Dirnhofer, Stefan

Hartmann, Karin

Kirsch, Mark

Konantz, Martina

Meyer, Sara C

Passweg, Jakob

Stehle, Gregor T

English

Bern Open Repository and Information System (BORIS)

Clinical CommunicationsPresence of neoplastic mast cellsin ascites in advanced systemicmastocytosisMark Kirsch, MDa, Gregor T. Stehle, MDb,Martina Konantz, PhDc, Jakob Passweg, MDb,Stefan Dirnhofer, MDd, Sara C. Meyer, MD, PhDb,c, andKarin Hartmann, MDc,eClinical ImplicationsIn patients with advanced systemic mastocytosis and ascites,neoplastic mast cells expressing CD25 and CD2 can bedetected in ascites. These mast cells in ascites might serve asa diagnostic and treatment response marker in advancedsystemic mastocytosis.Systemic mastocytosis (SM) is characterized by clonal expan-sion of mast cells (MCs) in different organs, usually in bonemarrow (BM) and variably also in other tissues like skin andgastrointestinal tract.1-3 Clinical manifestations are heteroge-neous and can include MC mediator symptoms and a dysfunc-tion of various organ systems, such as cytopenia,hepatosplenomegaly, ascites, and weight loss. Most patients withSM carry the activating mutation KIT D816V. Typically, pa-tients with SM are also characterized by elevated serum levels oftryptase, an enzyme produced by MCs, and aberrant expressionof CD25 and/or CD2 on MCs in BM and possibly other tis-sues.1-3 Advanced SM (advSM) is categorized into 3 subtypes:aggressive SM (ASM), SM with associated hematologicalneoplasm (SM-AHN), and MC leukemia. Here, we report 2patients with advSM with ascites, in whom we observedneoplastic MCs expressing CD25 and CD2 in the ascites.After institutional review board approval, routine diagnosticworkup of BM biopsies was performed to assess World HealthOrganization criteria of SM by histochemistry, immunohisto-chemistry using antibodies against CD117, tryptase, CD25, andCD2, and KIT mutational analysis using digital droplet poly-merase chain reaction.1,2 Serum tryptase levels were measuredusing a fluoroimmunoenzymatic assay (ImmunoCAP Tryptase;Thermo Fisher Scientific, Uppsala, Sweden). In addition, asciteswas collected and analyzed by flow cytometry using antibodiesagainst CD45, CD117, CD25, and CD2, by KIT mutationalanalysis, and by measurement of tryptase levels. The study wasapproved by the local ethics committee (ethics committee ofNorthwestern and Central Switzerland, Basel, Switzerland) andconducted in accordance with the Declaration of Helsinki.Informed consent was obtained in each case before BM andascites samples were analyzed.The first patient was a 72-year-old man presenting with fa-tigue, weight loss, hepatosplenomegaly, and ascites developingfor 5 months. Diagnostic workup of blood and BM as well asrecording of C-findings of advSM revealed the diagnosis of ASM(Table I). Additional next-generation sequencing (NGS) of BMcould detect mutations in SRSF2, TET2, and RUNX1. Rectalbleeding required endoscopy and abdominal contrast-enhancedcomputed tomography, which then also detected a locallyadvanced rectal adenocarcinoma. We ruled out significant heart,liver, and kidney diseases as causes of the ascites. Analysis ofascites revealed a transudate without metastatic cells, but with,interestingly, CD117-positive MCs expressing CD25 and CD2(Table I, Figure 1). Some of these MCs also showed a spindle-shaped phenotype (data not shown). KIT mutational analysisof ascites also showed the presence of the KIT D816V mutation.Treatment of ASM with midostaurin was started but could notstop progression of ASM with pancytopenia and symptomaticascites as well as persistent rectal bleeding. Therefore, cytore-ductive therapy with cladribine and palliative radiation of therectal adenocarcinoma was started, which resulted in initialimprovement of fatigue, weight loss, bleeding events associatedwith normalization of hemoglobin, platelets, alkaline phospha-tase, and a decrease of the elevated serum tryptase (Figure E1,available in this article’s Online Repository at www.jaci-inpractice.org). Of note, response to treatment was also associ-ated with a significant decrease of neoplastic MCs in ascites(Figure 1). The patient then relapsed and died 14 months later.The second patient was a 62-year-old man presenting withweight loss, recurrent nose bleeding, diarrhea, hep-atosplenomegaly, and ascites for 6 months (Table I). Diagnosticworkup revealed the diagnosis of SM. In addition, we observedsignificant anemia, thrombocytopenia, and monocytosis in pe-ripheral blood as well as increased and dysplastic myelopoiesis inBM, resulting in the final diagnosis of SM-AHN, with chronicmyelomonocytic leukemia (ASM-CMML-0). Additional NGS ofBM could detect mutations in ASXL1, TET2, and STSF2. Weagain ruled out significant heart and liver diseases as well assignificant albuminuria as causes for the ascites, although thepatient also suffered from chronic renal disease. Analysis of theascites revealed a transudate as well as CD117-positive MCsexpressing CD25 and CD2 (Figure 1). Monocytosis and spindle-shaped MCs were also found in the ascites. Furthermore, thetryptase level in ascites was 105 ng/mL, suggesting an increase intryptase resulting from the presence of MCs in ascites. Allogenicstem cell transplantation was performed on conditioning with aFLAMSA-RIC protocol. Unfortunately, SM-AHN persisted af-ter transplantation and was progressive over 12 months. Mid-ostaurin treatment had no significant effect on diseaseprogression and was also associated with side effects. Cutaneousgraft-versus-host reaction occurred and had to be treated withtacrolimus and prednisone. The patient is currently in evaluationfor a second allogenic transplantation.In summary, we report 2 patients with advSM and ascitescharacterized by the presence of neoplastic MCs expressingCD25 and CD2. As MCs develop from pluripotent precursorcells and usually leave the BM for further tissue-specific differ-entiation under physiologic conditions, the histopathologicpattern of aggregates of MCs infiltrating the BM serves as amajor criterion to diagnose SM.1,2 Although similar aggregates inother extracutaneous organs such as gastrointestinal tract andliver are valued equally as a major diagnostic criterion, thespecificity and sensitivity are still a point of discussion.3 Only few3035TABLE I. Characteristics of the 2 patients with advSM before therapyCharacteristics Patient 1 Patient 2Age at diagnosis (y) 72 62Sex Male MaleCategory of mastocytosis ASM SM-AHN (ASM-CMML-0)Diagnostic criteria of SMMajor criterionMultifocal dense infiltrates of MCs in BM þ þMinor criteriaSpindle-shaped MCs in BM, >25% þ þCD25/CD2 expression on MCs in BM þ/þ þ/þKIT D816V mutation in BM þ þTryptase level in serum, >20 ng/mL þ (171 ng/mL) þ (125 ng/mL)C-findingsCytopeniaHemoglobin <10 g/dL þ (7.5 g/dL) þ (6.7 g/dL)Platelets, <100,000 n/mm3 þ (53,000 n/mm3) þ (46,000 n/mm3)Neutrophils, <1000 n/mm3  Hepatomegaly with ascites þ þSplenomegaly with hypersplenism NK NKMalabsorption with weight loss þ þLarge-sized osteolysis with pathologic fractures  Life-threatening organ damage caused by MC infiltration  Additional neoplastic disease Rectal cancer Additional pathological laboratory findingsMonocytosis, >620 n/mm3  þ (1560 n/mm3)Elevated alkaline phosphatase, >130 U/L þ (403 U/L) þ (144 U/L)Characteristics of ascitesCD25 expression on MCs in ascites þ þCD2 expression on MCs in ascites þ þKIT D816V mutation in ascites þ NDTryptase level in ascites (ng/mL) ND 105 ng/mLAdditional mutations in the BM SRSF2, TET2, RUNX1 STSF2, ASXL 1, TET 2“þ” indicates presence and “” absence of characteristic.advSM, Advanced SM; ASM, aggressive SM; BM, bone marrow; CMML, chronic myelomonocytic leukemia;MC, mast cells; ND, not determined; NK, not known; SM, systemicmastocytosis; SM-AHN, SM with associated hematological neoplasm.J ALLERGY CLIN IMMUNOL PRACTNOVEMBER 20223036 CLINICAL COMMUNICATIONSstudies investigated ascites in SM, reporting on lymphocytichypoalbuminemic ascites associated with few MCs.4,5 In thepresent study, we report as a novel finding the presence ofneoplastic MCs in ascites in patients with advSM, characterizedby expression of CD25 and CD2. Thus, these ascites MCsclosely resembled MCs in BM in SM.Along with these findings, we also detected the KIT D816Vmutation and a tryptase level of 105 ng/L in ascites. Measure-ment of tryptase in serum is well established, whereas we couldonly find few reports in the literature on measurement of tryptasein peritoneal fluid. Berdún et al6 showed a baseline tryptase levelof 0.12 ng/mL with an increase to 4.99 ng/mL after colectomy.We hypothesize that the level of 105 ng/L in our second patientcould be indicative of mastocytosis and correlate with MCburden, similar to what has, for example, been shown for tryptaselevels in BM.7 To verify this hypothesis, however, further studiesand larger patient populations are needed.Taken together, we were able to demonstrate the presence ofneoplastic MCs in ascites in 2 patients with advSM. In bothpatients, these MCs were characterized by high expression ofCD25 and CD2. Under treatment, the number of CD25- andCD2-expressing ascites MCs decreased. These observationssuggest that ascites MCs and possibly also tryptase levels in as-cites might serve as additional diagnostic and treatment responsemarkers in patients with advSM when paracentesis is performedfor treatment of ascites. However, systematic studies on thepresence of MCs in the peritoneal cavity and their exact numbersare sparse, and further functional studies are needed to definewhether neoplastic MCs are always present in advSM with ascitesor whether they are just “bystanders” derived possibly also fromother tissues such as lymph nodes or serosal MCs from peritoneallining. In the future, it would also be interesting to explorewhether isolation and analysis of ascites MCs, as, for example,established for lung MCs,8 could represent a novel approach toFIGURE 1. Flow cytometric analysis of mast cells in ascites before and after therapy in the 2 patients with advanced systemic mas-tocytosis. After doublet elimination and selection for CD45þ, cells were analyzed for CD117bright, CD117brightCD45þ, CD117brightCD25þ,and CD117brightCD2þ (top to bottom). Percentages of gated cells from all events are shown. The table below shows the number of eventsfor each gate before and after therapy.J ALLERGY CLIN IMMUNOL PRACTVOLUME 10, NUMBER 11CLINICAL COMMUNICATIONS 3037J ALLERGY CLIN IMMUNOL PRACTNOVEMBER 20223038 CLINICAL COMMUNICATIONSevaluate in vitro individual diagnostic features and therapeuticresponses in advSM.9AcknowledgmentsM. Kirsch, G. T. Stehle, J. Passweg, and K. Hartmann pro-vided clinical care; M. Kirsch, G. T. Stehle, J. Passweg, S. C.Meyer, and K. Hartmann conceptualized the study; M. Kirsch,G. T. Stehle, J. Passweg, M. Konantz, S. Dirnhofer, S. C. Meyer,and K. Hartmann performed experiments and/or analyzed dataand/or visualized data; M. Kirsch, G. T. Stehle, M. Konantz, S.C. Meyer, and K. Hartmann prepared the original draft of themanuscript; all authors edited and reviewed the manuscript.aDivision of Internal Medicine, University Hospital Basel and University of Basel,Basel, SwitzerlandbDivision of Hematology, University Hospital Basel and University of Basel, Basel,SwitzerlandcDepartment of Biomedicine, University Hospital Basel and University of Basel,Basel, SwitzerlanddDepartment of Medical Genetics and Pathology, University Hospital Basel andUniversity of Basel, Basel, SwitzerlandeDivision of Allergy, Department of Dermatology, University Hospital Basel andUniversity of Basel, Basel, SwitzerlandNo funding was received for this work.Conflicts of interest: K. Hartmann has received research funding from Thermo Fisherand consultancy or lecture fees from Allergopharma, Germany, ALK-Abello,Blueprint, Deciphera, Leo Pharma, Menarini, Novartis, Switzerland, Pfizer,United States, Takeda, and Thermo Fisher. S. C. Meyer has consulted for andreceived honoraria from Celgene/BMS and Novartis. The rest of the authorsdeclare that they have no relevant conflicts of interest.Received for publication March 24, 2022; revised June 28, 2022; accepted forpublication July 13, 2022.Available online July 22, 2022.Corresponding author: Mark Kirsch, MD, Department of Internal Medicine, Uni-versity Hospital Basel and University of Basel, Petersgraben 4, 4031 Basel,Switzerland. E-mail: mark.kirsch@usb.ch.2213-2198 2022 The Authors. Published by Elsevier Inc. on behalf of the American Academyof Allergy, Asthma & Immunology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).https://doi.org/10.1016/j.jaip.2022.07.014REFERENCES1. Valent P, Akin C, Hartmann K, Alvarez-Twose I, Brockow K, Hermine O, et al.Updated diagnostic criteria and classification of mast cell disorders: a consensusproposal. Hemasphere 2021;5:e646.2. Valent P, Akin C, Metcalfe DD. Mastocytosis: 2016 updated WHO classificationand novel emerging treatment concepts. Blood 2017;129:1420-7.3. Doyle LA, Sepehr GJ, Hamilton MJ, Akin C, Castells MC, Hornick JL.A clinicopathologic study of 24 cases of systemic mastocytosis involving thegastrointestinal tract and assessment of mucosal mast cell density in irritablebowel syndrome and asymptomatic patients. Am J Surg Pathol 2014;38:832-43.4. Essner C, Thiefin G, Diebold MD, Pignon B, Caulet T, Zeitoun P. Lymphocyticascites revealing systemic mastocytosis [in French]. Gastroenterol Clin Biol1995;19:948-51.5. Addada J, Lloyd J, Bain B. Teaching cases from the Royal Marsden and StMary’s Hospitals: case 27, ascites and oedema in a patient with systemic mas-tocytosis. Leuk Lymphoma 2004;45:1713-5.6. Berdún S, Bombuy E, Estrada O, Mans E, Rychter J, Clavé P, et al. Peritonealmast cell degranulation and gastrointestinal recovery in patients undergoingcolorectal surgery. Neurogastroenterol Motil 2015;27:764-74.7. Proelss J, Wenzel J, Ko Y, Bieber T, Bauer R. Tryptase detection in bone-marrowblood: a new diagnostic tool in systemic mastocytosis. J Am Acad Dermatol2007;56:453-7.8. Ravindran A, Rönnberg E, Dahlin JS, Mazzurana L, Säfholm J, Orre A-C, et al.An optimized protocol for the isolation and functional analysis of human lungmast cells. Front Immunol 2018;9:2193.9. Landolina N, Zaffran I, Smiljkovic D, Serrano-Candelas E, Schmiedel D,Friedman S, et al. Activation of Siglec-7 results in inhibition of in vitro andin vivo growth of human mast cell leukemia cells. Pharmacol Res 2020;158:104682.J ALLERGY CLIN IMMUNOL PRACTVOLUME 10, NUMBER 11CLINICAL COMMUNICATIONS 3038.e1ONLINE REPOSITORY0 5 10 15050100150200250tryptase (ng/ml)weeks after start of treatment0 5 10 15050100150200250             weeks after start of treatmenttryptase (ng/ml)Patient 1 Patient 2Tryptase(ng/ml)Tryptase(ng/ml)Weeks W eksMidostaurin 400 mg/dCladribine 0.14 mg/d Allogenic SCTEngraftmentMidostaurin 400 mg/dParacentesisX X XXParacentesisXFIGURE E1. Course of serum tryptase levels upon therapy in the 2 patients with advanced systemic mastocytosis. Tryptase levels in theserum over several weeks after the start of therapy. The shaded areas represent the normal range (<11.4 ng/mL). Time course oftreatment with midostaurin (daily administration; black line), cladribine (X), bone marrow transplantation and engraftment (X), andparacentesis (black syringe). SCT, Stem cell transplantation.

Presence of neoplastic mast cells in ascites in advanced systemic mastocytosis.

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Presence of neoplastic mast cells in ascites in advanced systemic mastocytosis.

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