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Leukemia Research 31 (2007) 1749–1754

Case report

Distinct clonal origins of systemic mastocytosisand associated B-cell lymphoma

Young Kim a, Lawrence M. Weiss a, Yuan-Yuan Chen a, Vinod Pullarkat b,∗a Division of Pathology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA

b Division of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center,1500 East Duarte Road, Duarte, CA 91010, USA

Received 26 January 2007; received in revised form 4 April 2007; accepted 6 April 2007Available online 4 June 2007

bstract

Systemic mastocytosis (SM) may rarely be associated with lymphoproliferative disorders. In such cases, the relationship between theeoplastic mast cells and the malignant lymphocytes remains unclear. We describe a patient with indolent SM whose bone marrow showedvidence of low-grade B-cell lymphoma. By detecting the activating KIT mutation D816V in the microdissected bone marrow mast cells, butot in the neoplastic B-lymphocytes, we demonstrate the distinct clonal origins of the mastocytosis and lymphoma when these two entities

oexist. We also highlight the clinical and pathologic differences between SM associated with lymphoid as opposed to myeloid neoplasmsnd discuss their pathogenesis.

2007 Elsevier Ltd. All rights reserved.

eywords: Systemic mastocytosis; Hematologic malignancy; KIT mutation; Lymphoproliferative disease; Non-Hodgkin’s lymphoma

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. Introduction

Systemic mastocytosis (SM) is characterized by infil-ration of the bone marrow and extramedullary tissues byeoplastic mast cells. Activating mutations in exon 17 ofhe KIT gene are a hallmark of the neoplastic mast cellsf SM. SM may coexist with hematologic malignancies.his subset of SM termed systemic mastocytosis with asso-iated clonal hematologic non-mast cell lineage diseaseSM-AHNMD) in the WHO classification currently encom-asses both myeloid and lymphoid malignancies that coexistith SM [1]. Although the overwhelming majority of suchalignancies are of myeloid origin, a handful of cases of

ymphoid neoplasms associated with SM have been reported

2–6].

Unlike myeloid malignancies associated with SM inhich the neoplastic mast cells and the malignant myeloid

∗ Corresponding author. Tel.: +1 626 359 8111x65285;ax: +1 626 301 8116.

E-mail address: vpullarkat@coh.org (V. Pullarkat).

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145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2007.04.008

ells originate from the same clone, the relationship betweenhe neoplastic mast cells and the coexisting lymphoid malig-ancy is not well established. In this paper, we reportcase of indolent SM associated with low-grade B-cellon-Hodgkin’s lymphoma. Mast cells and B-lymphocytesicrodissected from the lymphoma-involved bone marrowere analyzed for activating KIT mutations in order toelineate their clonal relationship. The pathogenesis of SMssociated with lymphoproliferative disease, as well as thelinical and pathologic differences between SM associatedith myeloid as opposed to lymphoid neoplasms are alsoiscussed in light of current evidence.

. Materials and methods

.1. Case history

An 83-year-old man was diagnosed with indolent SM inovember 2003 when he presented with a 5-year history ofpruritic rash over his trunk and lower extremities. A skin

1750 Y. Kim et al. / Leukemia Research 31 (2007) 1749–1754

Fig. 1. Morphologic and immunohistochemical features of the mast cell infiltrate and B-cell lymphoma in the bone marrow. (A) Paratrabecular infiltrate ofatypical spindle-shaped mast cells typical of SM. (B and C) These mast cells aggregates are highlighted by tryptase and CD117 staining, respectively. (D) Themast cells show aberrant expression of CD25 consistent with their neoplastic phenotype. (E) Bone marrow clot section shows numerous lymphoid nodulesadmixed with pale staining mast cell aggregates. (F) Higher power view of a lymphoid nodule shows cluster of mast cells occupying the center. (G) B-cellcomponent of the lymphoid nodules are highlighted by CD20 staining. (H) Tryptase staining shows the mast cells within a lymphoid nodule. (I) The neoplasticB-cells show intense BCL-2 expression. (J) CD5 staining shows the T-cell component of the lymphoid nodule, but the neoplastic B-cells do not express CD5.(K and L) Absence of CD 10 and aberrant CD43 expression by the neoplastic B-cells, respectively.

Y. Kim et al. / Leukemia Research 31 (2007) 1749–1754 1751

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iopsy was consistent with urticaria pigmentosa. His periph-ral blood counts were within normal limits. Biopsy of bonearrow showed a hypercellular bone marrow (90% cellu-

arity) with clusters of atypical mast cells comprising about5% of marrow cellularity. These mast cells CD25 consistentith their neoplastic mast cell phenotype (Fig. 1). In addition

here were abundant non-paratrabecular nodular aggregatesf lymphocytes. These lymphocytes accounted for approx-mately 20% of the bone marrow cellularity and expressedD20, PAX-5 and BCL-2 and CD43, but were negative forD5, CD10, and BCL-1. Ki-67 was expressed in about 20%f the malignant B cells. Flow cytometry showed monoclonalappa light chain expression in the bone marrow B cells.mmunoglobulin heavy chain gene rearrangement confirmedonoclonality of B cells microdissected from the lymphoid

odules. The D816V KIT mutation was detected within theicrodissected bone marrow mast cells as described below.he patient’s serum tryptase level was 116 ng/ml (normal–10). Based on the immunohistochemical and moleculartudies a diagnosis of B-cell Non-Hodgkin’s lymphoma coex-sting with indolent SM was made. There was no evidencef lymphadenopathy, hepatosplenomegaly or systemic symp-oms at the time of initial diagnosis of the B-cell lymphoma.n November 2006, the patient developed abdominal pain

nd was found to have intrathoracic and intraabdominal lym-hadenopathy on CT scan with a large right external iliacymphnode measuring 10.4 cm × 5.5 cm. Core biopsy of thisymph node confirmed the diagnosis of low-grade B-cell

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on-Hodgkin’s lymphoma. The immunophenotype of theymphoma was similar to that of the lymphoid infiltrate inhe bone marrow. PET scan showed increased FDG uptaken the skin, mediastinum and a large focus of intense uptaken the right pelvis. He is currently being treated with ritux-mab alone. He is also is receiving antihistamines with goodymptomatic relief of pruritus and has not required any otherherapy for SM.

.2. Immunohistochemistry and laser microdissection

The paraffin tissue block from the patient’s bone mar-ow clot was cut into 5-�m sections and immunostainedith anti-tryptase and anti-CD20 antibodies for the iden-

ification of mast cells and B-lymphocytes. The tryptaseositive cells were compared with mast cells found H&stained slide for confirmation of mast cell identity. The

umerous B-lymphocytes were highlighted with anti-CD20ntibodies. The tryptase positive mast cells and the CD20 pos-tive B-lymphocytes were microdissected individually or asmall clusters into the separate tubes using the PALM lasericrodissection system (Carl Zeiss Microimaging, Thorn-ood, NY, USA).

.3. Molecular studies

Genomic DNA was extracted from microdissected bonearrow mast cells and B-lymphocytes. Exon 17 of the KIT

1752 Y. Kim et al. / Leukemia Researc

Fig. 2. Sequencing of KIT exon 17. (A) Analysis of genomic DNA from mastc(8f

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ells microdissected from the bone marrow shows an A → T point mutationarrow) resulting in an aspartate to valine amino acid substitution at position16 (D816V). (B) Analysis of genomic DNA from B-cells microdissectedrom lymphoid nodules shows the wild type sequence.

ene was amplified by semi-nested PCR separately withhe following primers: forward primer: 5′-AAG CAA CACAT AGT ATT AAA AAG TTA GTT TTC ACT-3′, reverserimer 1: 5′-TCG AAA GTT GAA ACT AAA AAT CCTTG-3′, and reverse primer 2: 5′-CAG GAC TGT CAACA GAG AAT GG-3′ and using Qiagen HotStart Taqolymerase (Qiagen, Valencia, CA). The first PCR was per-ormed using reverse primer 1 and the common forwardrimer. PCR conditions were an initial step of 95 ◦C for5 min, followed by 40 cycles of 95 ◦C for 20 s, 58 ◦Cor 30 s and 72 ◦C for 30 s and a final extension step of2 ◦C for 15 min. The second PCR was done using reverserimer 2 and the common forward primer. Amplificationas performed for 45 cycles using the same conditions as

or the first reaction. PCR products were analyzed on 2%garose gel and the amplified product was purified withieax II kit (Qiagen). PCR products were directly sequencedith ABI BigDye 3.0 sequencing kit and ABI 3700 Prism

equencer (ABI Biosystems). Immunoglobulin heavy chainene rearrangement analysis was performed on genomicNA extracted from microdissected B-cells using standardethods.

. Results

The activating KIT mutation D816V was detected in thelusters of mast cells that were microdissected from the bonearrow, consistent with the diagnosis of SM. This muta-

ion was not detected in the B-cells that were microdissected

rom multiple bone marrow lymphoid nodules after delin-ating them with CD20 staining (Fig. 2). Monoclonality ofhe neoplastic B-cells was demonstrated by flow cytometrynd immunoglobulin heavy chain gene rearrangement anal-

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h 31 (2007) 1749–1754

sis. These findings prove the distinct clonal origins of theeoplastic mast cells and the coexisting B-cell lymphoma.

. Discussion

Our results show that when SM coexists with lympho-roliferative disease, the neoplastic mast cells and malignantymphoma cells are clonally distinct. Similar results haveecently been described by Horny et al., who detected the816V KIT mutation in bone marrow mast cell aggregates butot in the neoplastic B cells in a case of SM associated withhronic lymphocytic leukemia [6]. These results are unlikehe case with SM associated with myeloid malignancy wherehe neoplastic mast cells and the coexisting myeloid malig-ant cells have been shown to have the same clonal origin asiscussed below.

Our findings are discordant with two studies that demon-trated the D816V KIT mutation in mast cells, T cells and Bells from the bone marrow of patients with SM. However,ifferences in methodology and the patient population stud-ed may account for this discrepancy. In the study by Akint al., in addition to bone marrow mast cells, the mutationas detected in the bone marrow B cells in 60% of patients

7]. In this study the various cell types were isolated by flu-rescence sorting. Hence the morphologic relationship ofhe bone marrow B cells carrying the KIT mutation to theast cell aggregates cannot be determined from this study.ince the activating KIT mutation occurs in a pluripotentematopoietic progenitor cell, the origin of some lymphoidells from the mutated clone is not surprising. In order toddress this issue, Taylor et al. microdissected mast cells, Tells and B cells from mast cell lesions and non-lesional areasf bone marrow of patients with systemic mastocytosis. The816V mutation was detected by PCR analysis in the B cells

nd T cells obtained from mast cell lesions but not from non-esional areas of the bone marrow thereby showing that onlyntralesional lymphocytes have the same clonal origin as theeoplastic mast cells [8]. None of the patients in both thesetudies were reported to have a malignant lymphoprolifera-ive disorder in association with SM.

It is important to appreciate that reactive lymphoid aggre-ates are a feature of bone marrow pathology in patients withndolent SM [9]. Bone marrow infiltrates in indolent SMan be comprised of predominantly mast cells, mixture ofast cells and lymphocytes or predominantly lymphocytes

10]. While the lymphocytes are admixed with the neoplas-ic mast cells in most cases, occasionally the lymphocytesan predominate and assume a nodular appearance arous-ng suspicion of a lymphoma associated with indolent SM.

hen the bone marrow is extensively infiltrated with bothast cells and lymphocytes, another possibility is that the

ast cell infiltrate is a reactive process associated with a low

rade lymphoma especially lymphoplasmacytic lymphoma9]. However these mast cells infiltrates can easily be clas-ified as reactive since they lack features of neoplastic mast

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ells namely dense mast cell aggregates, aberrant expressionf CD2 and/or CD25 and activating KIT mutations [1].

Although the majority of hematopoietic malignanciesssociated with SM are of myeloid origin, various lym-hoid malignancies have occasionally been described inssociation with SM. These include cases of Hodgkins andon-Hodgkin’s lymphoma, multiple myeloma and chronic

ymphocytic leukemia [2–6]. Precise classification of theymphoma in our patient was not possible due to the lim-ted amount of tissue available for morphologic analysis.owever the immunophenotype was most consisitent withlow-grade B-cell Non-Hodgkin’s lymphoma.

The role played by the neoplastic mast cells of SM inupporting the associated lymphoproliferative disorder is notnown. Using in vitro coculture experiments, Tournihac etl. have demonstrated the ability of the human mast celline HMC-1 (which carries the D816V KIT mutation) tonduce proliferation of the neoplastic lymphoplasmacyticells of patients with Waldenstrom’s macroglobulinemia.hese investigators further showed that this interactionetween mast cells and the neoplastic B-lymphocytes wasediated through CD154-CD40 signaling [11]. In a sim-

lar set of experiments, Molin et al. have demonstratedhe ability of HMC-1 to support the growth of humanodgkin’s disease-derived cell lines through CD30L-CD30

ignaling [12]. These experiments demonstrate the abil-ty of neoplastic MC to support the growth of malignantymphoproliferative disorders. It will be interesting tossess in our patient the response of the lymphoma in theone marrow to therapy with a tyrosine kinase inhibitorike dasatinib or nilotinib that inhibits the D816V KIT

utation.According to the current WHO classification, patients with

ither myeloid or lymphoid malignancies coexisting withM could be included under the subset of SM termed SM-HNMD. However, the clinical and pathologic features of

hese two entities are different. Unlike SM associated withyeloid malignancies where both the mastocytosis and theyeloid neoplasm are often diagnosed concurrently [13],

eported patients with SM and LPD have a long history ofndolent SM. This temporal profile suggests a different patho-enesis for these two entities and this conclusion is supportedy molecular studies. It has now been shown that in the casef SM associated with myeloid malignancies, the neoplas-ic mast cells and the malignant myeloid cells are clonallyelated. For instance, in the case of SM with acute myeloideukemia, the leukemic translocation can be detected in mastells as well [14,15]. Thus, the pathogenesis of these casesppears to be due to an activating KIT mutation that occurs in arimitive hematopoietic progenitor in addition to a transloca-ion resulting in acute myeloid leukemia and concurrent SM.s mentioned earlier, in a case of SM associated with CLL,

olecular analysis of the neoplastic MC and the malignant

ymphocytes obtained by microdissection for KIT mutationshowed the D816V mutation only in the MC, showing thathe malignant lymphocytes and not clonally related to the

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h 31 (2007) 1749–1754 1753

C [6]. The findings in our case lend further support to thisonclusion.

In summary, we report a case of lymphoproliferative dis-ase in a patient with indolent SM. By KIT mutation analysis,e demonstrate that the neoplastic B lymphocytes are not

lonally related to the coexisting neoplastic mast cells, as evi-enced by the absence of KIT mutations in the microdissectedymphoma cells. When malignant lymphoproliferative dis-ase coexists with indolent SM, we suggest classifying theseases as indolent SM with lymphoproliferative disease ratherhan combining them with myeloid malignancies under SM-HNMD, in recognition of the different pathogenesis and

linical features of these two entities.

cknowledgments

The authors have no conflicts of interest to declare.Contributions. Vinod Pullarkat was the physician for the

atient, planned the study and wrote the initial draft of theanuscript. Young Kim performed microdissection of bonearrow cells. Young Kim and Lawrence Weiss analyzed

one marrow and lymph node pathology. Yuan-Yuan Chenerformed KIT mutation analysis. All authors provided intel-ectual input and revised the final manuscript.

eferences

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[2] Travis WD, Li C-Y, Bergstralh EJ, et al. Systemic mast cell disease.Analysis of 58 cases and literature review. Medicine 1988;67:345–68.

[3] Stellmacher F, Sotlar K, Balleisen L, et al. Bone marrow mastocytosisassociated with IgM kappa plasma cell myeloma. Leuk Lymphoma2004;45:801–5.

[4] Saletti P, Ghielmini M, Scali G, et al. Hodgkin’s and Castle-man’s disease in a patient with systemic mastocytosis. Ann Hematol1999;78:97–100.

[5] King PHM, Hutchinson RM. Blood findings in generalized mas-tocytosis: coexistent Non-Hodgkin’s lymphoma. Br J Hematol1991;77:436–42.

[6] Horny H-P, Sotlar K, Stellmacher F, et al. An unusual case of sys-temic mastocytosis associated with chronic lymphocytic leukemia(SM-CLL). J Clin Pathol 2006;59:264–8.

[7] Akin C, Kirshenbaum AS, Semere T, et al. Analysis of the surfaceexpression of c-kit and occurrence of the c-kit Asp816Val activatingmutation in T cells. B cells and myelomonocytic cells in patients withmastocytosis. Exp Hematol 2000;28:140–7.

[8] Taylor ML, Sehgal D, Raffeld M, et al. Demonstration that mast cells.T cells and B cells bearing the activating kit mutation D816V occur inclusters within the marrow of patients with mastocytosis. J Mol Diagn2004;6:335–42.

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