Case Report: Treatment of systemic mastocytosis with sunitinib [version 1; referees: 2 approved]

Mast cell activation disease typically presents as chronic multisystem polymorbidity of generally inflammatory ± allergic theme.  Presently, treatment of the rare, cytoproliferative variant systemic mastocytosis employs empirically selected therapies to impede mast cell mediator production and action and, when necessary, inhibition of proliferation. Some tyrosine kinase inhibitors (TKIs) have been used successfully in uncommon cases of systemic mastocytosis not bearing that disease’s usual imatinib-resistant KITD816V mutation. Recently, sunitinib, a multi-targeted TKI, had been successful in a case of systemic mast cell activation syndrome. In addition, most allergy is principally a mast cell activation phenomenon, and sunitinib has been shown helpful in controlling a murine model of oral allergy syndrome. Here, we present the first use of sunitinib in systemic mastocytosis

Determination of plasma heparin level improves identification of systemic mast cell activation disease.

Diagnosis of mast cell activation disease (MCAD), i.e. systemic mastocytosis (SM) and idiopathic systemic mast cell activation syndrome (MCAS), usually requires demonstration of increased mast cell (MC) mediator release. Since only a few MC mediators are currently established as biomarkers of MCAD, the sensitivity of plasma heparin level (pHL) as an indicator of increased MC activation was compared with that of serum tryptase, chromogranin A and urinary N-methylhistamine levels in 257 MCAD patients. Basal pHL had a sensitivity of 41% in MCAS patients and 27% in SM patients. Non-pharmacologic stimulation of MC degranulation by obstruction of venous flow for 10 minutes increased the sensitivity of pHL in MCAS patients to 59% and in SM patients to 47%. In MCAS patients tryptase, chromogranin A, and N-methylhistamine levels exhibited low sensitivities (10%, 12%, and 22%, respectively), whereas sensitivities for SM were higher (73%, 63%, and 43%, respectively). Taken together, these data suggest pHL appears more sensitive than the other mediators for detecting systemic MC activity in patients with MCAS. The simple, brief venous occlusion test appears to be a useful indicator of the presence of pathologically irritable MCs, at least in the obstructed compartment of the body

ATZ11 Recognizes Not Only Z-α₁-Antitrypsin-Polymers and Complexed Forms of Non-Z-α₁-Antitrypsin but Also the von Willebrand Factor

<div><p>Aims</p><p>The ATZ11 antibody has been well established for the identification of α₁-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue.</p><p>Methods and Results</p><p>Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens.</p><p>ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes.</p><p>Conclusions</p><p>ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.</p></div

Sensitivities of heparin, tryptase, chromogranin A levels in blood and <i>N</i>-methylhistamine excretion into urine for indication of an increased mast cell activity.

<p>Sensitivities of heparin, tryptase, chromogranin A levels in blood and <i>N</i>-methylhistamine excretion into urine for indication of an increased mast cell activity.</p

Confocal imaging of VWF-transfected HEK293 cells: Pseudo-Weibel-Palade-Body (pseudo-WPB) granules formed after transfection of HEK293 cells using recombinant wild-type VWF (rVWF-WT) constructs: (A, B) Pseudo-WPB granules are shown in green (anti-VWF staining).

<p>(C) The same intracellular structures are stained with ATZ11 (red). (D) Small dot-like signals of less than .25 µm were found in very few HEK293 cells stained with anti-AAT (arrow). (E) Merged images of anti-VWF and ATZ11 stains highlight the co-localization of the antibody-binding sites. At a single cell level, small dot-like positive signals were found in the ATZ11 reaction, which were not co-localized with VWF staining (arrow). (F) Merged images of anti-VWF staining and anti-AAT signals demonstrated that the dot-like anti-AAT positive signals were not associated with pseudo-WPBs (arrow). Scale bar = 10 µm.</p

Recombinant von Willebrand Factor Expression in four Transfection Experiments in HEK293 cell lines.

<p>rVFW – recombinant von Willebrand factor.</p

Comparative immunostaining of temporary artery specimens.

<p>(A) Localization of AAT in the temporal artery. Specimen stained with polyclonal anti-AAT (1∶5000) showed immunoreactivity on the endothelial surface and a gradient of presumably soluble AAT within the vessel wall. (B) ATZ11 (1∶100) showed a distinct staining of the endothelial layer. (C) The endothelial layer is distinctly stained using anti-VFW (1∶500). (D) After saturation with anti-AAT antibody (1∶10), ATZ11 labeled a thin endothelial layer. (E) Blockade with anti-VWF antibody (1∶10) abolished ATZ11 staining (1∶100) of the endothelial layer. (F) Sequential blockade with anti-AAT (1∶10) and anti-VWF (1∶10) completely abolished ATZ11 staining (1∶100).</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

Protein-electrophoretic studies on VWF-transfected HEK293 cells and human serum samples:

<p>(A) <b>SDS-PAGE electrophoresis and subsequent western blotting (WB) and visualization using anti-VWF: (lane 1) cell lysates of recombinant wild-type VWF (rVWF-WT)-transfected HEK293 cells, (lane 2) mock-transfected HEK293 cells.</b> (B) SDS-PAGE electrophoresis and subsequent WB and visualization using ATZ11: (lane 1) of cell lysates of rVWF-WT-transfected HEK293 cells and (lane 2) mock-transfected HEK293 cells. A congruent single band of 225 kDa was detected in the VWF-transfected HEK293 cells using both anti-VWF (A) and ATZ11 (B). (C) SDS-PAGE electrophoresis and subsequent WB of human serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with anti-VWF. (D) SDS-PAGE electrophoresis and subsequent WB of serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with ATZ11. (E) Native PAGE electrophoresis and subsequent WB of a recombinant VWF (lanes 1–2) and serum samples of a non-Z healthy individual (lanes 3–5) stained with the anti-VWF antibody.</p