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NEOPLASIA
From the Department of Internal Medicine I, Division of
Hematology and Hemostaseology, Department of Internal Medicine III,
Division of Rheumatology, Institute of Histology and Embryology,
Department of Clinical Pathology, and Institute of Pathophysiology,
University of Vienna, Austria; Department of Internal Medicine,
Division of Rheumatology, Allergy and Immunology, Virginia Commonwealth
University, Richmond, VA.
Acute myeloid leukemia (AML) is characterized by
clonal proliferation of immature myeloid (progenitor) cells without
significant differentiation.1 The prognosis and clinical
picture in AML varies, depending on the genes that underwent
deregulation, cell type(s) involved, and the specific biological
properties of the clone(s).1-6 In the past few decades,
several disease-specific or lineage-associated markers indicating the
presence and/or type of AML have been established. Some of these
markers, like the breakpoint-specific gene-fusion products, represent
useful tools for the diagnosis and monitoring of AML.7-10
Other markers, like the CD molecules or distinct enzymes
(myeloperoxidase, lysozyme), are useful for determining the subtype of
AML.11-20
So far, Patients' characteristics
Measurements of tryptase and histamine
Immunohistochemistry and immunocytochemistry Immunohistochemistry was performed on bm biopsy specimens obtained from 23 patients (de novo AML, n = 20; secondary AML, n = 1; ALL, n = 2). Biopsies were taken from the iliac crest and fixed in ethanol (95%)/formaldehyde (37%) at 4:1, followed by fixation in 7.5% neutral-buffered formaldehyde and decalcification in EDTA. Paraffin-sections of 2 µm were cut, dewaxed, and treated with 0.3% methanol-H2O2 (vol/vol) (30 minutes) or 0.3% Tris-buffered saline (TBS)-H2O2 to block endogenous peroxidase. After each step, sections were rinsed twice in 0.05 M TBS at pH 7.5. Immunohistochemical staining was performed according to published techniques.21,40,41 In brief, sections were incubated with antibodies for 1 hour at room temperature (RT), washed in TBS, incubated with biotin-labeled horse antimouse antibody (30 minutes), washed, and then exposed to avidin-biotin-peroxidase complex (30 minutes at RT). The following first-step antibodies were applied: anti-tryptase monoclonal antibody (mAb) G3 (dilution, 1:5000; Chemicon, Temecula, CA), the basophil-specific mAb 2D742 (dilution, 1:1000), and anti-Kit mAb 1A2C543 (dilution, 1:300) kindly provided by Dr H.-J. Bühring (Tübingen, Germany). Antibody binding was made visible by 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer Hämalaun and mounted in Aquatex (Merck, Darmstadt, Germany). Control slides were similarly treated with the primary antibody being omitted. For immunocytochemistry, peripheral blood or bm mononuclear cells (MNCs) from 6 patients with AML were spun on cytospin slides, fixed in acetone, washed in TBS, and incubated with antitryptase mAb G3 (1:5000) for 60 minutes. Then, slides were washed and incubated with biotinylated horse antimouse immunoglobulin G (IgG; 30 minutes). Slides were again washed and exposed to streptavidin-biotin-alkaline phosphatase complex (DAKO, Glostrup, Denmark) for 30 minutes at RT. Antibody reactivity was made visible with vector red alkaline phosphatase substrate. In control experiments, CD34+ cells purified from MNCs of normal bm (n = 3; one bm transplant donor and 2 patients with suspected hematologic neoplasm) were analyzed by immunocytochemistry by using biotinylated anti-tryptase mAb G3.Immunoelectron microscopy Immunoelectron microscopy was performed on primary leukemic bm cells (de novo AML, n = 9) according to established techniques.44,45 In brief, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% CaCl2 in 0.1 M cacodylate buffer (pH 7.4) for 60 minutes. Then, cells were washed 3 times in 0.1 M cacodylate buffer, resuspended in 2% agar, and centrifuged. Agar pellets were postfixed in 1.3% OsO4 buffered in 0.66 M collidine, and stained en bloc with 2% uranyl acetate in sodium maleate buffer (pH 4.4) for 2 hours at RT. Pellets were then rinsed, dehydrated in alcohol series, and embedded in Eponate 812 (Serva, Heidelberg, Germany). Thereafter, ultrathin sections were cut and placed on gold grids (Plano, Marburg, Germany). Grids were etched 3 times in 3% sodium metaperiodate and washed in phosphate-buffered saline (PBS) containing 50 mM glycine (pH 7.4). Sections were then preincubated in PBS containing 10% fetal calf serum (FCS) and 0.5 µg/mL Tween at RT (pH 7.4) for 30 minutes, washed, and then incubated with anti-tryptase mAb G3 (10 µg/mL) in 1% bovine serum albumin (BSA) for 4 hours. After incubation, samples were washed 4 times in PBS containing 0.5 µg/mL Tween (pH 7.4) and once in PBS containing 1% BSA and 0.5 µg/mL Tween (pH 8.0). After washing, grids were incubated with a goat anti-mouse antibody conjugated with gold (10 nM gold particles) for 3 hours at RT. Thereafter, grids were washed 3 times in PBS containing 0.5 µg/ml Tween (pH 7.4) and then contrasted in 1% uranyl acetate (5 minutes) and lead citrate (30 minutes). Sections were viewed in a JEOL 1200 AX transmission electron microscope (Tokyo, Japan). The staining reaction was controlled by using mouse IgG (10 µg/mL) instead of G3. In selected experiments, CD34+ cord blood MNCs (blasts) were purified by magnetic cell sorting and flow cytometry46,47 and examined by immunoelectron microscopy.Flow cytometric evaluation of tryptase expression Cytoplasmic expression of tryptase was analyzed in AML blasts (tryptase+ AML, n = 3; tryptase AML,
n = 1; AML in complete remission [CR], n = 1), normal
CD34+/CD45+ bm cells (n = 3 donors), HMC-1
cells (positive control), and the epithelial cell line A431 (negative
control). In whole blood or bm samples (normal bm or AML),
CD34+ cells were analyzed by multicolor flow cytometry
using anti-tryptase mAb G3, a phycoerythrin (PE)-labeled CD34 mAb, and
a peridinin chlorophyll protein (PerCP)-labeled CD45 mAb (Becton
Dickinson, San Jose, CA). Prior to staining, erythrocytes were
lysed by FACS Lysing Solution (Becton Dickinson). Cytoplasmic staining
was performed according to published techniques.48,49 In a
first step, cells were fixed in formaldehyde solution (15 minutes at
RT). Cells were then washed in PBS and permeabilized with 0.1% saponin
(Sigma, St Louis, MO) dissolved in HEPES buffer. Then, cells were
incubated with anti-tryptase mAb G3 (diluted in saponin solution) for
30 minutes. After washing in saponin solution, a fluorescein
isothiocyanate-conjugated second-step antibody was applied for 30 minutes. Cells were then saturated with mouse IgG (Becton Dickinson),
washed once in saponin solution and once in PBS, and then were exposed
to PE-CD34 plus PerCP-CD45. After washing, cells were examined by
multicolor flow cytometry on a FACS Scan (Becton Dickinson). Isolated
AML blasts and cell lines were examined by single-color flow cytometry.
Staining reactions were controlled using isotype-matched antibodies.
Northern blot analysis In 23 patients (M0, n = 3; M1, n = 4; M2, n = 3; M3, n = 4; M4, n = 1; M4eo, n = 4; M6, n = 1; secondary AML, n = 2; ALL, n = 1), bm MNCs were isolated using Ficoll and were prepared for Northern blotting. Northern blot analysis was carried out essentially as described.50 Total RNA was extracted from MNCs by the guanidinium isothiocyanate/cesium chloride method.51 Total RNA (10 µg) was size-fractionated on 1.2% agarose gels and blotted onto nylon membranes (Hybond N, Amersham, United Kingdom) using 20 × SSC (1 × SSC, 150 mM NaCl and 15 mM sodium citrate, pH 7.0) overnight. Then, RNA was cross-linked to membranes by UV irradiation (UV Strata-linker 1800; Stratagene, La Jolla, CA). Prehybridization was performed at 65°C for 4 hours in 5 × SSC, 7% sodium dodecyl sulfate (SDS), 10 × Denhardt solution (DhS; 1 × DhS consists of 0.02% wt/vol BSA, 0.02% wt/vol polyvinyl pyrolidone, 0.02% wt/vol Ficoll), 10% wt/vol dextran sulfate, 20 mM sodium phosphate (pH 7.0), sonicated salmon sperm DNA (100 µg/mL), and t-RNA. Hybridization was performed using 32P-labeled synthetic oligonucleotide probes: -tryptase
(28-mer), 5'-CATGACCGTGTG GACGCGGCTGGAGATG-3' (413-440); -tryptase
(32-mer), 5'-CAGTCTGGATGATGTAG AACTGTGGGTGCACC-3' (341-372);
 -tryptase (30-mer), 5'-GATCTGGGCGGTGTAGAA CTGTGGGTGCAC-3' (342-371);
-tryptase (28-mer), 5'-GGTGACCGTGTGGACGTGGCTG GAGACC-3' (413-440);
-actin (34-mer), 5'-GGCTGGGGTGTTGAAGGTCTCAAACATGA TCTGG-3'. Blots
were washed in 5% SDS, 3 × SSC, 10 × DhS, and 20 mM sodium
phosphate (pH 7.0) for 30 minutes (65°C), and once in 1 × SSC,
1% SDS (30 minutes at 65°C). Bound radioactivity was visualized by
XAR-5 films at  70°C using intensifying screens (Eastman Kodak,
Rochester, NY).
Detection of tryptase messenger RNA by reverse transcriptase-polymerase chain reaction To confirm expression of tryptase messenger RNA (mRNA) in bm MNCs in AML (M1, n = 1; M2, n = 3; M3, n = 2; M4eo, n = 3) an established reverse transcriptase-polymerase chain reaction (RT-PCR) protocol47 was applied. The human mast cell line HMC-1 served as a positive control for tryptase, and cultured human fibroblasts (obtained from synovial tissue) served as a negative control. Total RNA was isolated by a modified guanidinium isothiocyanate-acid phenol extraction procedure using RNAzol B (Biotecx Laboratories, Houston, TX). Total RNA from 106 cells was reverse transcribed into complementary DNA (cDNA) using a cDNA Synthesis kit (First-Strand c-DNA synthesis-kit; Pharmacia Biotechnology, Brussels, Belgium). Aliquots (6 µL) of the cDNA were used for PCR amplification in a final volume of 50 µL containing 1 × PCR buffer (PerkinElmer/Cetus ), 1.25 U Taq polymerase (PerkinElmer), 25 pM of each upstream and downstream primers specific for tryptase gene (5' primer: 5'-GAGGCCCCCAGGAGCAAGTG-3'; 3' primer: 5'-ACATCGCCCCAGCCAGTGAC-3'), and-actin (5' primer: 5'-AGGCCGGC- TTCGCGGGCGAC-3'; 3' primer: 5'-CTCGGGAGCCACCAGCAGCTC-3'). Tryptase-specific primers were selected to amplify identical regions of - and -tryptase cDNA molecules that can be discriminated by restriction fragment polymorphism, ie, the
-tryptase-specific PCR product, but not -tryptase-specific PCR
product, contained a Dra III restriction site. Primers were purchased
from MWG Biotech (Ebersberg, Germany). Samples were amplified by 32 PCR
cycles (94°C for 1 minute, 63°C for 1 minute, and 72°C for 90 seconds; initial denaturation step at 95°C for 1 minute). To
differentiate between -tryptase and -tryptase gene species, PCR
products were digested with the restriction endonuclease Dra III
(Boehringer Mannheim, Germany). For control purposes, - and
-tryptase-specific PCR products were obtained from plasmid clones
containing the - and -tryptase cDNA. After digestion, PCR
products were subjected to gel electrophoresis and visualized by
ethidium bromide staining.
Culture experiments using AML blasts Isolated bm MNCs obtained from patients with de novo AML (n = 6) were cultured in RPMI 1640 medium supplemented with 10% FCS in 5% CO2 at 37°C for up to 30 days. Cultures were serially examined for the presence of total tryptase in cell lysates and cell-free supernatants. Lysates were prepared by centrifugation and freeze thawing. The amounts of tryptase generated/released by cultured AML cells were quantified by FIA.Statistical analysis To analyze the correlation between serum tryptase levels, whole blood tryptase levels, and other laboratory findings (white blood cell count [WBC], percentage of blasts, serum lactate dehydrogenase [LDH]), a linear correlation was applied.
Detection of elevated serum tryptase levels in a group of patients with AML The levels of serum tryptase were determined in 108 patients with de novo AML, in 25 with secondary AML, and in 17 patients with ALL. In healthy controls (n = 30), the median serum tryptase level amounted to 5.1 ng/mL (mean ± SD, 5.3 ± 2.2; range, 2.0-12.6). Elevated serum tryptase levels (> 15 ng/mL) were detected in 42 (39%) of 108 patients with de novo AML and 11 (44%) of 25 patients with secondary AML. By contrast, in patients with ALL (n = 17), tryptase levels were consistently normal. Among de novo AML, elevated tryptase levels were particularly detectable in FAB groups M0 (6 of 9), M2 (9 of 14), M3 (4 of 6), and M4eo (7 of 7). The highest tryptase concentrations were found in AML-M4eo (up to 881 ng/mL) (Figure 1). In M5 and M6, the majority of the patients had normal or near normal tryptase levels. In M1 (7 of 20), M4 (6 of 26), and M7 (1 of 3), a subgroup of patients had enhanced tryptase levels. No significant correlations between serum tryptase levels and other laboratory parameters (WBC, percentage of blasts, LDH) were found (r < 0.2). In 15 patients with AML in whom significantly elevated total tryptase levels (> 20 ng/mL) were detected, samples were also examined for the presence of-tryptase
by ELISA. In the majority of these patients (9 of 15), serum
-tryptase levels were < 1 ng/mL. In 6 of 15 patients, however,
-tryptase levels were also detectable (range, 1-14 ng/mL). Those
-tryptase+ AML cases (particularly those with AML-M4eo)
were found to exhibit very high levels of total serum
tryptase.
Detection of tryptase in leukemic blood cells In healthy controls (n = 30) whole blood tryptase levels amounted to 4.6 ng/mL (range, 0.8-17.2) and thus showed a similar range compared with serum enzyme levels. In patients with AML, whole blood tryptase concentrations were elevated (> 20 ng/mL) in a group of patients (58 of 114 = 50.9%) and showed a significant correlation with serum enzyme levels (r = 0.86) (Figure 2). Thus, in the majority of the (tryptase+) cases, whole blood enzyme levels were slightly higher or in the same range as compared with serum tryptase levels. By contrast, in patients with SM (n = 10) in whom the enzyme is derived from tissue mast cells (but not produced or stored in circulating blood cells) serum tryptase levels always exceeded whole blood tryptase levels (ratio whole blood tryptase/serum tryptase = 0.5-0.7). The presence of tryptase in circulating AML cells could be demonstrated by measuring the enzyme in isolated blood MNCs in patients with serum tryptase levels > 50 ng/mL. In those patients, circulating AML blasts were found to express significant amounts of tryptase (range, 0.03-7.7 ng/105 cells). No significant correlation between whole blood tryptase and whole blood histamine levels could be substantiated (r = 0.36). In fact, we were able to identify AML patients with high tryptase and high histamine levels, patients with high tryptase but low histamine levels, and also patients with high histamine but low tryptase levels (not shown).
Detection of the tryptase protein in AML blasts To further analyze tryptase expression in AML, immunohistochemistry, immunocytochemistry, and flow cytometry were performed by using the anti-tryptase mAb G3. Immunohistochemical staining experiments were conducted on bm sections (AML, n = 21; ALL, n = 2). As exemplified in Figure 3A,B, an increase in diffusely scattered tryptase+ cells was found in AML cases with significantly elevated serum tryptase levels (Table 2). Most of the tryptase-reactive material was localized in the cytoplasm of immature myeloid cells. The distribution of tryptase+ cells (blasts) in the bm always showed a diffuse pattern. In contrast, no focal (dense) accumulations of tryptase+ cells (like seen in mastocytosis) were found. In normal bm and cases with tryptase AML (serum tryptase
levels, < 15 ng/mL), only a few if any tryptase+ cells
were detectable. Also, in cases with ALL, bm sections did not contain
significant numbers of tryptase+ cells (Figure 3C). To
evaluate lineage relationships, we also applied a basophil-specific
antibody (2D7) and anti-Kit antibody (1A2C5). Interestingly, in cases
with tryptase+ AML, a variable expression of 2D7 and/or Kit
was demonstrable, whereas in most cases of tryptase AML,
leukemic blasts were also negative for 2D7 and Kit (Table 2).
Immunocytochemical staining experiments (cytospin preparations) of bm
MNCs confirmed the presence of tryptase-reactive material in
myeloblasts in all patients with AML with clearly elevated serum
tryptase levels (> 50 ng/mL) analyzed (n = 4) (Figure 3D). In
contrast, no reactivity was found in AML blasts in patients with normal
levels ( 15 ng/mL) of serum tryptase (Table 2). Also, normal
CD34+ bm cells (normal bm; n = 3) were
tryptase (0 of 200 blast cells counted). Flow cytometric
examination of tryptase expression disclosed corresponding results. In
fact, in all cases with elevated enzyme levels (n = 3), blast cells were found to react with anti-tryptase mAb G3 whereas no reactivity was
seen in a patient with tryptase AML, a patient with
tryptase+ AML in CR, and in
CD34+/CD45+ progenitor cells obtained from
normal bm (n = 3) (Figure 4).
Detection of tryptase in leukemic blasts by immunoelectron microscopy Immunoelectron microscopy was performed on bm MNCs obtained from 6 patients with tryptase+ AML (serum tryptase, > 15 ng/mL) and 3 with tryptase AML ( 15 ng/mL). In cases
with enhanced serum tryptase, a significant proportion of blasts were
found to contain tryptase-immunoreactive material in their cytoplasmic
compartment (Figure 5A). In most cells,
the reactive material was located in granulelike structures (Figure
5A). Sometimes, tryptase was also found loosely spread in the
cytoplasm. The intensity of immunogold staining in blasts varied from
donor to donor. In particular, in cases with high tryptase levels
(> 100 ng/mL), AML blast cells were found to contain substantial
amounts of tryptase (Figure 5A). In those with lower tryptase levels
(< 100 ng/mL), we detected fewer tryptase+ particles in
blast cells by immunogold labeling (Figure 5B). In the blast cells of
patients with AML who exhibited normal serum tryptase levels, no
tryptase-immunoreactive material could be detected in bm blasts. Also,
no tryptase-reactive material was found in normal purified
CD34+ (cord blood-derived) blasts.
Detection of tryptase mRNA in bm MNCs To analyze whether tryptase is expressed at the mRNA level in AML blasts, Northern blot experiments were performed using oligonucleotide probes specific for- and -tryptase. For this purpose, RNA of bm
cells from 22 patients with AML and one patient with ALL was analyzed.
Using oligonucleotide probes specific for -tryptase, expression of
mRNA was found in all AML cases in whom elevated serum tryptase could
be detected (n = 16), whereas no transcripts for -tryptase were
detected in patients with AML with normal serum tryptase (n = 6) and
in the patient with ALL. Figure 6 shows the results from 17 (of the 23) patients (ALL, n = 1;
tryptase+ AML, n = 12; tryptase AML,
n = 4). With the use of probes specific for -tryptase, only the 2 patients with AML-M4eo expressing very high serum tryptase concentrations (395 and 881 ng/mL, respectively) had detectable -tryptase mRNA (Figure 6).
To further demonstrate the presence of mRNA specific for tryptase
subtypes (
The specificity of the RT-PCR/RFLP protocol was demonstrated using
plasmids containing the cDNA coding for Production of tryptase by isolated bm MNCs Cultures were generated by using bm MNCs obtained from 6 patients with tryptase+ de novo AML. Cells were cultured in RPMI 1640 plus 10% FCS for up to 30 days. Production and release of tryptase in cultured cells were analyzed by FIA. Baseline levels of tryptase were detected in both cell lysates and supernatants on days 2 and 5. During a first phase (days 2-18) in culture, the cellular levels of tryptase (pg/cell) increased in all cases (1.2- to 3.1-fold) with a maximum level of 1.2 pg/cell on day 23 measured in a patient with AML-M4eo. Accompanying this increase in cellular tryptase, there was a parallel increase in released tryptase detected in supernatants (1.2- to 5.1-fold increase comparing day 5 and day 23 levels). After day 25, cellular levels of tryptase decreased, whereas, in supernatants, tryptase remained at a high level (Figure 8).
Correlation between clinical course and elevated tryptase levels In a first step, tryptase levels measured at diagnosis and after achieving hematologic CR were compared. As visible in Figure 9, induction treatment resulted in a decrease in serum tryptase levels in all cases. In the majority of patients with initially high levels, the serum tryptase concentrations were within the normal range at CR. Similarly, the whole blood tryptase levels decreased during therapy. In a smaller subset of patients, however, tryptase remained elevated despite hematologic CR. In most patients the time course of tryptase levels was analyzed. In patients who remained in CR during the observation period, tryptase levels remained 15 ng/mL (Figure 10A). By
contrast, in cases with blast cell persistence or regrowth shortly
after CR, tryptase levels remained elevated (Figure 10B). Figure 10C
shows a patient with recurrence of disease during consolidation
treatment. In this patient, tryptase levels decreased during induction
treatment but remained above normal range at CR. The regrowth of blasts was associated with a marked increase in tryptase levels. The CR rate
was similar in patients with normal serum tryptase compared with those
with enhanced enzyme levels.
Alpha- and Two major mast cell tryptase genes have been
identified and cloned, To further examine tryptase production in AML, culture experiments were performed on isolated AML blasts. In these experiments, a spontaneous increase in extracellular tryptase levels was found, suggesting that AML blasts produce and release tryptase in vitro in a constitutive manner. Furthermore, we were able to detect the tryptase protein in isolated AML cells by immunocytochemistry and immunoelectron microscopy. As assessed by electron microscopy, tryptase could be localized to the (small) cytoplasmic compartment of AML blasts. An interesting aspect was that most of the immunoreactive material was detected in smaller or larger granulelike structures. These granules did not exhibit typical morphologic features of granules detected in mast cells or basophils. In fact, neither the scrolls and grating/lattice structures seen in mast cells53,54 nor the particulate-granula structures seen in basophils53 were detected in AML blasts. All in all, no definitive morphologic or ultrastructural signs of mast cell or basophil maturation could be detected in tryptase+ AML cells. To analyze the distribution of tryptase+ cells in the bm of our AML patients, immunohistochemistry was performed. In these experiments tryptase was detectable in AML cells in bm sections of all cases with elevated serum tryptase, whereas no reactivity of leukemic cells was seen when serum levels of tryptase were normal. In all cases examined, the tryptase+ AML cells exhibited a diffuse infiltration pattern without focal dense infiltrates characteristic of mastocytosis.41 Thus, by histologic and immunohistochemical criteria, a primary mast cell disease was not present in our tryptase+ AML patients. An interesting observation was that elevated serum tryptase levels cluster in distinct FAB groups of AML. In particular, high levels of the enzyme were detected in (most) patients with AML M0, M2, M3, and AML-M4eo, whereas most patients with M4, M5, and M6 had normal tryptase levels. The highest tryptase levels were detectable in AML-M4eo. In this regard it is noteworthy that the genes coding for human tryptases cluster on the short arm of chromosome 16.27,28,30 It is also of interest that a subgroup of patients with M4eo, M2, and M3 reportedly exhibit c-kit point mutations at position 816,55-57 a defect that is otherwise found specifically in SM. Finally, M4eo blasts express the CD2 antigen,58,59 a T-cell and natural kill cell marker that is also expressed specifically on neoplastic (but not normal) mast cells in patients with SM.60 Therefore, it may be tempting to speculate that tryptase expression in AML-M4eo is indicative of minimal differentiation along the mast cell pathway. The maturation arrest, however, would not allow for terminal differentiation and maturation. To further examine lineage relationships, we also examined expression of histamine and other differentiation antigens (2D7, Kit) in our AML cases. In these experiments it was found that histamine is variably expressed in AML blasts without a significant correlation to tryptase expression. In particular, there were cases exhibiting high levels of histamine but normal tryptase levels and also cases of tryptase+ AML without significant expression of histamine. In a smaller group, however, both mediators were detectable. This finding is of particular interest because both mast cells and basophils express histamine, but only mast cells are capable of synthesizing larger amounts of tryptase.22,26 Thus, it is tempting to speculate that those AMLs with high amounts of both mediators involved a mast cell-committed progenitor and those expressing histamine in excess over tryptase involved a basophil-committed cell. It is also noteworthy that tryptase+ AMLs were sometimes labeled by 2D7 mAb that detects a basophil-related antigen.42 Another possibility would be aberrant expression of mediators and antigens. This may especially be true for cases expressing only one specific mediator (tryptase). Physiologic expression of tryptase in early development of (normal) myeloid progenitor cells seems unlikely, however. Notably, normal CD34+ bm progenitor cells were consistently tryptase negative by immunocytochemistry and flow cytometry without any evidence of subpopulations of tryptase-positive cells. The functional or biologic significance of tryptase expression in AML remains unknown. Previous observations have shown that tryptases are potent mitogens for fibroblasts and endothelial cells.61-63 Because such cells are well known to produce hemopoietic growth factors (like granulocyte-macrophage colony-stimulating factor or stem cell factor) on activation, one may speculate on a paracrine function of tryptases produced by AML blasts. Another possibility could be an effect of AML-derived tryptases on bm angiogenesis known to be up-regulated in AML.64,65 A direct effect of tryptases on proliferation of AML blasts (autocrine loop) would be a third possibility. Studies are in progress to clarify the pathophysiologic role of tryptases expressed in AML blasts. The most interesting aspect of our study was that whole blood and serum tryptase levels in AML showed a significant correlation with disease. In particular, a significant decrease in serum tryptase levels was seen during induction treatment, and at the time of CR the majority of the cases returned to normal values, whereas blast cell persistence was associated with a persistently elevated enzyme level. Moreover, in patients with hematologic CR and persistent elevation of serum tryptase, a hematologic relapse occurred in all cases. Therefore, tryptase may also be useful as a marker of disease in AML monitoring. The actual prognostic value of tryptase as an AML marker will be clarified in forthcoming studies. In summary, we show that significant amounts of tryptase are expressed in blast cells in a group of patients with AML. Tryptase appears to be a myeloid-specific marker in acute leukemias and may be useful to detect and monitor minimal residual AML especially in patients who do not have another reliable (genetic) disease-related marker.
We thank Stefanie Wessel for skillful technical assistance.
Submitted December 20, 2000; accepted May 25, 2001.
Supported by Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich (FWF); grants P-14031 and F0506 by the ICP program of the Austrian Federal Ministry for Education, Science and Culture; and by grants AI20487 and AR45441 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wolfgang R. Sperr, Department of Internal Medicine I, Division of Hematology and Hemostaseology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria; e-mail: wolfgang.r.sperr{at}univie.ac.at.
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© 2001 by The American Society of Hematology.
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  H-P Horny, K Sotlar, F Stellmacher, M Krokowski, H Agis, L B Schwartz, and P Valent The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers. J. Clin. Pathol., March 1, 2006; 59(3): 298 - 302. [Abstract] [Full Text] [PDF]
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W. R. Sperr, M. Mitterbauer, G. Mitterbauer, M. Kundi, U. Jager, K. Lechner, and P. Valent Quantitation of Minimal Residual Disease in Acute Myeloid Leukemia by Tryptase Monitoring Identifies a Group of Patients with a High Risk of Relapse Clin. Cancer Res., September 15, 2005; 11(18): 6536 - 6543. [Abstract] [Full Text] [PDF]
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 A. D. Klion, P. Noel, C. Akin, M. A. Law, D. G. Gilliland, J. Cools, D. D. Metcalfe, and T. B. Nutman Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness Blood, June 15, 2003; 101(12): 4660 - 4666. [Abstract] [Full Text] [PDF]
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 L. B. Schwartz, H.-K. Min, S. Ren, H.-Z. Xia, J. Hu, W. Zhao, G. Moxley, and Y. Fukuoka Tryptase Precursors Are Preferentially and Spontaneously Released, Whereas Mature Tryptase Is Retained by HMC-1 Cells, Mono-Mac-6 Cells, and Human Skin-Derived Mast Cells J. Immunol., June 1, 2003; 170(11): 5667 - 5673. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and supernatants (
)
were measured serially by FIA. As visible, significant levels of
tryptase were detected in these cultures. Cellular tryptase levels
(pg/cell) as well as the tryptase levels in the supernatants increased
during a first phase of culture. Thereafter, cellular levels of
tryptase decreased, whereas, in the supernatants, tryptase remained at
a high level.
procedural guide to specimen handling for the ultra-structural pathology service laboratory.
J Electron Microsc Technol.
1987;6:255-259