KIT D816V Positive Acute Mast Cell Leukemia Associated with Normal Karyotype Acute Myeloid Leukemia

Introduction Mast cell (MC) leukemia (MCL) is extremely rare. We present a case of MCL diagnosed concomitantly with acute myeloblastic leukemia (AML). Case Report A 41-year-old woman presented with asthenia, anorexia, fever, epigastralgia, and diarrhea. She had a maculopapular skin rash, hepatosplenomegaly, retroperitoneal adenopathies, pancytopenia, 6% blast cells (BC) and 20% MC in the peripheral blood, elevated lactate dehydrogenase, cholestasis, hypoalbuminemia, hypogammaglobulinemia, and increased serum tryptase (184 μg/L). The bone marrow (BM) smears showed 24% myeloblasts, 17% promyelocytes, and 16% abnormal toluidine blue positive MC, and flow cytometry revealed 12% myeloid BC, 34% aberrant promyelocytes, a maturation blockage at the myeloblast/promyelocyte level, and 16% abnormal CD2−CD25+ MC. The BM karyotype was normal, and the KIT D816V mutation was positive in BM cells. The diagnosis of MCL associated with AML was assumed. The patient received corticosteroids, disodium cromoglycate, cladribine, idarubicin and cytosine arabinoside, high-dose cytosine arabinoside, and hematopoietic stem cell transplantation (HSCT). The outcome was favorable, with complete hematological remission two years after diagnosis and one year after HSCT. Conclusions This case emphasizes the need of an exhaustive laboratory evaluation for the concomitant diagnosis of MCL and AML, and the therapeutic options.


Introduction
Mastocytoses are rare neoplasms de ned by an abnormal expansion/accumulation of clonal mast cells (MC) in one or more organs or tissues [1,2]. e 2016 revision to the "World Health Organization (WHO) Classi cation of Tumors of the Hematopoietic and Lymphoid Tissues" divides the disease into cutaneous mastocytosis (CM), systemic mastocytosis (SM), and localized mast cell tumors [1,2]. Cutaneous mastocytosis includes maculopapular CM, also known as urticaria pigmentosa, di use CM, and mastocytoma of skin. Systemic mastocytosis is further divided into indolent SM (ISM), smoldering SM (SSM), and advanced SM variants; the latter includes aggressive SM (ASM), mast cell leukemia (MCL), and SM with associated hematological neoplasm (SM-AHN), previously known as SM with associated clonal hematological non-MC lineage disease (SM-AHNMD), and mast cell leukemia (MCL) [1,2]. Associated hematological neoplasms may consist of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and also acute myeloid leukemia (AML).
In the last years, serum tryptase levels [3], immunophenotypic characterization of BM mast cells by ow cytometry (FCM) [4,5] and KIT mutation analysis [6], have proved to be useful for establishing the diagnosis, subclassifying and evaluating the prognosis of SM, adding important information to conventional cytomorphology and histopathology. Mast cells, and often other BM cells, from most patients with SM usually harbor the activating KIT D816V mutation. In addition, they regularly have abnormal morphology, from atypical MC type I, usually observed in ISM, to more immature atypical MC type II and metachromatic blasts, more frequently found in ASM and MCL, and they exhibit abnormal phenotypic features, from which the most frequent and more extensively studied are positivity for CD25 and/or CD2.
Mast cell leukemia accounts for <1% of all mastocytosis, it may appear de novo or secondary to a previously diagnosed MC disorder (usually ASM or SM-AHN), and it may associate with other hematological neoplasms (MCL-AHN) [7,8]. Diagnosis is based on the presence of ≥20% atypical MC in the bone marrow (BM) and/or ≥10% in the peripheral blood (PB); an aleukemic variant with less than 10% of MC in the PB also exists. e European/American Consensus Group on Mastocytosis (EU/US-CGM) proposed a subclassi cation that distinguishes acute from chronic MCL based on the presence or absence of organ damage, respectively [9]. e neoplastic MC usually have an immature morphology and an abnormal immature and/or activated phenotype, although they often fail to have the abnormal CD2+CD25+ pattern encountered in most forms of SM; in addition, the KIT D816V mutation is detected in less than 50% of cases, and most patients have a normal karyotype. Symptoms of MC activation and involvement of the liver, spleen, peritoneum, digestive tract, and bones are relatively frequent, and skin lesions occur in only 1/3 of the cases. Treatment usually fails, and the median survival time is short. Due in part to the rarity, no standard therapy exists, and the role of hematopoietic stem cell transplantation (HSCT) needs further investigation.
We present a case of MCL diagnosed concomitantly to AML, given emphasis on the role of the laboratory exams, specially immunophenotyping and genetic testing, in establishing the di erential diagnosis with MML. In addition, we compare the clinical and laboratory features of our patient with those described in previously published series of patients with MCL, ISM, ASM, and SM-ANH. Finally, we describe the success of the therapeutic strategy used in this patient, which included cladribine, anthracycline in combination with cytosine arabinoside, and HSCT.

Case Report
A 41-year-old Caucasian woman was admitted at the hospital with one-month history of asthenia, anorexia, fever, abdominal pain, early postprandial surfeit, and diarrhea. She had past history of an intermittent migratory pruritic maculopapular rash and mild episodes of ushing that had never been investigated, hypothyroidism, an anxiety disorder, and emotional instability, and she had smoked 20 cigarettes a day since the age of 13. ere was no history of allergies or other pathologies.
When rst observed at the hospital, she had a fever, a dark spot on the tongue, a slightly pruritic brownish erythematous maculopapular skin rash predominantly in the upper limbs, hepatomegaly (the left lobe of liver was enlarged and extended to epigastric region and the right lobe was four ngers below the right costal margin in the midclavicular line), and splenomegaly (5 ngers below the left costal margin).
ere was no peripheral lymphadenopathy.
Bone marrow trephine biopsy revealed a hypercellular marrow with increased proportions of immature MPO+ myeloid cells and morphologically atypical CD117+ fusiform MC forming perivascular dense aggregates, and grade 2 brosis ( Figure 5). Skin biopsy was not performed. Cytogenetic analyses of at least 20 Giemsa-banded BM cell metaphases obtained from unstimulated 24 hour cultures disclosed a 46,XX karyotype, without numerical or structural abnormalities. Genetic studies using probes for relevant targets, Abdominopelvic computerized tomography scan a rmed hepatomegaly (18.5 cm) and mild splenomegaly (13 cm) with small hypodense nodules (maximum diameter 10 mm) and revealed retroperitoneal adenopathies forming a conglomerate extending from the lesser gastric curvature and involving the large vessels; the largest adenopathy was in the hepaticduodenal ligament and had 2.4 cm of major diameter. ere was also a lamina of peritoneal liquid in pelvic cavitation.
Digestive endoscopy revealed slight reduced distensibility of the gastric body, which had a congestive mucosa Circulating mast cells (MC) had variable morphological features, from metachromatic blasts of medium to large cell size, round to oval shape, ne/immature nuclear chromatin, high nuclear : cytoplasmic ratio, and cytoplasmic metachromatic granules, to atypical MC type 2 with variable, sometimes spindle, cell shape, bilobated nucleus with ne to moderately condensed chromatin, variable nuclear : cytoplasmic ratio, and hypogranulated cytoplasm. Please note that in spite of these heterogeneous morphological features, all the MC present in the peripheral blood had an aberrant immature phenotype, corresponding to abnormal MC precursors ( Figure 4). with foci of erythema, and the duodenum had a congestive and micronodular mucosa. Biopsies were not performed due to severe thrombocytopenia. Skeleton radiography did not reveal osteolytic lesions.
orax radiography had no evidence of mediastinal enlargement, lung consolidations, or pleural e usions.
According to the WHO criteria [1,2], and to the consensus recommendations of the EU/US-CGM and the ECNM [9], the patient was diagnosed with KIT D816V+ MCL associated with AML with normal karyotype. She was immediately started with oral corticosteroids (prednisolone, 60 mg/day for one week, tapered to 20 mg/day over 1 month, and then maintaining 20 mg/day) and disodium cromoglycate (200 mg capsules, 4 times daily), and H1 (cetirizine, 10 mg/day, orally) and H2 (ranitidine, 150 mg twice a day, orally) antihistamines, which ameliorate the symptomatology. en, she received two cycles of cladribine (0.14 mg/kg/day, administered over a 2-hour infusion for 5 days) with one month of interval, and the serum tryptase levels transiently decreased to 41 μg/L ( Figure 6).
As complication of treatment she had bartholinite, treated with piperacillin plus tazobactam, and CD30 CITO:APC-A CD117+CD34+ myeloid precursor cells CD117+CD34-mast cell precursors CD117++CD34-mast cells Other bone marrow cells Case Reports in Hematology metronidazole; oral mucositis grade II controlled with tramadol; febrile neutropenia with bacteremia by Escherichia Coli treated with piperacillin plus tazobactam; pneumonia without respiratory insu ciency, which was responsive to imipenem plus vancomycin, and pseudomembranous colitis by Clostridium di cile, treated with metronidazole. Two months after the second course of consolidation chemotherapy, the patient received isogroup HLA-identical related allogeneic HSCT from her sister (10/10 match) (5.09 × 10 6 /kg nonmanipulated peripheral blood CD34+ cells, totalizing 322 × 10 6 CD34+ cells). e reduced-intensity conditioning regimen included udarabine (30 mg/m2/day for 5 days) and busulfan (4 mg/kg/day for 2 days). As acute complication, she had febrile neutropenia treated with meropenem. On day 30 after HSCT, she had recovery of the hematological counts, and no myeloblasts or MC were seen in the PB. Unfortunately, the BM aspirate was hypocellular and results from BM studies were unevaluable. Abdominal echography revealed stable hepatomegaly (17.5 cm), without splenomegaly, or adenomegalies. ree months after HSCT a complete chimerism was documented in PB and BM neutrophils, monocytes, and lymphocytes. She developed a chronic graft versus host disease with cutaneous manifestations, controlled with cyclosporine A and mycophenolate mofetil. By the time of this report (24 months after the diagnosis, 15 months after HSCT), she maintains normal serum tryptase levels, complete hematological remission, and complete chimerism in PB ( Figure 6).

Discussion
According to the WHO classi cation, this complex case ful ls the criteria for the diagnosis of SM-AHN, more precisely, KIT D816V+ MCL associated with normal karyotype AML [1,2]. Criteria for MCL include not only the conditions for SM, such as BM in ltration by morphologically and phenotypically abnormal MC forming dense aggregates (>15 MC), increased serum tryptase levels (>20 ng/ml), and the KIT (D816V) mutation in BM cells, but also 16% MC in the BM and 20% MC in the PB; criteria for AML were more than 20% MPO+ myeloblasts in the BM by cytomorphology. Curiously, FCM studies showed that the cells of the MC lineage were phenotypically heterogeneous, with both aberrant MCP and more mature MC being identi ed in the BM, and only the former being present in the PB. Interestingly, FCM studies also revealed that the granulocytic cells had an almost complete blockage at the promyelocyte stage, despite the fact that t(15;17) was negative. us, the MC and the granulocytic cell lineages were both compromised by the leukemic process. In accordance to this multilineage involvement, the KIT Other bone marrow cells D816V mutation was found in all BM cell populations tested, except in T cells. ese ndings are in line with previous studies, indicating that the occurrence of KIT mutations in an early progenitor cell results multilineage involvement, MC maturation blockade, immature MC phenotype, and aggressive disease [17][18][19]. e possibility of a secondary MCL arising in the context of a previously undiagnosed ISM is plausible, as the patient had a chronic maculopapular rash that had never been investigated. Unfortunately, skin biopsies were not performed, and thus, cutaneous in ltration by MC was not formally documented. It should however be mentioned, that secondary MCL usually occurs in patients with SM-AHN or ASM, and direct evolution from ISM to MCL is exceptionally rare [20][21][22].   Figure 3, same color code). Please also note that the relatively mature mast cells observed in BM (red dots, Figure 3) were not present in the peripheral blood. a lower time from symptoms to diagnosis and a more severe neutropenia and thrombocytopenia, the clinical features observed in our patient with MCL + AML did not di er substantially from those usually found in patients with other advanced MC neoplasms, such as ASM and SM-AHN, as described in the largest series of patients with SM (342 cases), published by Lim et al. in 2009 (Table 1) [20]. In this series, ISM was the predominant SM subtype (46%), followed by SM-AHN (40%, subtype not speci ed) and ASM (12%), and only 4 cases were MCL (1%).
e clinical and laboratory features of our patient were those expected to occur in cases of MCL, as previously described in the literature (Table 2). In 2013, Georgin-Lavialle et al. revised all the MCL cases that had been published in scienti c journals indexed in the MedLine [8]. In total, they provided data from 51 cases, including a series of 10 cases of MCL, published by Valentini et al. in 2008 [21], several cases reported individually from 1950 to 2012, and 4 personal unpublished cases; in 41 cases, they had enough data to classify them as de novo MCL (n � 30) or as secondary MCL (n � 11), and to compare the clinical and biological features of these entities (   Rare cases of MCL have been diagnosed previously, subsequently or concomitantly to AML, as occurring in this patient [23][24][25]. Mast cell leukemia-AML cases exhibit a substantial increase (>20%) in myeloblasts in the BM, and they must be distinguished from MML where an AML may also be diagnosed, but criteria for SM are not met (Table 3).
Myelomastocytic leukemia is a very rare type of leukemia that is not yet incorporated in the WHO classi cation of the tumors of lymphoid and hematopoietic tissues [9,16]. It is characterized by an expansion (>10%) of atypical MC together with BC with metachromatic granules (identi ed as MC precursors by immunophenotyping) in the BM and/or PB, concomitantly with criteria for an advanced myeloid neoplasm, which may have features of AML, MDS with excess of blast cells, or accelerated/blast phase of a MPN or a MDS/MPN. By de nition, MML carries no speci c (recurrent) molecular and immunophenotypic markers for SM. Speci cally, activating point mutations at codon 816 of KIT are not found, and the aberrant CD2+/CD25+ MC immunophenotype, which is typically found in the majority of SM cases, is rare in MML. In addition, in patients with MML, the karyotype usually re ects the underlying disease (e.g., MDS, MPN, MDS/MPN, and AML) and chromosomal aberrations are frequently complex; in contrast, no recurrent chromosome abnormalities are known for patients with MCL [9,16]. In addition, the neoplastic MC in MCL usually express CD117, low tryptase, and low FcεRI, and are often CD25+, whereas in MML, MC also express CD117 and tryptase, but they habitually stain negative for CD25. CD2 is usually negative in both cases, being frequently detected in MC from patients with indolent or smoldering SM (Table 4). Some other markers, including HLA-DR, CD30, and CD123, may also be positive in MCL cells, as observed in our patient. us, the clinical and laboratory ndings observed in this case would favor the diagnosis of MCL associated to AML, instead of MML.
Management of patients with AML relies on genetic tests that allows for the diagnosis, informs about prognosis, and predicts response to therapy, and the value of genetics is reinforced in the WHO classi cation scheme for AML. For instance, cytogenetic aberrations have long been recognized as important prognostic variables in AML patients [26]. However, patients with AML and normal karyotype have had a very heterogeneous outcome, and previous studies have indicated that many other molecular aberrations do in uence the response to treatment as well as in the risk of relapse [27]. For example, AML with normal cytogenetics may carry poor prognostic genetic lesions, such as FLT3 mutations, overexpression of BAALC (brain and acute leukemia cytoplasmic), ERG (ETS/E26 transformation-speci c-related gene), or MN1 (meningioma 1) genes, or they may have aberrations that predict better prognosis as are cases with isolated NPM-1 or CEBPA (CCAAT/enhancer binding protein alpha) mutations [27]. Among them, FLT3 and NPM-1 mutations were found to be absent in this patient. Also negative were studies for other relevant targets including t(15;17) PML-RARA, t(8;21) RUNX1-RUNX1T1, inv(16) CBFB-MYH11, and t(9,22) BCR-ABL. As mentioned before, SRSF2, ASXL1,    and RUNX1 mutations, described to be present in around 50% of MCL patients and found to a ect adversely response to treatment and to predict lower OS [22], were not examined in this case. Due fundamentally to the extreme rarity of MCL, no standard treatment exists, and therapies frequently consist on those commonly used in aggressive SM, such as cladribine (2-chloro-deoxy-adenosine, 2-CDA), interferon alpha 2a (IFN-α2a), and tyrosine kinase (TK) inhibitors (TKI). As MCL is presumably a clonal disorder of hematopoietic myeloid stem cells, chemotherapy with drugs proven successful in AML, such as anthracyclines in combination with AraC, have also been used to treat patients with MCL. If hematological remission is achieved, additional therapy with curative intent involving HSCT might be attempted, as in this patient [7,8]. e rationale behind the use of cladribine, which was the rst therapy tried in our patient, is based on the value of this drug in the treatment of aggressive SM, which has been con rmed in several studies [28,29]. However, 2-CDA had no or little activity in this case, as in most previously reported cases of MCL treated with this purine analogue [22,30], although in rare cases transient or prolonged partial response has been observed [21,31,32]. Reports on the use of IFN-α2a in MCL are scarce, and the results obtained were also not encouraging [21,31].
Tyrosine kinase inhibitors have been actively investigated for the treatment of patients with mastocytosis because KIT mutations often cause constitutive activation of TK activity of the KIT receptor [33]. Imatinib is e ective in patients with increased mast cells and eosinophils associated  with FIP1L1/PDGFRA (Factor Interacting with PAPOLA and CPSF/Platelet-derived growth factor receptor A) fusion gene (e.g., myeloid neoplasm with eosinophilia and rearrangement of PDGFRA) [34] or rare patients with SM associated with KIT mutations outside of exon 17 [30]. However, the results from imatinib and other TKI, such as masitinib and dasatinib, in MCL and other D816V+ SM have been disappointing [21,[35][36][37]. Midostaurin (PKC412), a multitarget TKI, is a promising agent for patients with advanced SM, as it inhibits the growth of neoplastic MC exhibiting various mutant forms of KIT, including KIT D816V [38]. In contrast to other KITtargeting drugs, midostaurin also impedes IgE-dependent release of histamine [39,40]. Midostaurin has been reported to be e cacious in patients with advanced SM, including ASM and MCL [22,41,42]. Data from a Phase 2 single-arm open-label trial (CPKC412D2201), which included 89 with mastocytosis-related organ damage (16 with aggressive SM, 57 with SM-AHN, and 16 MCL), revealed that treatment with midostaurin 100 mg twice daily resulted in an overall response rate of 60% with a median duration of response of 24 months and a median overall survival of 29 months [42]. Unfortunately, midostaurin was not yet approved for patients with advanced SM, including MCL, and the drug was not available for compassionate use at the time the MCL was diagnosed in our patient. Brentuximab vedotin has also been considered for the treatment of patients with advanced SM. e results obtained in a small series of 4 patients with SM have suggested that this anti-CD30 monoclonal antibody-drug conjugate can induce durable responses with a manageable toxicity pro le [43], and a clinical trial in patients with CD30 positive ASM and MCL is currently going on in the United States (NCT01807598). us, midostaurin and brentuximab vedotin may be considered alternative adjuvant therapies in case of disease relapse in our patient. Induction chemotherapy has been used in some patients with MCL. According to the review performed by Georgin-Lavialle et al., AML-type chemotherapy was used in 6 of 51 MCL patients reported till 2013; the median survival time was 7 months, and all patients died between 2 and 29 months, because of disease progression or multiorgan failure [8]. A few cases of patients with MCL who received HSCT have also been reported, but sustained remission was not achieved in any of them [21,[44][45][46]. Ustun et al. have reported on a retrospective series of 57 patients with SM, including SM-AHN (n � 38), ASM (n � 7) and MCL (n � 12), who received allogeneic HSCT either after myeloablative (n � 36) or nonmyeloablative reduced-intensity (n � 21) conditioning regimens [47]. Responses were observed in 70% of the patients, with complete remission in 28%. Twenty-one percent of patients had stable disease, and 9% had primary refractory disease. e best responses were obtained in SM-AHN (AML/MDS), and the worst were obtained in MCL patients. Overall survival at 3 years was 57% for all patients, 74% for patients with SM-AHN, 43% for those with ASM, and 17% for those with MCL, and the strongest risk factor for poor overall survival was MCL. Survival was also lower in patients receiving nonmyeloablative compared with myeloablative conditioning regimens and in patients having progression compared with patients having stable disease or response. Although allogeneic HSCT may be considered a potentially curative treatment for advanced SM, including MCL, its de nitive role needs to be determined by prospective clinical trials. Our patient maintains hematological remission of both diseases (MCL and AML) with complete chimerism, 15 months after HLA-identical HSCT.
In summary, we described an extremely rare case of an adult female with KIT D816V+ MCL associated with normal karyotype AML without FLT3 and NPM-1 mutations, who was refractive to cladribine and who achieved complete hematological remission after receiving induction chemotherapy with idarubicin and AraC, followed by allogeneic HLA-identical sibling HSCT preceded by a reducedintensity conditioning regimen.
Ethical Approval e patient gave informed consent for publication of this case report and the accompanying images. O -label prescription of cladribine was performed after obtaining authorization of the Ethical Committee and the Pharmaceutical Committee of the Hospital.

Conflicts of Interest
e authors declare that they have no con icts of interest.

Authors' Contributions
Marta Lopes collected data and contributed to manuscript writing. Patrícia Seabra, Vanessa Mesquita, Cláudia Casais, Renata Cabral, and Jorge Coutinho provided clinical support and were responsible for the therapeutic decisions. José-Palla Garcia and José Ramón Vizcaíno performed analysis of bone marrow trephine biopsy and immunohistochemical staining. Maria dos Anjos Teixeira contributed to ow cytometry data analysis and interpretation, and case discussion. Catarina Lau participated in ow cytometry data interpretation. Inês Freitas performed analysis of the bone marrow smears, cytomorphology, and cytochemical staining. João Rodrigues performed cytogenetic and molecular genetic studies. Maria Jara-Acevedo performed cell sorting and KIT mutation analysis. Luís Escribano was involved in data interpretation and case discussion. Alberto Orfao was involved in ow cytometry data analysis and interpretation, and in case discussion. Margarida Lima participated in ow cytometry data analysis, data interpretation and case discussion, and gave major contributions to manuscript writing. All authors read, discussed, and approved the nal version of the manuscript.