive BM MC: n=918, 95%; KIT D816V positive: n=910, 86%), without differences between the three groups. As a consequence, in 31% of BMM and 11% of ISM cases the diagnosis was made on the basis of minor criteria only. BMM pts had significantly higher hemoglobin levels compared to pts with ISM/SSM (median 14.5 vs 13.9 g/dL, respectively, p BMM pts had less frequent and less severe (grade 0-2) mediator-related symptoms as compared to ISM/SSM, except for hypotension, mainly due to the association of BMM with severe allergic reactions. Indeed, 76% of the BMM pts had a history of anaphylaxis, a significant difference compared to ISM or SSM pts (34% and 31%, respectively; p Long-term prognosis was similar for all groups, even though SSM tended to a less favourable course: EFS at 10 years was 88.9% (95%CI: 73.6-95.6) for BMM, 88.1% (95%CI: 83.4-91.6) for ISM and 78.8% (95%CI:55.7-90.8) for SSM (p=0.2). Conclusions. BMM is a distinct subgroup of non-advanced SM characterized by male predominance, low burden of neoplastic MC, low level of serum tryptase and AP, limited frequency and severity of mediator-related symptoms, except hypotension, and a striking association with anaphylaxis, mainly triggered by hymenoptera. These data support the proposal of considering BMM as a distinct variant of mastocytosis. Disclosures Bonifacio: Pfizer: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Hadzijusufovic: Novartis: Honoraria. Gotlib: Promedior: Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Promedior: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding. Sperr: Meda: Research Funding; Phadia: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Other: Register; Teva: Honoraria. Valent: Teva: Honoraria; BMS: Honoraria; Deciphera: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Blueprint: Research Funding; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Incyte: Honoraria. Zanotti: Deciphera: Consultancy.

An Intriguing Coexistence of Cutaneous and Systemic Mastocytosis with Atypical Chronic Myeloid Leukemia

Systemic mastocytosis (SM) characterized by focal or diffuse infiltrates of neoplastic mast cells can range from indolent cutaneous disease to aggressive systemic disease. Skin involvement is usual in indolent systemic mastocytosis, less frequent in aggressive SM, and rare in mast cell leukemia. Here, we present a rare coexistence of cutaneous mastocytosis and SM with an associated hematologic neoplasm SM-associated hematologic neoplasm [AHN] (atypical chronic myeloid leukemia [CML]-BCR/ABL negative), the first case being reported in the literature so far to the best of our knowledge. An elderly male with leukocytosis, myeloid hyperplasia, and mast cell infiltrates in the marrow was diagnosed with atypical CML and cutaneous mastocytosis. Approximately 40% of cases of SM are associated with a hematologic neoplasm (SM-AHN) encompassing all defined myeloid neoplasms, such as myelodysplastic syndrome, myeloproliferative neoplasm, acute myeloid leukemia, CML, chronic myelomonocytic leukemia, or plasma cell myeloma. The prognosis of SM-AHN is dependent on the associated hematologic neoplasm (AHN).

Systemic mastocytosis in adults: 2021 Update on diagnosis, risk stratification and management.

Systemic mastocytosis (SM) results from a clonal proliferation of abnormal mast cells (MC) in extra-cutaneous organs. The major criterion is presence of multifocal clusters of spindled MC in the bone marrow. Minor diagnostic criteria include elevated serum tryptase level, abnormal MC CD25 expression, and presence of KITD816V mutation. Establishing SM subtype as per the World Health Organization classification system is an important first step. Broadly, patients either have indolent/smoldering SM (ISM/SSM) or advanced SM, the latter includes aggressive SM (ASM), SM with associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL). Identification of poor-risk mutations (ie, ASXL1, RUNX1, SRSF2, NRAS) further refines the risk stratification. Recently, clinical and hybrid clinical-molecular risk models have been developed to more accurately assign prognosis in SM patients. Treatment goals for ISM patients are primarily directed towards anaphylaxis prevention/symptom control/osteoporosis treatment. Patients with advanced SM frequently need MC cytoreductive therapy to ameliorate disease-related organ dysfunction. High response rates have been seen with small-molecule inhibitors that target mutant-KIT, including midostaurin (Food and Drug Administration approved) or avapritinib (investigational). Other options for MC cytoreduction include cladribine or interferon-α, although head-to-head comparisons are lacking. Treatment of SM-AHN primarily targets the AHN component, particularly if an aggressive disease such as acute myeloid leukemia is present. Allogeneic stem cell transplant can be considered in such patients, or in those with relapsed/refractory advanced SM. Imatinib has a limited therapeutic role in SM; effective cytoreduction is limited to those with imatinib-sensitive KIT mutations.

Clinical Impact of Skin Lesions in Mastocytosis: A Multicenter Study of the European Competence Network on Mastocytosis

Mastocytosis is a rare neoplasm characterized by the expansion and accumulation of mast cells in various organ systems. Systemic mastocytosis (SM) may or may not present with cutaneous lesions. To examine the frequency and clinical impact of cutaneous involvement, data on 1,510 patients with mastocytosis collected in the registry of the European Competence Network on Mastocytosis were analyzed. Cutaneous involvement was found in 1,195 of 1,510 patients (79.1%). Of these, 286 had cutaneous mastocytosis, and 721 had SM with skin involvement. Adult patients with skin involvement who did not have a bone marrow examination (n= 188) were defined as having mastocytosis in the skin. In 315 patients, SM without skin involvement was found. The percentage of cases with cutaneous involvement was higher in indolent SM (100%) and smoldering SM (87.9%) compared to aggressive SM (46.8%) or mast cell leukemia (38.5%). After a median follow-up of 5.6 years, no patient with cutaneous mastocytosis had died, but 2.6% of the patients with mastocytosis in the skin, 5.7% of the patients with SM with skin involvement, and 28.95% of the patients with SM without skin involvement had died. Overall survival was longer in patients with skin involvement (cutaneous mastocytosis and/or mastocytosis in the skin and/or SM with skin involvement) than in patients with SM without skin involvement (P < 0.0001). These data argue for a thorough examination of both the skin and bone marrow in adult patients with mastocytosis.