Neoplastic Mast Cells In Patients Research Articles

Activation of Siglec-7 results in inhibition of in vitro and in vivo growth of human mast cell leukemia cells

Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in “preventive” and “treatment” settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.

Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis

AbstractThe Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30+ MCPV-1.1 cells (10 µg/mL) compared with CD30− HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30+ MC lines tested as well as in primary neoplastic MCs in patients with CD30+ SM, but did not induce apoptosis in neoplastic MCs in patients with CD30− SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE–induced histamine release in CD30+ MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30+ advanced SM.

CD30-targeting drugs: cure for mastocytosis?

CD30 is an established therapeutic target of the CD30-targeting antibody-drug conjugate brentuximab-vedotin in patients with Hodgkin lymphoma2 and anaplastic large cell lymphoma.3 Recent reports have also demonstrated brentuximab-vedotin efficacy in relapsed/refractory diffuse large B-cell lymphoma4 and primary effusion lymphoma.5 Valent and colleagues have recently shown that CD30 is aberrantly expressed in the cytoplasm of neoplastic mast cells in patients with advanced systemic mastocytosis (SM).6 In the current study, the authors asked whether CD30 is expressed on the surface of neoplastic mast cells in advanced systemic mastocytosis and whether this surface structure may serve as a therapeutic target in mastocytosis. The authors found that CD30 is expressed on the surface of neoplastic mast cells in 3/25 patients (12%) with indolent SM (ISM), 4/7 patients (57%) with aggressive SM (ASM) and 4/7 patients (57%) with mast cell leukemia (MCL). The immature RAS-transformed human mast cell line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed human mast cell line HMC-1.2 expressed no detectable CD30. In most patients, CD30 expression in mast cells was confirmed by immunohistochemistry on bone marrow sections. Using flow cytometric analysis, the authors demonstrated a correlation between the type of systemic mastocytosis and the surface CD30 expression on mast cells. CD30 levels on mast cells in patients with advanced disease (ASM and MCL) were higher than in ISM patients (median CD30 MFI: ASM/MCL 4.24 vs ISM 1.88, P < .05).

Correlation of CD30 Expression on Neoplastic Mast Cells in Systemic Mastocytosis Assessed By Immunohistochemistry Versus Multiparameter Flow Cytometry and Correlation to Clinical Parameters

Background: Systemic mastocytosis (SM) is rarely diagnosed and presents with highly variable clinical manifestation from taking rather indolent to very aggressive courses, mast cell leukemia being the most aggressive variant. Expression of CD30 (Ki-1 antigen) has been detected on some neoplastic mast cells (MC) by immunohistochemical staining (IHC) in bone marrow (BM) biopsies and presence of CD30 has been reported to be correlated to more aggressive variants of SM. Assessmentof CD30 expression by multiparameter flow cytometry (MFC) might not only contribute to improved diagnostic accuracy but correlation of the hereby detected CD30 expression with clinical SM parameters might also give further insights into disease biology.Aims: Comparison of CD30 expression detected by either MFC or IHC on MC of patients with SM and correlation of results with patient and disease characteristics. Correlation of MFC detected CD30 expression with cytogenetics (CG) determined by chromosome banding analysis and molecular genetics (MG).Methods: For this study, CD30 expression was analyzed in BM samples from 93 patients with SM by MFC using a five color staining assay with monoclonal antibodies against CD30, CD45, CD117, CD2 and CD25. We identified MC based on CD45 positivity and bright expression of CD117. Based on aberrant coexpression of CD2 and/or CD25 neoplastic MC were identified. On those MC, mean and median fluorescence intensities (MFI, medFI) of CD30 were determined and related to CD30 MFI and medFI in lymphocytes to derive CD30 index. Moreover, data on MC infiltration and CD30 expression by IHC was assessed in 22 patients and examination of CG and MG was done in 44 and 80 patients, respectively. KIT D816V mutation was analyzed using melting curve-based DNA mutation analysis applying PNA-mediated PCR clamping according to Sotlar et al. [Am J Pathol. 162: 737-746, 2003]. Results of MFC detected CD30 expression was correlated to those of CD30 expression examined with IHC and to the results of CG and MG.Results: 42 patients were female and 51 male. Median age was 59 years (20-87 years). While we found normal karyotypes in 41 patients, 3 patients showed aberrant karyotypes. KIT D816V mutation was seen in 74/80 patients (93%). Diagnosis of concurrent hematological non-mast cell disease (AHNMD) was made in 14/93 patients (15%). Mean (±SD) MC infiltration was 20%±26% (range, 1.5%-85%) by IHC and 0.4%±1.8% (range, 0.01%-17%) by MFC. Mean (±SD) CD30 index was 19±20 (range, 3-154), mean (±SD) CD30 expression by IHC was 9%±17% (range, 0%-70%). Percentages of MC infiltration detected by IHC and MFC correlated significantly (p=0.002, r=0.819). No correlation of MFC CD30 index (MFI and medFI) with age, sex, concomitant AHNMD, grade of MC infiltration or percentage of CD30 positive MC by IHC was found. Interestingly, a significantly higher medFI CD30 index and a trend to higher MFI CD30 index were seen in patients with normal karyotype (12.2±6.2 vs 6.3±2.7, p=0.037 and 15.1±9.4 vs. 11.6±10.3, n.s., respectively) versus those with an aberrant karyotype. We also detected a trend to higher MFI and medFI CD30 index in patients with KIT D816V mutation (19.8±21.8 vs. 10.2±7.4, n.s. and 24.0±89.0 vs 10.0±5.8, n.s., respectively).Conclusions: Assessment of CD30 expression as a dynamic parameter on neoplastic MC in patients with SM can be reliably performed by MFC. A stronger expression of CD30 expression on neoplastic MC harbouring an aberrant karyotype was found. CD30 expression seems also stronger on MC from patients harbouring KIT D816V mutations compared to those who do not. CD30 expression on neoplastic MC in patients with SM should be further analyzed combining analyses by MFC and IHC to substantiate the present findings. DisclosuresBellos:MLL Munich Leukemia Laboratory: Employment. Sotlar:Ludwig-Maximilians-University: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

CD52 is a molecular target in advanced systemic mastocytosis.

Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH-1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV-1, by lentiviral immortalization of cord blood-derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52-targeting antibody alemtuzumab induced cell death in CD52(+) primary neoplastic MCs obtained from patients with SM as well as in MCPV-1 cells. NSG mice xenotransplanted with MCPV-1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.

Identification Of The Ki-1 Antigen (CD30) As a Novel Marker and Potential Therapeutic Target In Neoplastic Mast Cells In Advanced Systemic Mastocytosis

Systemic mastocytosis (SM) is a hematologic neoplasm characterized by abnormal expansion and accumulation of neoplastic mast cells (MC) in various organs, including the bone marrow, skin, liver, spleen and gastrointestinal tract. In a majority of all patients, the transforming KIT mutation D816V is detectable. The clinical picture and prognosis in SM vary, depending on the variant of SM and the presence of an associated hematologic non-MC-lineage disease (AHNMD). The WHO classification discriminates between indolent SM (ISM), SM-AHNMD, aggressive SM (ASM) and MC leukemia (MCL). Recent data suggest that neoplastic MC in ASM and MCL express substantial amounts of cytoplasmic Ki-1 (CD30), a lymphoid antigen otherwise expressed in neoplastic cells in anaplastic large cell lymphoma and Hodgkin lymphoma, where CD30 serves as a therapy target. In the present study, we examined the expression of CD30 in various human MC-lines (HMC-1, MCPV) and primary neoplastic MC in patients with ISM (n=20) and advanced SM (ASM/MCL; n=10) and asked whether CD30 may serve as potential therapeutic target in SM. We found that CD30 is expressed abundantly in the cytoplasm of neoplastic MC in ASM and MCL in almost all patients, whereas in a majority of patients with ISM, MC expressed only low amounts or did not express cytoplasmic CD30. As assessed by flow cytometry, neoplastic MC in SM were also found to express cell surface CD30. In contrast to cytoplasmic expression, no clear correlation was found between the type of SM and CD30 surface expression on MC. Notably, MC expressed surface CD30 in 8/9 ISM patients and in 2/6 ASM/MCL patients. The immature human RAS-transformed MCL line MCPV (KIT D816V negative) also expressed cell surface CD30, whereas the MCL line HMC-1.1 (lacking KIT D816V) expressed only low levels, and the HMC-1.2 subclone (KIT D816V+) was found to stain CD30 negative. To examine whether CD30 may serve as a therapy target in SM, we applied brentuximab vedotin (provided by Millennium: The Takeda Oncology Company, Cambridge, MA, USA), which is an antibody-drug conjugate consisting of a chimeric anti-CD30 antibody and the microtuble inhibitor monomethyl auristatin E (MMAE). Brentuximab vedotin has recently been described to induce dose-dependent growth arrest in human CD30+ lymphoblastic cell lines. In the present study, brentuximab vedotin was found to inhibit the proliferation of MCPV and HMC-1 cells in a dose-dependent manner, with lower IC50 values found in CD30+ MCPV and HMC-1.1 cells (5 µg/ml) than in CD30− HMC-1.2 cells (10 µg/ml). As assessed by AnnexinV/PI staining and staining for active-caspase-3, brentuximab vedotin also induced dose-dependent apoptosis in CD30+ MCPV and HMC-1.1 cells, but did not induce substantial apoptosis in CD30− HMC-1.2 cells. Brentuximab vedotin was also found to induce growth inhibition and apoptosis in the CD30+ canine-mastocytoma cell line C2. We next examined the effects of brentuximab vedotin on in vitro survival (apoptosis) of primary neoplastic MC in patients with CD30− SM (n=3) and in patients with CD30+ SM (n=3). In these experiments, brentuximab vedotin was found to induce dose-dependent apoptosis at pharmacologically relevant concentrations in neoplastic MC in patients with CD30+ SM, whereas no effects were seen in patients with CD30− SM (Figure). Finally, we examined the effects of a drug combination consisting of brentuximab vedotin and the KIT D816V targeting drug PKC412 (midostaurin). We found that both drugs synergize with each other in inhibiting the in vitro proliferation of CD30+ MCPV cells. In conclusion, our data provide evidence that the target-antigen CD30 is expressed on the surface of neoplastic MC in patients with indolent and advanced SM. In addition, our data show that the CD30-targeting antibody brentuximab vedotin induces growth arrest and apoptosis in neoplastic MC and synergizes with midostaurin in inhibiting the growth of CD30+ neoplastic MC. Whether these effects also occur in vivo in patients with advanced mastocytosis remains to be determined in clinical trials. [Display omitted] Neoplastic MC from patients with CD30− SM (n=3, grey bars) and CD30+ SM (n=3, black bars) were incubated in control medium (Co) or in various concentrations of brentuximab vedotin (37°C). After 96 hours, the percentage of AnnexinV+ viable DAPI-negative MC was determined by flow cytometry. Results represent the mean±S.D. of 3 independent experiments. Asterisk: p<0.05 compared to control. Disclosures:Sotlar:Nanostring: Honoraria; Novartis: Consultancy. Reiter:Novartis: Consultancy, Honoraria. Horny:Novartis: Consultancy, Honoraria. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Identification of Campath-1 Antigen (CD52) As a Novel Therapeutic Target in Advanced Systemic Mastocytosis.

Abstract 2866▪▪This icon denotes a clinically relevant abstractSystemic mastocytosis (SM) is a neoplasm of mast cells (MC) and MC progenitor cells. The clinical picture in SM ranges from an indolent course to highly aggressive cases with short survival time. In a majority of all patients with SM, the KIT mutation D816V is detectable. In addition, activating RAS mutations have recently been identified in patients with advanced SM. So far, no curative therapy for advanced SM is available. To identify molecular targets in neoplastic MC, we have generated novel human MC lines by lentiviral immortalization of cord blood-derived MC progenitors with RAS G12V, SV40 large T antigen, and hTERT. These cell lines, designated MCPV1 through MCPV4, display a morphology and ultrastructure resembling immature MC progenitors. MCPV cells also express a number of MC differentiation antigens, such as KIT and tryptase, and produce an aggressive MC disease when injected into NOD-SCID IL-2RG−/− (NSG) mice. As assessed by flow cytometry, MCPV cells were found to express a number of different surface antigens, including CD13, CD30, CD33, CD44, CD52, CD54, CD63, and CD95. In consecutive analyses, we studied the cell surface target CD52, also known as CAMPATH-1 antigen. As assessed by FACS, CD52 was found to be expressed on primary neoplastic MC in patients with aggressive SM (ASM, n=3), whereas in all patients with indolent SM (ISM, n=6), neoplastic MC stained negative or only slightly positive for CD52. Normal MC stained negative for CD52 in these experiments. Expression of CD52 in neoplastic MC was also demonstrable by immunohistochemical staining of bone marrow biopsy sections. We then asked whether the putative stem cells in ISM and ASM would express CD52. Indeed, in all patients examined, including cases with ISM and ASM, the immature CD34+/CD38- cells were found to express CD52. Next, we studied the regulation of expression of CD52 in neoplastic MC. Since activating RAS mutants have been described to be expressed in neoplastic MC in patients with advanced SM, we asked whether oncogenic RAS would promote expression of CD52 in MC. Indeed, lentiviral-mediated expression of activated RAS mutants was found to upregulate mRNA expression and to promote surface expression of CD52 in the human MC line HMC-1 as well as in various other myeloid cell lines. To validate CD52 as a potential target in neoplastic MC, we applied the CD52-targeting drug alemtuzumab. In these experiments, alemtuzumab exerted only minimal direct effects on viability of MCPV cells after IgG-mediated crosslinking. However, incubation of MCPV cells with alemtuzumab induced rapid and dose-dependent cell death via complement-dependent cytotoxicity (Figure 1). Furthermore, alemtuzumab was found to induce complement-dependent cytotoxicity in CD52+ primary neoplastic MC obtained from patients with advanced SM (n=2). In conclusion, we have established a new powerful model for studying the biology of advanced SM. Moreover, our study has identified CD52 as a novel promising therapeutic target in neoplastic MC and MC progenitor cells. Whether targeting of neoplastic (stem) cells in advanced SM using alemtuzumab will improve therapy by eradicating malignant cells remains to be determined in clinical trials. [Display omitted] Disclosures:Valent:Phadia: Research Funding.

Identification of Oncostatin M as a STAT5-Dependent Mediator of Bone Marrow Remodeling in KIT D816V-Positive Systemic Mastocytosis

Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.

Identification of Oncostatin M as a Novel KIT D816V-Dependent Cytokine in Neoplastic Human Mast Cells.

Abstract 213Systemic mastocytosis (SM) is a neoplastic disease of mast cells (MC) and their bone marrow-derived progenitors. The clinical picture in SM is variable ranging from an indolent course to highly aggressive variants with short survival time. The pathologic hallmark in SM is the multifocal dense infiltrate of MC in the bone marrow. Other typical features of SM include alterations of the bone marrow microenvironment such as increased angiogenesis and fibrosis. In a majority of patients, MC display the KIT mutation D816V which affects the activation loop at the entrance to the enzymatic pocket of the KIT kinase. As a consequence, KIT D816V exhibits constitutive tyrosine kinase activity and promotes cytokine-independent differentiation of MC. However, so far, little is known about KIT D816V-dependent expression of pathogenetically relevant molecules in neoplastic MC. Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family which is produced mainly by activated T cells and monocytes. OSM has been shown to inhibit cell growth in cell lines derived from solid tumors but to stimulate proliferation of fibroblasts and endothelial cells. Recently, it has been reported that OSM produced by activated MC promotes growth of human dermal fibroblasts. Moreover, it has been suggested that OSM stimulates growth of murine bone marrow-derived mast cells in a mast cell/fibroblast coculture. However, expression of OSM in neoplastic MC or a potential pathogenetic role of OSM in SM have not been examined so far. The aim of the present study was to analyze expression of OSM in neoplastic human MC and to determine the role of KIT D816V in OSM expression. As assessed by immunohistochemistry performed on bone marrow sections of patients with SM, typical spindle-shaped neoplastic MC were found to express OSM. Serial section-staining confirmed that tryptase-positive MC co-express OSM. Expression of OSM was found in neoplastic MC in all patients investigated (n=15) and in all variants of SM (indolent SM as well as aggressive variants) with comparable staining intensities. Preincubation of anti-OSM antibody with a specific blocking peptide resulted in a negative stain. In Ba/F3 cells, doxycycline-inducible expression of KIT D816V led to a substantial upregulation of OSM mRNA and OSM protein, whereas expression of wild type KIT did not affect expression of OSM. In addition, the KIT D816V-positive HMC-1.2 mast cell line was found to express OSM at high levels, whereas the KIT D816V-negative HMC-1.1 subclone expressed only baseline levels of OSM. Correspondingly, the KIT D816V-targeting drug midostaurine (PKC412) decreased the expression of OSM in HMC-1.2 cells as well as in KIT D816V-expressing Ba/F3 cells in a dose-dependent manner. To investigate signaling pathways involved in KIT D816V-dependent expression of OSM, we applied pharmacologic inhibitors and dominant negative-acting signaling molecules. We found that KIT D816V-dependent expression of OSM is inhibited by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Expression of dominant negative mutants of signal transducer and activator of transcription 5 (STAT5) did not affect expression of OSM in KIT D816V-expressing cells. In summary, our data identify OSM as a novel cytokine expressed in neoplastic MC in patients with SM and show that KIT D816V directly promotes expression of OSM through activation of the mitogen-activated protein-kinase pathway. OSM may be an important KIT D816V-dependent effector promoting angiogenesis and fibrogenesis/sclerosis in patients with SM. Disclosures:No relevant conflicts of interest to declare.

CD203c is Overexpressed on Neoplastic Mast Cells in Systemic Mastocytosis and is Upregulated upon IgE Receptor Cross-Linking

The ectoenzyme E-NPP3 (CD203c) has recently been identified as a novel activation-linked cell surface antigen on basophils. In the present study, we examined expression of CD203c on normal mast cells (MC)and bone marrow (bm) MC derived from 85 patients with systemic mastocytosis (SM), including cases with indolent SM (ISM, n=72), SM with associated clonal hematologic non-MC-lineage disease (SM-AHNMD, n=6), aggressive SM (ASM, n=3), and mast cell leukemia (MCL, n=4). Surface expression of CD203c was analyzed by multicolor flow cytometry. In patients with SM, bm MC expressed significantly higher amounts of CD203c compared to normal bm MC (median MFI in controls: 260 versus median MFI in SM: 516, p<0.05). Slightly lower amounts of CD203c were detected on MC in SM-AHNMD and ASM compared to ISM. To demonstrate CD203c expression in MC at the mRNA level, neoplastic MC were highly enriched by cell sorting, and were found to express CD203c mRNA in RT-PCR analysis. Cross-linking of the IgE receptor on MC resulted in a substantial upregulation of CD203c, whereas the KIT-ligand stem cell factor (SCF) showed no significant effects. In conclusion, CD203c is a novel activation-linked surface antigen on MC that is upregulated in response to IgE receptor cross-linking and is overexpressed on neoplastic MC in patients with SM.

Systemic Mastocytosis Mimicking Inflammatory Bowel Disease: A Case Report and Discussion of Gastrointestinal Pathology in Systemic Mastocytosis

Gastrointestinal (GI) symptoms are present in up to 80% of patients with systemic mastocytosis (SM). GI symptoms include mainly abdominal pain, diarrhea, nausea, and vomiting. It is believed that most of the GI symptoms are due to the secondary effect of mast cell mediators on the GI tract. Direct involvement of the GI tract by neoplastic mast cell infiltration has not been well documented. We report a case of SM that initially mimicked inflammatory bowel disease based on clinical, radiographic, endoscopic, and histopathologic findings. On routine histologic sections of small bowel and colonic mucosal biopsies, there was expansion of the lamina propria by mononuclear inflammatory cells, foci of erosions with associated acute inflammation, and evidence of chronic mucosal injury with architectural distortion and gland foreshortening. Only on repeat biopsies and with ancillary tests for mast cells was a diagnosis of SM made, with extensive involvement of the GI tract. This is the first reported case of SM presenting as and mimicking inflammatory bowel disease. It is critical that clinicians and pathologists are aware that neoplastic mast cells in patients with SM can infiltrate the mucosa throughout the GI tract and that this infiltration can lead to symptoms and findings that can mimic inflammatory bowel disease.

Immunohistochemical detection of histidine decarboxylase in neoplastic mast cells in patients with systemic mastocytosis

Synthesis of histamine in hematopoietic progenitor cells may be one of the earliest events in mastopoiesis. We therefore asked whether the key enzyme involved in histamine production, histidine decarboxylase (HDC), can be used as an immunohistochemical marker for the detection of immature neoplastic mast cells (MC) in patients with MC-proliferative disorders. To address this question, we examined bone marrow biopsy specimens in a cohort of 102 patients with mastocytosis using an antibody against HDC. Independent of the maturation stage of MC, the anti-HDC antibody produced clear diagnostic staining results in all patients with systemic MC disease examined including those with MC leukemia and MC sarcoma, in which MCs are particularly immature. In these patients, expression of HDC was reconfirmed at the messenger RNA level by reverse transcriptase polymerase chain reaction analyses performed with RNA of highly enriched CD117(+) MC. In summary, HDC is expressed in neoplastic MC in patients with systemic mastocytosis independent of the maturation stage of cells or the variant of disease. Histidine decarboxylase should therefore be considered as a new MC marker in the screen panel of antigens used to diagnose high-grade MC malignancies.

Inhibition of Growth of Neoplastic Mast Cells by CD44 mAb A3D8 Is Associated with G1 Cell Cycle Arrest and Apoptosis.

Recent data suggest that CD44 may serve as a new therapeutic target in AML and possibly also in other myeloid neoplasms. Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in one or multiple organs. We have previously shown that normal tissue MC express CD44. In the present study, we asked whether CD44 is expressed on neoplastic human MC and whether CD44-ligation by the monoclonal antibody (mAb) A3D8 would be associated with inhibition of growth of neoplastic MC. As assessed by flow cytometry, primary neoplastic MC were found to express CD44 in all patients with SM analyzed (n=10). The human mast cell leukemia (MCL) cell line HMC-1 was also found to express CD44. As assessed by 3H-thymidine incorporation, the CD44 mAb A3D8 decreased the proliferation of HMC-1 cells in a dose-dependent manner (A3D8, 5 μg/ml: 46±26% of control=100%, p<0.05). Similar effects of A3D8 were observed with primary neoplastic MC obtained from a patient with MCL (A3D8, 5 μg/ml: 68±20%) and one with smouldering SM (A3D8, 2.5 μg/ml: 42±7% of control, p<0.05). To analyze the mechanism of A3D8-induced growth inhibition, cell survival and cell cycle distribution were analyzed. We found that CD44-ligation induces an approximately 3-fold increase in apoptotic HMC-1 cells compared to control. As assessed by flow cytometry, we were also able to demonstrate that A3D8 induces cell cycle arrest in the G1-phase. Moreover, as assessed by Western blotting, we found that incubation of HMC-1 cells with mAb A3D8 results in an increase in the cyclin-dependent kinase-inhibitor p27Kip1. In summary, our results suggest that CD44-ligation is followed by inhibition of growth of neoplastic human MC through induction of apoptosis and G1 cell cycle arrest. The latter is probably mediated by stabilization of p27Kipl. Whether targeting of CD44 in neoplastic MC in patients with high grade MC disorders is of clinical significance remains to be determined in forthcoming studies.

Detection of Activated STAT5 in Neoplastic Mast Cells in Patients with Systemic Mastocytosis: Role of c-kit D816V.

Recent data suggest that activated STAT5 contributes to growth and differentiation of mast cells (MC) and that STAT5-knock out mice are MC-deficient. We have recently shown that constitutively activated STAT5 acts as a potent oncogenic signaling molecule in hematopoietic progenitor cells (Cancer Cell 2005; 7:87–99). In the present study, we examined the expression of activated STAT5 in neoplastic MC in systemic mastocytosis (SM) and asked whether the SM-related oncogene c-kit D816V is involved in STAT5-activation. For the immunohistochemical detection of activated tyrosine phosphorylated STAT5 (P-Y-STAT5), we used the specific monoclonal antibody AX1 (Advantex) which does not react with inactive STAT5. In all patients with SM tested (indolent SM, n=11; smouldering SM, n=2; aggressive SM, n=1; mast cell leukemia, n=1; all exhibiting c-kit D816V), MC were found to display P-Y-STAT5. Expression of activated STAT5 was also demonstrable in the c-kit D816V-positive mast cell leukemia-derived cell line HMC-1. The reactivity of HMC-1 cells with AX1 antibody was abrogated by a STAT5-specific blocking-peptide. To define the role of c-kit D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of c-kit D816V (Ton.kit) were employed. In these cells, induction of c-kit D816V was followed by a massive increase in phosphorylated STAT5 as determined by a specific DNA-binding assay, whereas the total amounts of STAT5-mRNA and of the STAT5-protein showed only a slight increase or remained unchanged. In summary, these data show that neoplastic MC in SM express activated STAT5 (P-Y-STAT5), and that the transforming c-kit mutation D816V leads to persistent activation of STAT5 in these cells.

Differential expression of CD2 on neoplastic mast cells in patients with systemic mast cell disease with and without an associated clonal haematological disorder.

Recently, aberrant coexpression of CD2 and CD25 has been reported to reliably distinguish neoplastic mast cells from normal or so-called reactive mast cells. Such expression is included in the consensus diagnostic criteria for systemic mast cell disease (SMCD). In our study of patients with SMCD, we found CD2 expression to be more prevalent on mast cells from patients without an associated haematological disorder (P = 0.04). Furthermore, no correlation was found between mast cell CD2 expression and other clinicopathological features in these patients.