Case 3 -

Systemic Mast Cell Disease with an Associated Clonal Hematologic Non Mast Cell Lineage Disease (SM-AHNMD) Vishnu V. B Reddy University of Alabama at Birmingham Birmingham, AL

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Clinical History: This 73 year old female presented to family practice clinic in June of 2005 complaining increasing weakness and weight loss. A routine CBC showed anemia (Hgb 10.4 gm/dl), mild thrombocytopenia and mild leukocytosis (WBC 10.7 k/ul) with monocytosis (absolute monocyte count 2140 / cmm). Later, she was evaluated by hematologist and underwent bone marrow biopsy. Physical examination was negative for skin rash and hepatosplenomegaly. Later, serum tryptase levels are found to be elevated. Case 3 - Figure 1 - Bone marrow aspirate (Wight-Giemsa) showing marrow elements and clusters of mast cells. Case 3 - Figure 2 - Bone marrow aspirate (Wight-Giemsa) showing marrow elements and clusters of mast cells (100x oil). Case 3 - Figure 3 - Bone marrow aspirate (Wight-Giemsa) containing several clusters of abnormal myeloid/monocytic precursors and some with dysgranulation and marked left-shift (blasts ~ 8%). Case 3 - Figure 4 - Bone marrow aspirate (Wight-Giemsa) showing focus of immature myeloid cells / left-shift. Case 3 - Figure 5 - Bone marrow biopsy was hypercellular (85-90%) cellular and contained mixtures of early and large hematopoietic elements in varying stages of maturation. Also, are found several collections of mast cells with central round pale nuclei and pale granular cytoplasm. Case 3 - Figure 6 - Bone marrow biopsy (20x Obj) shows mild para-trabecular fibrosis and mast cells / hematopoietic elements. Case 3 - Figure 7 - Immunohistochemical stain CD117 (c-kit) was positive in most of mast cells (10x Obj). Case 3 - Figure 8 - Immunohistochemical stain CD117 (k-kit) was positive in most of mast cells. High power examination (40x Obj) confirms that most are mast cells with strong membrane / cytoplasmic staining and pale nuclei. Rare myeloid precursors are staining. Case 3 - Figure 9 - Immunochemical stain tryptase was positive in few of the mast cells. Morphology / Histology : Peripheral blood smear showed moderate monocytosis and some of them were immature appearing. Few hypogranular / hyposegmented neutrophils, rare early myeloid cells (promyelocytes and myelocytes) were found. The platelets are decreased. Bone marrow aspirate was cellular and all three cells lines are identified with some myeloid left-shift, few blasts (~5%) and minimal dyserythropoiesis. Dysmorphic granules (eosinophilic / basophilic) are noted in some of the myeloid precursors. Mast cells are markedly increased (>20%) with clustering and as well as individually scattered among hematopoietic elements. Bone marrow biopsy was hypercellular (cellularity ~ 70-90%) with multifocal infiltrates of mast cells mixed with other hematopoietic elements. In some areas mast cells predominate, and focally clusters of early myeloid precursors were also noted. Few normal appearing megakaryocytes are seen. Bony cortices are irregular and mild para-trabecular fibrosis was noted on reticulin stain. Immunophenotypic Studies: IPOX stains - Mast cells are positive for CD117, tryptase, CD21, CD14. MPO and NSE were positive in myelomonocytic cells. Flow cytometry analysis - Mast cells are positive for dim CD117, dim CD33 and CD13. Negative for CD25, CD34 and CD15 Differential Diagnosis: Myelodysplastic syndrome / CMML Reactive mastocytosis Systemic mast cell disease / associated MDS (RAEB-1) Diagnosis: Systemic Mast Cell Disease with an Associated Clonal Hematologic Non Mast Cell Lineage Disease (SM-AHNMD). Discussion Background: Mast cells (MCs) originate / derived from hematopoietic progenitor cells (Valent et al). Several factors and cytokines contribute to mast cell development (mastopoiesis). Stem cell factor (SCF) induces development of mast cells from uncommitted progenitors and promote terminal differentiation / maturation. SCF acts through KIT, transmembrane tyrosine kinase-type receptor encoded by the c-kit proto-oncogene. Morphologically, 4 stages of maturation have been described: the non-granulated blast cell (tryptase+), the metachromatic blast, the pro-mastocyte (atypical MC type II, MC with bilobed or multilobed nuclei), and the mature MC. Mast cells are widely scattered in the body, and are more often found near vascularized tissues, and release pro-inflammatory and vasoactive mediators. These mediators are released from MCs in response to IgE receptor / specific allergen interaction or other stimuli. Some of these mediator release can be measured e.g. serum MC tryptase levels which aid in the diagnosis of mastocytosis and represent an indirect measurement of total MCs in the body. Pathogenesis: SCF and KIT are critical molecules in the regulation of MC development (Akin et al), proliferation, and survival. Mutations in the c-kit gene are associated with enhanced growth / proliferation of MCs. These transforming mutations, particularly c-kit D816V (Asp-816-Val), are frequently identified in patients with systemic mastocytosis (SM) when the appropriate tissues are examined. Morphology of the mast cells vary considerably, immature MCs (promastocytes and metachromatic blasts) are frequently detected in bone marrow smears of patients with aggressive SM (ASM) or mast cell leukemia (MCL). In indolent SM, the bone marrow smear usually contains more mature appearing MCs. However, in most cases of SM MCs show morphological abnormalities / atypical MCs, including (1) prominent cytoplasmic extensions, (2) oval nuclei, and (3) a hypogranular / degranulated cytoplasm. In some patients with SM, the majority of MCs are round and do not show significant morphologic abnormalities. MCs are known to display a characteristic antigenic phenotype in normal tissues, as well as in SM. In contrast to normal MCs, MCs in SM usually express CD25 and CD2. Immature MCs might express additional antigens. The most immature MC progenitors (both normal and neoplastic) express CD34 and CD13 together with CD117. Classification (WHO, 2001) Systemic mast cell disease is recognized by the World Health Organization (WHO) as a separate group of myeloproliferative disorders (Valent et al) among hematopoietic neoplasms. This is based on the observation of mast cells (MC) developing from hematopoietic progenitor cells and associated hematologic (clonal) none MC lineage disease (AHNMD). The current WHO classification is based on clinical and pathologic findings, and useful in the identification of MC subtypes and in the discriminating between various modes of clinical presentation; e.g. SM and CM, SM and myelo-mastocytic disorders, and SM and reactive increase in MCs. Mastocytosis - Variants / Subtypes   Cutaneous mastocytosis CM • Maculopapular CM MPCM • Diffuse CM DCM • Mastocytoma of skin   Indolent systemic mastocytosis I ISM • Smoldering SM SSM • Isolated bone marrow mastocytosis BMM Systemic mastocytosis with an associated clonal hematologic non MC lineage disease SM-AHNMD Aggressive systemic mastocytosis ASM • With eosinophilia   Mast cell leukemia MCL • Aleukemic MCL   Mast cell sarcoma MCS Extracutaneous mastocytoma     WHO Criteria Major Multifocal dense infiltrates of MCs in bone marrow or other extracutaneous organs (>15 MCs aggregating) Minor MCs in bone marrow or other extracutaneous organs show an abnormal (spindling) morphology (>25%). Codon 816 c-kit mutation D816V in extracutaneous organs MCs in the bone marrow express CD2, CD25, or both Serum tryptase >20 ng/mL (does not count in patients who have an associated hematopoietic clonal non MC lineage disease (= AHNMD). Note: For diagnosis at least one major and one minor criterion or 3 minor criteria are fulfilled, the diagnosis of SM can be established. Other activating mutations at codon 816 of c-kit also count as a minor criterion. Diagnostic criteria (morphology / immunophenotypic / molecular) The most significant criteria is the presence of mast cell clusters / aggregates (>15 MCs) and positive staining with tryptase. Minor criteria include atypical morphology (spindle shaped cells or atypical morphology), aberrant immunophenotype (CD25 / CD2 aberrant expression), raised serum tryptase levels and demonstration of mutations of codon 816 of c-kit. Rarely reactive mastocytosis may present with significant mast cell clustering, and in these situations molecular studies are crucial in establishing the diagnosis. In most cases, the diagnosis of SM is made on the bone marrow examination alone. However, other organs, such as the liver or the gastrointestinal tract, might also be involved. Some pediatric CM cases may spontaneously regress. Systemic mast cells disease (SM) is considered a persistent clonal disease. In majority of the patients with SM, the c-kit mutation D816V is demonstrated. In addition, in smoldering variant of SM c-kit mutations are found both in MCs and other hematopoietic cells as well. Elevated serum tryptase levels may be found in other conditions without evidence of mastocytosis or SM. Higher levels of tryptase are reported in AML, MDS, MPD and hypereosinophilic syndromes. Bone marrow mast cells are often increased in many lymphoproliferative conditions, including chronic lymphocytic leukemia and myeloproliferative conditions (MPD). IPOX stains CD2, CD25, CD117 and CD15 on the fixed tissues are most helpful in separating reactive increase vs. neoplastic mast cell entities. Diagnostic Workup in Mastocytosis: Tissue biopsies (Bone marrow / Skin and other tissue biopsies) - Routine H&E, Wright Giemsa stains and toluidine blue (pH 1.5-25). Immunohistochemistry- CD117, CD2, CD25, CD35, CD14, CD15 and CD33 Flow cytometry (Bm and other tissues) – 0.5 – 1.5 cc BM aspirate in EDTA or heparin. Quick processing (same day), stain and lyse method is preferred over Ficoll separation. Double step data acquisition i.e. all nucleated cells followed by live gate containing only CD117+ cells. Pathologic MCs express CD2, CD25, hi CD11c / CD35 and low levels of CD117 (Escribano et al). Serum - Tryptase levels (> 20 ng/mL). Note: Pediatric ranges are higher. Molecular studies – c-kit (D816V). bcr/abl and FIPL1-PDGFRA may be needed to exclude MPD/ hypereosinophilic syndromes in some cases. Workup of the patients depends on the age, in pediatric patients a skin biopsy, tryptase levels (higher normal ranges due to MC granule volume/whole body volume ratio) , c-kit D816V are more than sufficient for the diagnosis. In adults, bone marrow biopsy, serum tryptase levels and c-kit mutation analysis are often necessary. Clinical Outcome : Clinical course in SM is variable and range from indolent to aggressive. Aggressive systemic mastocytosis (ASM) will present with organomegaly / multi-organ failures, cytopenias and bone fractures. Organomegaly may also be seen in indolent SM, however, organ failure is more indicative of aggressive course. Mast cell leukemia (MCL) is a rare subentity of SM characterized by a leukemic infiltration of various organs by immature / blastic MCs. Typically, the bone marrows are packed with immature MCs (>20%) and circulating mast cells (>10%). The prognosis is poor in patients with MCL. The differential diagnosis includes other myeloid leukemias including myelomonocytic leukemias. Survival in patients with SM-AHNMD is also short, and depends on the associated MPD/MDS type. Urticaria and skin lesions may not be present in aggressive SM or SM-AHNMD (mastocytosis may be missed in these conditions). Mediator-related symptoms may be found in any subtype of SM. Cutaneous mast cell diseases are most common in children (<6 months) and it may be present at birth. Adult mast cell diseases are less common. In one study (Horny et al), n=64 (0.3%) cases if SM found in a review of 20, 000 bone marrow biopsies. Indolent SM including cutaneous forms (n=35), SM-AHNMD (n=20), ASM (n=7), MCL (n=2). References Akin, C., Clonality and molecular pathogenesis of mastocytosis. Acta Haematol, 2005. 114(1): p. 61-9. Akin, C. and D.D. Metcalfe, Systemic mastocytosis. Annu Rev Med, 2004. 55: p. 419-32. Bain, B.J., Relationship between idiopathic hypereosinophilic syndrome, eosinophilic leukemia, and systemic mastocytosis. Am J Hematol, 2004. 77(1): p. 82-5. Ben-Amitai, D., A. Metzker, and H.A. Cohen, Pediatric cutaneous mastocytosis: a review of 180 patients. Isr Med Assoc J, 2005. 7(5): p. 320-2. Bernd, H.W., et al., Acute myeloid leukaemia with t(8;21) associated with "occult" mastocytosis. Report of an unusual case and review of the literature. 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