Intensity of expression of the CD117 antigen (c-kit) on myelomatous plasma cells

Intensity of expression of the CD117 antigen (c-kit) on myelomatous plasma cells
MARIA KRAJ, RYSZARD POGŁÓD, JOANNA KOPEĆ-SZLĘZAK, URSZULA SOKOŁOWSKA,
JOLANTA WOZNIAK, BARBARA KRUK
Institute of Haematology and Blood Transfusion, Warsaw, Poland





Correspondence: prof. dr hab. med. Maria Kraj, Department of Haematology, Institute of Haematology and Blood Transfusion, 5 Chocimska Str., 00-957 Warsaw, Poland. Phone: +48 22 849 84 32, fax: +48 22 849 84 32, e-mail: mkraj@ihit.waw.pl


Introduction
C-kit (CD117), a product of c-kit protooncogene, is a 145 kD transmembrane protein, with activity of class III receptor tyrosine kinase. Expression of c-kit can be detected by immunostaining with monoclonal antibody clustered under CD117. A ligand for c-kit is stem cell factor (SCF). Binding of c-kit receptor with its ligand leads to dimerization of receptor, tyrosine phosphorylation and activation of signal transduction cascade controlling cell proliferation, adhesion and differentiation [1, 2]. C-kit is functionally important protein involved in normal haematopoiesis, melanogenesis and gametogenesis. C-kit is expressed by a small proportion of normal human bone marrow cells (0.5-4%). Among normal bone marrow cells, CD117 expression has been found in the majority of the CD34⁺ precursors including progenitors committed to the myeloid, erythroid and megakaryocytic cell lineages. In addition, strong CD117 expression is detected in bone marrow mast cells as well as in a small subset of NK cells displaying strong reactivity for CD56, and in early T-cell precursors [3-5].
C-kit expression has been demonstrated on a wide range of non-haematopoietic cell types including: melanocytes, vascular endothelial cells, interstitial cells of Cajal, astrocytes, breast glandular epithelial cells as well as in testis, renal tubules and sweat glands [6].
Abnormal activity of c-kit and altered signal transduction coming from this receptor may play a substantial role in oncogenesis process. Increased expression of CD117 was found in some myelo- and lymphoproliferative diseases such as acute myeloblastic leukemia [7, 8], myelodysplastic syndromes, anaplastic lymphomas [9, 10] as well as in some solid tumours including small cell lung carcinoma, glioma, neuroblastoma, germinoma, gastrointestinal stromal tumours (GISTs), some types of breast cancer and melanoma [11-14]. Altered c-kit expression may result from c-kit gene point mutation in enzymatic or regulatory portion of c-kit protein that occurs in early steps of oncogenesis. Such mutation maintains cell stimulation status independently from the receptor ligand suggesting autocrine pathway of activation of the receptor cytoplasmic part [15]. However, in tumours in which no mutations of c-kit gene were found an increase c-kit expression may be a result of receptor stimulation by its ligand. In this context, multiple myeloma is an interesting model. Multiple myeloma is a B-lineage neoplasm characterized by the accumulation in the bone marrow of a clonal population of B-cells in the last stage of differentiation-plasma cells. A special role of SCF (c-kit ligand) in myeloma oncogenesis was revealed by Lemoli et al. [16, 17] who showed the stimulatory effect of SCF on proliferation of fresh myeloma bone marrow cells and some myeloma cell lines with expression of CD117. It was clearly demonstrated that SCF is able to stimulate proliferation of myeloma cell lines in the cultures both with and without presence of cytokines [16-18]. mRNA transcripts for SCF and its receptor, c-kit, have been detected by reverse transcriptase-polymerase chain reaction amplification in myeloma cell lines RPMI 8226, MT3, JJN3, U266B1, NCI-H929, ARH77 and HS-Sultan [17, 19, 20]. It suggests that c-kit may play a special role in myelomatous plasma cells by influencing their proliferation. We have previously shown the expression of CD117 on plasma cells of some multiple myeloma patients [21, 22].
The recent encouraging therapeutic experience with tyrosine kinase inhibitors in chronic myeloid leukemia and c-kit⁺ GISTs [23, 24] prompted interest in application of these drugs in the treatment of other tumours with overexpression of c-kit. It implies, that in selected multiple myeloma patients in whom expression of c-kit on plasma cells is unquestionable increased a targeted therapy with tyrosine kinase inhibitors may be a new, promising treatment option [25]. The aim of the present study was an analysis of the frequency and intensity of c-kit expression on bone marrow myelomatous plasma cells. The results has already been presented at several international hematology meetings and XI Polish Society for Immunology Congress [26, 27].
Material and methods
Patients and controls
The study included 109 newly diagnosed and untreated multiple myeloma patients, hospitalised in the Department of Haematology of the Institute of Haematology and Blood Transfusion in Warsaw.
In each patient the following parameters were estimated at the time of diagnosis: age, sex, complete blood examination, percentage of plasma cells in the bone marrow (at collection of bone marrow for FACS analysis there were performed regular bone marrow smears stained with May-Grünwald-Giemsa method), monoclonal protein isotype (using Beckman Paragon Immunofixation Electrophoresis Kit), urine immunoglobulin/24 hours, serum concentrations of monoclonal protein, calcium, creatinine and b2-microglobulin (using Beckman ARRAY 360 autoanalyser), complete X-ray skeletal survey, stage of disease according to Durie and Salmon classification [28]. Characteristics of analysed patients are presented in the table 1. The patient’s informed consent was obtained prior to performing bone marrow aspiration or biopsy.

The control group consisted of 10 healthy volunteers.
Immunophenotypical analysis of CD117 expression was done by means of flow cytometry using monoclonal antibodies.
Sample preparation and immunofluorescence
Immunophenotyping was performed on fresh bone marrow samples taken at diagnosis. The volume of 2 ml of bone marrow aspirate was collected into the tubes with heparine concentration – 20 U/ml was diluted with an equal volume of phosphate buffered saline (PBS) and centrifugated at 1300 r.p.m. Supernatant was removed and sample was washed with warm PBS, stirred for 2 min and diluted with aliquot of warm PBS.
Cell surface antigens were evaluated by means of direct immunofluorescence. Rinsed bone marrow samples were inserted in disposal glass tubes and stained with simultaneous triple labeling by using mouse anti-human monoclonal antibodies conjugated with respective fluorochroms: fluorescein isothiocyanate (FITC), phycoerythrin (PE) and phycoerythrin/Cyanin 5 (PE-Cy5). In the sudy monoclonal antibodies against following antigens were used: anti-CD117 (PE) obtained from Becton-Dickinson (USA), anti-CD38 (PE-Cy5) from DAKO (Denmark), anti-CD138 (FITC) obtained from Serotec (UK). Isotype specific FITC, PE-Cy5 and PE-conjugated control antibodies were used as controls.
After addition 10 ml of antibody the sample was incubated at 4oC for 30 min. in the dark. In order to remove red cells after incubation with antibodies a special red cell lysis reagent was added and the tube was incubated in the dark for 10 min at a room temperature. Then the sample was centrifugated at 1300 r.p.m. for 5 min and supernantant was removed. The volume of 2.5 ml of cold PBS was added into each tube, the sample was stirred and centrifugated. The supernatant of PBS was removed from the tube and finally sample was resuspended in 0.5 ml of cold PBS, ready for analysis.
Flow cytometry analysis
Flow cytometry analysis was performed immediately after staining by using flow cytometer Cytoron Absolute ORTHO and FACSCalibur Becton-Dickinson. The data acquisition was done by using softwares: Immunocount II for Cytoron Absolute and CELLQuest for FACSCalibur cytometers. At each time 10000 cells were analysed. At first, the gate according to linear forward angle scatter (FSC) and linear scatter (SSC) was set. On CD38/SSC dot plots the gate around cells with strong expression of CD38 (cells CD38⁺⁺) was drawn. The subject of analysis were plasma cells identified on the basis of strong CD38 expression, expression of CD138 and their typical light scatter distribution. The CD117 antigen expression was analysed on CD38⁺⁺ and CD138⁺ cells. The percentage of positive events in plasma cell subset was calculated and compared to the isotype control The results were presented as a percentage of cells with expression of CD117. The plasma cells were determined as CD117⁺ if at least 20% of cells were labeled with anti-CD117 antibody.
Determination of antigen intensity
The CD117 antigen expression intensity was evaluated using two different and independent methods: indirect and direct. The CD117 expression intensity was quantified by measuring mean values of PE fluorescence.
In an indirect quantitative analysis of antigen expression intensity the histograms of CD117 fluorescence and isotype control were plotted, mean fluorescence intensity defined by mean channel peak of fluorescence was determined and Relative Fluorescence Intensities (RFI) were calculated in arbitrary units (AU) according to the equation described by Miwa et al [29, 30]. This formula is based on the values of mean channel of fluorescence of analyzed antigen and that of isotypic control: RFI=10x3.5 mean channel of the sample/number of channels – 10x3.5 mean channel of the control/number of channels.
Fluorescent beads (DAKO fluoresphers) were used for standardization.
For a direct quantitative analysis the QuantiBRITE test (Becton Dickinson) was applied [31-33]. Briefly, the analysis Kit (Phycoerythrin Fluorescence Quantitation Kit) contains tubes with lyophilized pellet of beads coated with phycoerythrin in 4 known concentrations. These enable determination of quantitative bounding phycoerythrin linked antibody to a particular cell defined as Antibodies Bound per Cell (ABC). At first, Quantibrite PE tubes were run and 10,000 events was collected. The gate was set around bead singlets and bead bet singlets population was analyzed using a histogram plot of fluorescence (FL2) in linear values; four bead peak concentration of PE fluorescence: low, medLow, medHigh, and high were displayed. Using the same instrument settings for fluorescence and compensation there was made acquisition of cellular assay samples. When running Quantibrite PE at the same instrument settings as the assay the axis showing fluorescence (FL2) can be converted into the number of PE molecules bound per cell. Using known ratios of PE to antibodies one can convert PE molecules per cell to antibodies per cell.
The mean channels of PE fluorescence were defined and antibodies bound per cell were calculated using QuantiCALC software. In order to determine the number of PE molecules adhering to the cell the calibration curves were plotted on the basis of read values of mean fluorescence channel for 4 afore-mentioned various phycoerythrin densities using Quantitative Calibration Software. Mean channel of PE fluorescence for CD117 was determined and the values of ABC were read from calibration curve using QuantiCALC software.
All parameters of cytometer and determination of ABC and RFI were identical for both methods.
Statistical analysis
The different clinical and biological disease characteristics were considered individually, depending on their relationship with the CD117 expression by Mann-Whitney test. For correlation analysis Spearman’s correlation coefficient (r) was calculated.
Results
In bone marrow of healthy persons mean proportion of CD117⁺ cells was 2.4±0.7%, median 1.8%. Normal plasma cells from healthy persons were negative for the CD117 antigen. In 37 patients (=34%) myelomatous plasma cells showed CD117 expression, in 72 (66%) patients did not (Fig. 1, Table 2).

In myeloma CD117 positive out of all nucleated bone marrow cells the mean proportion of plasma cells with CD117 expression was 25.7±20.3%, median 21%. In myeloma CD117 negative the mean proportion of bone marrow CD117 positive cells was 3.0±2.0%; median 2.7% (Table 3).

The results of morphological and immunophenotypic analysis of bone marrow plasma cells in c-kit positive and c-kit negative cases were presented in table 4. The mean proportion of bone marrow plasma cells in patients with
c-kit positive plasma cell proliferation was 35.8±24.1%, median 35% and in patients with c-kit negative plasma cell proliferation was 41.1±23.2%, median 38%.
In patients with CD117 positive myeloma a correlation was found between proportion of CD117⁺ cells in FACS bone marrow analysis and percentage of plasma cells in bone marrow smears stained with May-Grünwald-Giemsa (r=+0.95).
In myeloma patients with CD117 positive plasma cell proliferation the majority of CD138⁺ and CD38⁺⁺ bone marrow cells coexpressed CD117 antigen independently on the bone marrow tumour infiltration (Fig. 2, 3, 4), but in some CD117⁺ myelomas expression of this antigen was weak but exceeding accepted in this paper threshold of positivity (20%) (Fig. 5).

The results of the analysis of CD117 antigen expression intensity on plasma cells of 11 multiple myeloma patients were presented in the Table 5. Determinations were done by using the method enabling measurement of the number of antigen molecules on the cell defined as Antibodies Bound per Cell (ABC) and the method of calculation of Relative Fluorescence Intensity (RFI).
CD117 expression intensity estimated by using both indices (RFI and ABC) showed heterogeneity. The RFI values ranged from 8,4 to 20 (mean: 12.4±3.2; median 11.6) while the ABC values from 1443 to 6217 (mean: 3262±1410; median 3109). Low ABC values corresponded to low values of RFI determinations and high values of ABC – to high RFI indices. A correlation coefficient between the CD117 expression intensity values determined by using of both indices was high (r=0.87) (Fig. 6, 7).

Table 6 presents comparison of clinical-laboratory characteristics parameters in both myeloma patients’ groups: c-kit positives and c-kit negatives. In the group of c-kit⁺ positive patients there was found more elderly persons compared to the c-kit- negative group (age medians 66 and 60 years, respectively). In the c-kit⁺ group compared to that of c-kit– there was revealed also a higher proportion of patients with less advanced disease (a percentage of patients at disease stage III in the group c-kit⁺ was 57% while in the c-kit– amounted 81%) and lower proportion of patients with the presence of lambda light chain (17% vs 47%). The differences, however, were not significant statistically.
Discussion
To the most important findings revealed in this study belongs the demonstration of the lack of c-kit expression on normal plasma cells and presence of this antigen expression exclusively on myeloma plasma cells in one third of patients. Importance of performed analysis consists also in applying two independent methods (ABC and RFI) to determine antigen expression intensity. One of them, ABC, is a relatively new method that makes possible a direct determination of the number of analysed antigen molecules (antigen density).
Our study on antigen expression intensity showed the heterogeneity of c-kit expression intensity on myeloma plasma cells. The presence of this phenomenon was revealed by using both methods. It reflects the existence of actual heterogeneity of c-kit expression intensity in multiple myeloma and confirms comparability and equivalence of two applied determination methods.
The studies on establishing CD117 expression pattern in multiple myeloma are not numerous and a main research is being done by Spanish authors. The results of those studies [34, 35] in a part devoted specificity of c-kit expression for multiple myeloma and frequency of occurrence of this phenomenon in myeloma patients are concordant with the results of our analysis. However, afore-mentioned authors did not deal largely with the problem of CD117 expression intensity and its heterogeneity but rather focused their attention on the question of plasma cell autofluorescence and its effect on antigen expression [35]. But they did demonstrate that immunophenotyping CD117 makes possible the detection of expression of this antigen at very high cell dilution. They also proved that the sensitivity of immunophenotyping CD117 used as a myeloma marker is similar to the sensitivity of polymerase chain reaction (PCR) in detection of small amount of tumour cells. In their opinion, immunophenotyping compared to PCR analysis is a method less time consuming and enables performance of quantitative estimation of tumour cells. It opens new perspectives for diagnosing minimal residual disease in multiple myeloma.
On the other hand, the association of tyrosine kinase properties of c-kit molecule with its specific expression on myeloma cells creates unusually important clinical implication – a possibility of therapeutical application of that tyrosinase inhibitors in the treatment targeted to deletion of specific pathogenetic disturbance on the molecular level (molecular targeting).
Altered activity of kinases, especially tyrosine kinases, in multiple myeloma raises a special interest and hopes also from the clinical point of view. The results both of experimental studies and first clinical trials revealed antiproliferative activity of tyrosine kinase inhibitors in some solid tumours including kidney, lung and gastrointestinal stromal tumours [12-14, 24]. Moreover, a tyrosine kinase inhibitor – imatinib (STI 571) showed to be an efficient drug in the treatment of chronic myeloid leukemia, even in definitely prognostically poor phases of this disease [23, 36].
In the experiments with myeloma cell cultures there were successfully applied inhibitors of some Janus kinases (JAK2) that block signal transduction at the stage of originating STAT (Signal Transducer and Activator of Transcription) proteins [37, 38]. Tyrphostin, an inhibitor of JAK2 tyrosine kinase, diminishes the activity of MAPK (Mitogen Activated Protein Kinase) and transcription factors (STAT) activated by phosphorylation leading to stimulation of myeloma cell apoptosis. This is a signal transduction pathway dependent on interleukin-6 and activation of gp 130 – a signal transduction protein. Intervention in this signal transduction pathway may lead to overcoming myeloma cell resistance to dexamethasone and radiotherapy [39, 40].
The question of application of a derivative of phenylaminopyrimidine, STI 571 (imatinib), a tyrosine kinase inhibitor showing its efficacy in the treatment of chronic myeloid leukemia, for the therapy of multiple myeloma remains open.
As it is known, the therapeutic effect of STI 571 in chronic myeloid leukemia results from inhibiting bcr-abl kinase activity. The rationale for application of STI 571 in the treatment of multiple myeloma is based on belonging of c-kit to the same receptor kinase family as bcr-abl, c-abl and platelet derived growth factor receptor (PDGFR), a close receptor homology between PDGFR and c-kit domains as well as a broad enough spectrum of tyrosine kinase inhibitors [36, 41, 42]. Moreover, there was unequivocally demonstrated that STI 571, apart from inhibiting afore- mentioned three tyrosine kinases, inhibits also the activity of c-kit [36, 41]. STI 571 acts as competitive ATP inhibitor and binding of this inhibitor with ATP leads to kinase blocking [43]. Pandiella et al. [44] have evaluated the action of STI 571 (imatinib mesylate) on different MM cell lines. Addition of STI 571 to cultures of MM1S, MM 144, RPMI 8226, and U266 cell lines caused a decrease in the proliferation of these cells by arresting cell cycle progression. STI 571 inhibited the proliferation of MM cells resistant to dexamethasone or melphalan and had an additive effect when combined with dexamethasone. A phase II trial of GleevecTM (=imatinib) in patients with refractory/relapsed myeloma is going [25].
Studies on the expression of c-kit and its relationship with SCF lead to two important clinical implications. The first is the necessity of determination of this antigen expression individually in each patient prior to the intended treatment with tyrosine kinase inhibitor due to the prevalence of c-kit negative myeloma proliferations. The second implication is the renunciation of SCF administration for cell mobilisation in multiple myeloma because of stimulating properties of this growth factor on myeloma cell proliferation.
Conclusions
In one third of multiple myeloma patients CD117 antigen could be considered as a „tumour associated marker” and it may be of value for the identification of the malignant clone in minimal residual disease as it was first shown by Spanish authors [34]. The intensity of c-kit expression on plasma cells varies among particular c-kit positive multiple myeloma patients and the differences in expression level may be as big as many times. It may be rationale to consider usefulness of therapy with tyrosine kinase inhibitors in the management of myeloma patients with c-kit positive plasma cell proliferation.
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