In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies. Bone Marrow Transplantation (2000) 26, 525–531.
Hematopoietic reconstitution after myeloablative chemotherapy and stem cell transplantation depends upon the quality and quantity of the re-infused stem cells, the microenvironment in the bone marrow, and the different growth factors affecting homing, proliferation and differentiation of the ‘new’ stem cells in a sequential manner.1 Stem cell factor (SCF) and Flt3 ligand (Flt3) have been shown to be of major importance for the proliferation and differentiation of early stem cells, and for the expansion of the progenitor cell compartment, acting in synergy with later acting growth factors. The early acting growth factors are intimately related to the stromal microenvironment. SCF exerts its action in small niches of proliferating, self-renewing stem cells between stromal fibroblasts and endothelial cells, excreting growth factors.2 Like SCF, Flt3 stimulates proliferation and differentiation of stem and progenitor cells in synergy with other growth factors.3 FA1 is the soluble product of delta-like (dlk) – a mRNA encoding a transmembrane protein that contains six epidermal growth factor (EGF)-like domains. FA1 includes all six EGF domains but lacks the juxtamembrane, transmembrane and cytoplasmic regions of dlk.4 Recently, in vitro experiments have demonstrated that FA1 can act as a hematopoietic regulator by promoting cobblestone area colony formation in stromal monolayers. The resulting stem cells are transplantable and repopulate in animal models.5 Erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (Tpo) as well as the corresponding receptor positive progenitors are considered essential for development of the lineage-specific progenitors.6789 Recently, Tpo has been demonstrated to be of importance in the early development of the stem cell.10
The successful outcome of high-dose therapy depends upon early engraftment and blood cell recovery, since morbidity and mortality are closely related to the duration of neutropenia and thrombocytopenia.11 Prediction of patients at risk of late hematopoietic recovery would make it possible to target growth factor treatment in a more rational manner.
Concentrations of growth factors in the blood are influenced by the interaction between the growth factors and their target cells via the specific receptor binding.1 Production, distribution and elimination of the growth factor and proliferation, maturation and apoptosis of the target cells affect the blood concentration in different ways.121314 In this context, information about concentrations of growth factors in peripheral blood alone might be more confusing than informative. Consequently, we have analyzed growth factor concentrations during the course of autologous stem cell transplantation as well as the autograft content of growth factor receptor positive CD34+ progenitor cells, aiming to identify new important factors for early blood cell recovery. The results obtained from this pilot study are meant to provide a basis for larger, prospective studies.
Materials and methods
Patients and treatment
Thirty-two patients were included in the study, 16 with intermediate/high-grade non-Hodgkin's lymphoma and 16 with multiple myeloma. The patients were selected on the basis of diagnosis and a full set of available blood samples before and during high-dose chemotherapy followed by autologous transplantation at the Department of Hematology, Herlev Hospital during the years 1993–1998. The two disease groups were comparable with respect to age, gender and courses of prior chemotherapy and/or radiation therapy (Table 1). All patients exhibited normal kidney and liver function at the time of transplantation.
The conditioning regimen before transplantation was BEAM for the NHL patients (BCNU 300 mg/m2 day −7, etoposide 200 mg/m2 and ara-C 100 mg/m2 twice daily for 4 days (day −6 to day −3) melphalan 140 mg/m2 once on day −2). Patients with multiple myeloma were treated with high-dose melphalan (200 mg/m2) on day −3. Stem cells were collected earlier by leukapheresis after priming with high-dose cyclophosphamide (4 g/m2) and G-CSF (filgrastim, 5 μg/kg bodyweight) from day 8 after cyclophosphamide and until leukapheresis had been completed. Leukapheresis was carried out using a CS 3000 Plus cell separator (Baxter, Deerfield, IL, USA) as previously described.15 The product was evaluated by enumeration of CD34+ cells according to the Nordic Protocol.16 The stem cells were re-infused on day 0. Four patients with NHL and eight patients with MM received exogenous, recombinant human G-CSF at some time during the course of transplantation after clinical judgment or according to an ongoing Nordic Myeloma Protocol. These patients were analyzed separately for endogenous G-CSF and neutrophil behavior.
All patient-related data, including the peripheral blood cell counts, were retrospectively obtained from the patient records. In the case of reticulocyte counts, the relative rather than absolute number of reticulocytes was used because red cell numbers were unavailable.
Sample preparations and analyses
Blood samples were obtained at least once a week for all patients from 1 to 2 weeks before and up until 4 weeks after transplantation (day 0). The samples were immediately centrifuged for 10 min at 3000 g. Plasma and/or serum was separated and stored at −20°C. Plasma from eight healthy donors, treated the same way, served as a control. In the case of FA1, the reference range had previously been established from the sera of 75 healthy adults.17 After thawing, plasma or serum (or both) were analyzed for growth factor content by the ELISA technique, using sensitive and specific, commercially available kits (R&D Systems, Minneapolis, MN, USA). In the case of FA1, the concentrations were analyzed by a newly developed ELISA technique as described.17 The sensitivity of this assay is <0.4 ng/ml. According to the manufacturer's information, the sensitivity of the commercial kits is for EPO <0.6 mIU/ml, for G-CSF 7 pg/ml, for Tpo <15 pg/ml, for SCF <4 pg/ml and for Flt3 <7 pg/ml. Serial dilutions were used if concentrations were higher than the maximum detection level. All measurements were performed in duplicate and the mean used for comparisons.
Samples of mononuclear cells, separated by density centrifugation from the leukaphereses, were re-suspended in a medium containing 95% fetal calf serum and 5% DMSO under sterile conditions, frozen and kept at −80°C. After thawing, the samples were separately analyzed for receptor concentrations in eight NHL patients and seven MM patients (eight MM patients were analyzed for SCF and G-CSF receptors) by using fluorescence-marked growth factors and monoclonal anti-CD34 antibodies, and subsequent detection by flow cytometry.
Flow cytometry for progenitor subsets
The growth factor receptor positivity for Tpo, SCF and Flt3 receptors was detected in the leukapheresis product by double-staining the cells with a phycoerythrin (PE)-conjugated monoclonal antibody against CD34 (Anti-HPCA-2 PE, Becton Dickinson, San Jose, CA, USA, 10 μg/ml in 50 μl) and biotinylated growth factors and fluorescein isothiocyanate (FITC)-conjugated avidin. In the case of the G-CSF receptor, FITC-conjugated anti-CD34 was used (anti-HPCA-2 FITC, Becton Dickinson, 10 μg/ml in 50 μl) together with directly PE-conjugated G-CSF. All the growth factor receptor concentrations were analyzed using modified flow cytometry kits (Fluorokine from R&D Systems). Approximately 1 × 106 cells in 50 μl were incubated with the biotinylated or PE-conjugated growth factor for 60 min at 4°C. The second step, with no prior wash, was incubation with avidin-FITC (10 μg/ml in 50 μl) and the CD34 antibody for 30 min at 4°C in the dark. Since we analyzed receptor positivity on a minor fraction of frozen mononuclear cells, we used more cells and more conjugated growth factor than recommended by the manufacturer (50 μl of each: SCF 1 μg/ml, Flt3 and Tpo 5 μg/ml and G-CSF 7.5 μg/ml). Accordingly, this was one of the reasons for choosing the biotin–avidin system. Another reason was that Tpo was unavailable in a conjugated form for flow cytometry. As a negative staining control for the biotin-conjugated growth factors, it was important to use an irrelevant molecule, biotinylated like the growth factors. A soybean trypsin inhibitor that was biotinylated to the same degree as the growth factors (5 μg/ml in 50 μl) was included as suggested by the manufacturer. For the G-CSF receptor, we used IgG1 PE (Becton Dickinson), CD34 FITC as a negative control. In all cases, the fractions of marked cells were subsequently analyzed in a CD34+ acquisition gate, using a FACScan Flow Cytometer equipped with an argon laser (Becton Dickinson). First, a wide, rectangular live gate was set on the isotype control (IgG1 FITC, IgG1 PE from Becton Dickinson, each 5 μg/ml in 50 μl) in a forward (FSC) − side light scatter (SSC) plot, to exclude cellular debris. After acquisition of 50 000 events from an IgG1/CD34 sample, a rectangular CD34-positive acquisition gate was set in a fluorescence against SSC plot. Most samples were also run without acquisition gate to enable establishment of the best negative control afterwards. Most of the leukapheresis products were analyzed with propidium iodide (PI; Sigma, St Louis, MO, USA) also, in order to estimate the number of viable cells (PI in PBS was used at a concentration of 0.5 mg/ml).
The leukapheresis products were pre-treated in the following manner: after careful thawing, the product was suspended in cold PBS and remaining erythrocytes were lysed in Ortho-mune Lysing Reagent (Ortho Diagnostic Systems, Raritan, NJ, USA). The samples were washed twice (centrifugation by 1500 r.p.m. at 4°C for 5 min) and re-suspended in a phosphate-buffered saline solution containing natriumazide 0.5 g/l (FACS-PBS). Finally, the samples were filtered, and the cells counted on a Coulter MDII (Coulter, Miami, FL, USA), centrifuged again and incubated with growth factors and/or antibodies after re-suspension in 0.25–0.5 ml FACS-PBS.
Hematopoietic recovery, definitions
The endpoints we used for analyses of erythropoiesis were: the number of days before the relative reticulocyte count exceeded 0.2%, the exact number of days with reticulocyte counts below or equal to 0.2%, and the number of days with a hemoglobin concentration less than or equal to 10.5 g/dl. The number of red blood cell transfusions was also recorded.
Analyses of neutropenia were made with the following endpoints: the number of days before the absolute neutrophil count (ANC) exceeded 0.5 × 109/l, the absolute number of days with ANC below or equal to this figure, and the absolute number of days with ANC less than or equal to 0.2 × 109/l. Thrombocytopenia was analyzed with the following endpoints: the number of days before the platelet count exceeded 20 × 109/l, the absolute number of days below or equal to this figure, and the number of days with platelet counts less than or equal to 10 × 109/l.
Patient characteristics were analyzed by Fisher's exact test and groups compared by non-parametric methods (Mann–Whitney U test) with median values as the best representation. All relationships between engraftment and growth factor/growth factor receptor concentrations were analyzed by survival statistics with comparison of Kaplan–Meier curves for different groups by the logrank test. Data from two patients who died before engraftment were censored. In all tests a P value <0.05 was considered significant. All statistical analyses were carried out using the software StatView 4.0 (Abacus Concepts, Berkeley, CA, USA).
Growth factor fluctuations and hematopoietic recovery
All patients reached a nadir of peripheral blood values at day +7 with reticulocyte counts close to 0%, absolute neutrophil counts of 0.0 to 0.1 × 109/l and platelets below 20 × 109/l. Two patients died in the nadir period; one patient with a highly malignant, anaplastic NHL in second PR on day 15, and one patient with progressive MM on day 37.
No significant differences were found between growth factor concentrations in the two disease groups during or after transplantation, except on day 0. This difference was most likely caused by a difference in peripheral blood cell decline due to different conditioning. For the early acting growth factors SCF, FA1 and Flt3, the pre-transplantation level was also higher in the patients with MM. No differences were found between the disease groups in the receptor analyses. In the following post-transplant analyses, data from all patients are pooled.
An inverse relation was observed between the peripheral blood cell counts and the concentrations of progenitor-specific growth factors (Figure 1). The median growth factor concentrations for patients and controls are shown in Table 2. The duration of nadir was analyzed by the method of Kaplan and Meier. The patients were divided in two groups: those with peak growth factor concentrations (day +7) above the median value and those with concentrations below or equal to the median value.
Regarding erythroid engraftment, we found no differences in duration of erythroid nadir between patients with high or low median peak values of erythropoietin, no matter how the endpoint was defined. For neutrophil engraftment, we compared those who had absolute neutrophil counts of ⩽0.5 × 109/l with patients who had achieved neutrophil counts above this value on day +14. G-CSF was significantly different in these two groups, with median concentrations day +7 at 885 and 3680 pg/ml (P = 0.025), respectively, the group with high G-CSF concentrations being more likely to experience fast engraftment. The survival analyses were, however, unable to demonstrate significant differences in neutropenia for G-CSF high/low level patients.
As regards the recovery of thrombopoiesis, patients with platelet counts ⩽20 × 109/l were compared to patients who had achieved platelets above this level on day +14. Median Tpo concentrations day +7 were highest in the group with late engraftment (platelets ⩽20 × 109/l), but the difference was not significant (median 2000 vs 1587, P = 0.17). This relationship was evident and significant in the survival analysis as well, no matter how the period of nadir was defined (Figure 2).
None of the early-acting growth factors SCF, Flt3 or FA1 emerged as significantly important factors for erythroid, neutrophil or platelet recovery in the survival analyses.
Receptor positive CD34+ subsets and hematopoietic recovery
After subtraction of background staining, SCF receptors were determined on 0 to 28%, Flt3 receptors on 0 to 22%, Tpo receptors (c-mpl) on 0 to 18% and G-CSF receptors on 3 to 44% of the CD34+ cells (Figure 3).
Survival statistics on receptor positivity and engraftment identified Tpo and Flt3 receptor positivity as having a significant impact on the duration of thrombocytopenia, for any chosen endpoint of platelet nadir (Figure 4). The Tpo receptor positivity was high in all patients with fast recovery and low in those with slow recovery, regardless of the reinfused number of CD34+ cells (Table 3).
In six patients treated with recombinant human G-CSF, G-CSF receptor positivity correlated by simple regression analysis to the number of days with deep neutropenia (r = 0.90, P = 0.02 for days with ANC ⩽0.2 × 109/l), i.e. patients with many receptor positive CD34+ progenitors had a less profound neutropenia than patients with a low G-CSF receptor positivity when treated with rhG-CSF. No other receptor analysis correlated with hematopoietic recovery following transplantation.
Autologous stem cell transplantation preceded by high-dose chemotherapy exerts the maximal stimulus for hematopoietic recovery. We know that the reconstitution of hematopoiesis requires a precise and complex coordination of growth factors. When this process takes place after stem cell transplantation, recovery may correlate with the growth factor and growth factor receptor status in the patient. The problem is that the interactive nature of this process makes it difficult to determine the relevant parameters to analyze. It would, however, be of obvious benefit to know the patients at risk of late engraftment at an early stage and perhaps stratify the administration of recombinant growth factors to these patients.
In the present pilot study, we have examined growth factor concentrations and receptor positivity in the re-infused stem cells and related these concentrations to the depth and duration of hematological nadir, aiming to improve prediction of the risk of late recovery. It seems possible to define a group of patients at risk of prolonged thrombocytopenia before the transplantation by flow cytometric detection of growth factor receptor representation in the stem cell product, analyzing the megakaryocytic CD34+/c-mpl+ and/or CD34+/Flt3+ progenitors, corresponding to what has been demonstrated for CD34+/CD61+ progenitors.1819 The prediction may be confirmed by analyzing the concentration of thrombopoietin in blood day +7 after re-infusion of the stem cells. A high Tpo concentration and a low relative number of Tpo receptors and Flt3 receptors in the autograft correlated in our study with a slow recovery of platelets. The inverse relation between Tpo concentration and Tpo receptor positive, CD34+ progenitor cells is probably caused by elimination of Tpo from the blood by binding to the target cells.20
The present study is not large enough to determine whether receptor analysis provides a more precise prediction than traditional CD34+ enumeration, but it does provide a basis for evaluation of new parameters in quality assessment of autografts in a larger context.
We did not find any convincing predictive parameters for recovery of erythropoiesis, being restricted by the retrospective and sometimes insufficient nature of our data. Obviously, red blood cell transfusions might disturb the picture as well. It is possible that a well-designed prospective study with flow cytometric quantification of median reticulocyte reticulation will be more successful in answering this question.21
Our results suggest a relationship between a high G-CSF concentration and fast recovery of neutrophils. Several studies have been performed concerning this matter and most studies find some relationship of G-CSF concentrations with engraftment.612 Only a few studies have been unable to demonstrate this relationship, and they included patients with predominantly myeloid diseases, mostly CML, undergoing allogeneic transplantation.2223 Apart from different study set-ups, including different statistical methods, it could very well be that the endogenous G-CSF response is dependent on more variables, like for example severe infections, than is Tpo. An interesting finding was that the number of G-CSF receptor positive CD34+ cells in the autograft seemed to be important for the outcome of recombinant human G-CSF treatment.
If these observations are confirmed they will provide predictive information on the outcome of treatment with recombinant growth factors in the peripheral blood stem cell transplantation setting.
There are several parameters that we have not been able to take into consideration. Among them are the density of the growth factor receptors on the stem cell and the affinity of the ligand binding to the receptor. These factors are not very well described and might be of equal importance.
Technical problems related to the method of flow cytometric quantification of growth factor receptors are far from solved. First, analyses on frozen material are difficult, since the behavior of the cells is clearly different from that of ‘fresh’ cells. Defining relevant negative controls is also a matter of great importance and controversy. In our study, we used the biotin–avidin system and with the amount of unspecific staining in this system, we chose to use an irrelevant protein marked the same way rather than ‘cold’ growth factor. Which procedure is more correct is difficult to determine and may, to a large extent, depend on the nature of the study material.
The present study adds to the accumulating evidence supporting the theory that measurements of endogenous growth factor and growth factor receptor profiles in the course of autologous transplantation in hematological patients may provide significant information on engraftment in the individual patient.
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This work was supported by grants from Dagmar Marshall's Foundation, Anders Hasselbalch's Foundation for Defeating Leukemia and Director Jacob Madsen and Wife Olga Madsen's Foundation.
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Schiødt, I., Jensen, C., Kjærsgaard, E. et al. Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery. Bone Marrow Transplant 26, 525–531 (2000). https://doi.org/10.1038/sj.bmt.1702554
- autologous stem cell transplantation
- cytokine receptors
- flow cytometry
Supplementation of Conventional Freezing Medium with a Combination of Catalase and Trehalose Results in Better Protection of Surface Molecules and Functionality of Hematopoietic Cells
Journal of Hematotherapy & Stem Cell Research (2003)