Since the first bone marrow transplantation went into clinical practice in 1968, the application of stem cell transplantations were widely applied in the treatment of haematological malignancies for the last four decades. Autologous stem cell rescue after myeloablative therapy is a preferable option in disease treatment of hematological malignancies including multiple myeloma (MM), Non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL) (Pusic et al., 2008). Mobilized peripheral blood stem cell is replacing bone marrow as a major source of stem cell in autologous transplantation in the last two decades due to its rapid hematologic reconstitution and avoidance of general anaesthesia in bone marrow aspiration (Leung and Kwong, 2010). Furthermore, mobilized progenitor cells are less tumour cell contamination compared to bone marrow (Alegre et al., 1997).
An infusion of significant number of stem cells is paramount to restore haematopoiesis in autologous stem cell transplantation. Ideal dose for rapid haematopoietic recovery is 4 - 6 x 106 CD34+ cells /kg recipient body weight and 2 x 106 CD34+ cells/kg is defined as minimum threshold for successful engraftment. Any infused dose below this level will classify as failure and will result in risk of delayed or non-engraftment after myeloablative chemotherapy (Leung and Kwong, 2010). However, as stem cells normally reside in bone marrow, thus only a small number of progenitor cells are found in the blood circulation. Stem cells were previously separated by blood separator, these circulating stem cells after transfusing enabled to restore haematopoiesis after myelosuppressive chemotherapy (McCredie et al., 1971). These findings postulated a new era of using peripheral blood stem cells in replacing bone marrow as a major stem cell source in autologous and allogeneic transplantation. However, multiple leukapheresis from non-mobilized steady-state peripheral blood is not practical in order to collect sufficient stem cells. Therefore, mobilization regimen is required to mobilize progenitor cells from bone marrow into peripheral blood that can be collected during leukapheresis procedure. Richman et al. (1976) demonstrated that a combination of adriamycin and cyclophosphamide increased approximately 20-folded above the baseline of circulating stem cells. These findings suggested that haemopoietic stem cells could be mobilized by chemotherapy. Later studied by Socinski et al. (1988) and Duhrsen et al. (1988) showed that circulating colony-forming unit granulocyte macrophages (CFU-GM) and progenitor cells increased remarkably by administration of granulocyte-macrophage-colony stimulating factor (GM-CSF) and granulocytes-colony stimulating factor (G-CSF). This formed the foundation of using hemopoietic growth factors (HGFs) in autologous stem cell transplantation. Although, chemotherapy was the first mobilizing agent for clinical use, hemopoietic growth factor (HGFs) is also being used alone or in combination with chemotherapy.
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Different mobilization regimen
Cyclophosphamide (CY) is the most commonly chemotherapeutic agent in chemotherapy. Lie et al. (1996) found that yield of CFU-GM as well as CD34+ yield increased significantly by adjusting CY dose from 4g/m2 to 7g/m2. Using chemothrapy in mobilization regimen enjoyed benefit of tumour cytoreduction of mobilized tumour cells. However, high dose CY as mobilizing agent also resulted in higher marrow toxicity. Morbidity and hemorrhagic cystitis were also reported which was associated with high dose CY. Besides marrow toxicities, apheresis session was difficut to schdule because recovery from cytopenic phase after chemotherapy was unpredictable. Hence, it is less common to use chemotherapy alone nowadays.
Hemopoietic growth factors (HGFs)
Another mobilization regime is by using hemopoietic growth factors (HGFs) alone. Granulocyte-colony stimulating factor (G-CSF, Filgrastim) and granulocyte-macrophages colony stimulating factor (GM-CSF, Sargarmostim) are the two commonly use mobilizing agents. Advantage of using HGFs avoid cytotoxic side effect caused by chemotherapy. Administraion of HGFs is also applicabe to mobilizing normal healthy donors in allogeneic stem cell transplantation. A 40- to 80-fold increase of circulating CFU-GM over steady-state level after 4-5 days of HGFs administration had been observed. In a comparative studies of mobilization kinetics between G-CSF and GM-CSF under same dose (5Âµg/kg/day), the yield of CD34+ cells under GM-CSF was 3-folded lower than G-CSF and more side effects had been observed in GM-CSF as mobilizating agent (Fischmeister et al., 1999, Bensinger et al., 2009). Hence, the application of GM-CSF as mobilizating agent is rarely use nowadays. However, for patients who were poorly mobilized by G-CSF in their primary mobilization, a combination of G-CSF with GM-CSF seems to be efficacious in re-mobilization (Cottler-Fox et al., 2003).
However, the optimal dose of G-CSF and GM-CSF are varied. The administrative dose of G-CSF alone mobilization regimen is ranging from 10Î¼g/kg/day to high dose (32Î¼g/kg/day). Normally, G-CSF was administrated at 10Î¼g/kg/day for 4 days before the start of apheresis and continued until the end of apheresis session (Bensinger et al., 2009). Kroger et al.(2000) suggested that a significant improvement in the number of CD34+ collected if G-CSF was administrated subcutaneously twice daily in an interval of 12 hours at 5Î¼g/kg. For those patients who were failed in primary mobilization, adequate number of stem cells might be collected in re-mobilization by administrated at high dose G-CSF (32Î¼g/kg/day) (Gazitt et al., 1999).
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In a review of Bensinger et al. (Bensinger et al., 2009), it was found that around 20% patients failed in G-CSF alone stem cell mobilization. Therefore, based on the drawbacks of the above two mobilization regimens, a combination of HGFs plus chemotherapy also known as chemomobilization is being used for stem cell mobilization. Chemomobilization serves several advantages over the above two regimens: It shortened the duration of cytopenia and hospital stay. Apheresis sessions scheduling was more predictable and manageable. Higher stem cell yields in combination therapy than HGFs alone as a result of fewer apheresis sessions were needed (Gertz et al., 2009). Most importantly, synergistic effect in chemomobilization suggested that low dose chemotherapy can be applied in the combination of HGFs in less marrow toxicity, in addition to collecting sufficient stem cells. In chemomobilization regimen, chemotherapeutic agent was administrated follow by G-CSF. On contrary to HGFs mobilization, even with daily G-CSF support, blood leucocytes count will continue to fall after chemotherapy for 7 to 10 days. Leukapheresis will start when the circulating CD34+ cells exceed 20 x 106/L when leucocytes count started to rebound from chemotherapy-induced nadir. Same as HGFs mobilization regimen, the optimal dose of CY in chemomobilization regimen is still unknown.
For patients who were poorly mobilized by either G-CSF alone or chemomobilization, the administration of novel agent MozobilÂ® (Plerixafor, Genzyme Corporation, Cambridge, MA, USA) showed a significant improvement in stem cell yield. Randomized phase III trial (DiPersio et al., 2009) showed that 71.6% myeloma patients achieved â‰¥ 6 x 106 CD34+ cells/kg cells in â‰¤ 2 apheresis after administration of MozobilÂ® together with G-CSF whereas only 34.4% under G-CSF alone regimen reaching target. In the same study, the successful rate in collecting â‰¥ 2 x 106 CD34+ cells/kg in â‰¤ 4 apheresis sessions after addition of MozobilÂ® versus G-CSF alone were 95.3% and 88.3% respectively (p = 0.031). The number of circulating CD34+ cells increased 4.8-fold after addition of Mozobil while only increased 1.7-fold with G-CSF alone. Patients normally received MozobilÂ® on Day 4 approximately 11 hours prior to apheresis on Day 5. Extensive study is required on the impact on stem cell dose after addition of MozobilÂ®.
Gertz et al. (2009) reported that by lengthening the period for over 30 days between completion of apheresis and autologous stem cell transplantation shows no significance difference in term of haemopoietic reconstitution between chemomobilization and G-CSF alone regimen. On contrary, Narayanasami et al. (2001) reported that no significance difference in the duration of neutrophil and platelet engraftment between G-CSF alone and CY + G-CSF but they all agreed that more apheresis procedures were required to collect adequate stem cell under G-CSF alone regimen. Dingli et al. (2006) argued about the usefulness of intermediate dose CY (3g/m2) chemomobilization in the relation to antimyeloma effect in the improvement of disease outcome after autologous stem cell rescue and concluded that complete response rate did not improve when compared to G-CSF alone regimen.
Base on the above observations and research findings, mobilization regimens varied among different centres. The effectiveness of the role of CY in mobilization regimen is controversial. The impacts of different mobilization regimens in relation to stem cell yield as well as hematologic reconstitution after autologous stem cell transplantation are necessary for further studies.
Multiple Myeloma (MM)
Multiple Myeloma (MM) is one of the hematologic malignant disorders that comprises of about 10-15% of hematologic neoplasm and 1% of all cancers (Kyle and Rajkumar, 2008). It is characterized by accumulation of plasma cells in bone marrow and producing immunoglobulin (mostly IgG or IgA) or only immunoglobulin light chains (Harousseau and Moreau, 2009). Plasma cells are important components in the immune system because of its ability in producing specific antibodies to fight against infections. Under normal situation, the population of plasma cells will eliminate to very low level after infection has been eradicated. In myeloma, this self-controlled mechanism has been dysregulated but instead, plasma cell is continuous producing antibodies. This abnormal plasma cell is called myeloma cell. Clinical features of multiple myeloma include bone lesion, anaemia and renal failure and susceptible to infection (Harousseau and Moreau, 2009). Diagnosis of myeloma are based on the presence of monoclonal protein in blood serum and urine, abnormal population of myeloma cell in bone marrow and skeletal lesions (Kyle and Rajkumar, 2009).
Myeloma is commonly found in elderly patients with a median age group of 65 to 70 years (Harousseau and Moreau, 2009). Mechanism of how multiple myeloma developed into plasma cell is still poorly understood. It is believed that progression of myeloma is originated from an asymptomatic premalignant growth of plasma cell in the blood marrow termed "monoclonal gammopathy of unknown significance" (MGUS). The change of progression to multiple myeloma is increasing by 1% amongst the 3% MGUS population (Kyle and Rajkumar, 2008). Another factor causing myeloma is alteration of genetic material (translocation, insertion or deletion) within chromosomes (Harousseau and Moreau, 2009).
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Choices of treatment rely on conventional chemotherapy by the use of alkylator and steroid-based combination regimens or stem cell transplantation. The median survival rate if disease untreated is 2 years. The life expectancy is about 3 years with conventional chemotherapy (Blade and Rosinol, 2008, Harousseau and Moreau, 2009). High dose chemotherapy (HDC) followed by stem cell rescue is considered outcome benefit as compared to conventional therapy alone in prolonging event free survival, overall survival and in higher complete response (CR) rate (Siddiqui and Gertz, 2008, Attal et al., 1996, Child et al., 2003). Autologous hematopoietic stem cell transplantation after high dose chemotherapy (HDC) is preferable because of faster hematopoietic recovery and ease of collection than bone marrow. However, patients who are over 65 years of age or impediments to have intensive chemotherapy are not recommended for autologous or allogeneic stem cell transplantation (Blade and Rosinol, 2008, Nakasone et al., 2009, Mehta and Singhal, 2007).
Studies showed that high dose chemotherapy (HDC) followed by autologous stem cell rescue for younger patient is better than conventional chemotherapy in improving overall survival and increase complete response rate, event-free survival (EFS) are still controversial (Blade and Rosinol, 2008). Some of the researchers demonstrated that complete remission (CR) and OS rate improved by double transplantation of multiple myeloma patients but these were only benefited when the patients had poor response in their first transplantation (Barlogie et al., 1999).
However, up until now, the optimal mobilization regimen in stem cell mobilization has not yet been defined. Therefore, we would like to investigate the impacts of different mobilization regimens on stem cell yield as well as hematologic recovery after autologous stem cell rescue through a retrospective study of a monocentric experience, in order to find out the feasible regimen to be used. Myeloma was selected of choice in this study other than hematological malignancies because of its uniqueness in treatment method. Despite that, the impact of mobilization regimen on stem cell dose in regard to hematopoietic recovery and disease outcomes would not be interfered by other treatment options and facilitate for comparisons
Aims, objectives and testable hypotheses
The main aim of this study is to investigate the impact of different mobilization regimens on stem cell dose and hematopoietic recovery in myeloma patients undergoing peripheral blood stem cell harvest and autologous stem cell transplantation.
The most practical and effective mobilization regimen for stem cell mobilization.
The role of cyclophosphamide in stem cell mobilization in addition to hematologic reconstitution
The role of infused dose of CD34+ and CFU-GM in the prediction of engraftment kinetic
The effect of novel agent (MozobilÂ®) for the improvement of stem cell dose in mobilization
The stem cell yield, as enumerated by CD34+ cells, in peripheral blood stem cell harvest depends on the dosage of cyclophosphamide used in mobilization.
Hematopoietic recovery after stem cell transplantation is related to the dose of CD34+ cells infused to the patients.
The percentage of CD34+ cells in the stem cell harvest is correlated with the frequency of colony forming unit- granulocytes macrophages (CFU-GM).
We analyzed the stem cell yield and clinical outcome of patients undergoing stem cell mobilization and autologous transplantation, with particular reference to the impact of different mobilization regimens. Multiple myeloma patients who underwent stem cell mobilization followed by autologous stem cell transplant at Queen Mary Hospital between Jan-2000 and Oct-2010 would be included in this retrospective study. Patients were divided into two groups for analysis. In group 1: patients received G-CSF (2 x 5Âµg/kg/day) alone. In group 2: patients received G-CSF (2 x 5Âµg/kg/day) plus cyclophosphamide at 1.5g/m2, 2.5g/m2, 3g/m2 and 4g/m2 respectively for stem cell harvesting. The impact of stem cell dose and hematologic recovery among different CY dosage were compared. Personal particulars, clinical and laboratory parameters that were related to stem cell mobilization and autologous stem cell transplantation were abstracted.
Stem cell collection
Informed consents have been obtained from patients undergoing stem cell mobilization, harvesting and transplantation according to the Hong Kong Hospital Authority (HA) regulation. Complete blood picture (CBP) was being monitored everyday after the commencement of mobilization. Circulating CD34+ count in peripheral blood was enumerated by flow cytometry (Beckman Coulter) before stem cell harvesting according to ISHAGE protocol.
Patients receiving CY + G-CSF mobilization regimen: CY was administrated on day 1 followed by G-CSF (2 x 5Âµg/kg/day) on day 2 onwards until the end of LK. CD34+ count in peripheral blood would be enumerated by flow cytometry (Beckman Coulter) daily after chemotherapy from Day 8 according to ISHAGE protocol. Patients receiving G-CSF alone were treated with G-CSF (2 x 5Âµg/kg/day) daily for four days and circulating CD34+ count in peripheral blood would be enumerated on day 4 by flow cytometry (Beckman Coulter) before stem cell harvesting according to ISHAGE protocol.
Leukapheresis (LK) started when the circulating peripheral blood CD34+ was greater than 20/Âµl otherwise delay. Daily G-CSF administration would be continued until the end of the LK procedure. LK was considered satisfactory when the CD34+ count reaches 2 x 106/kg of patient body weight. Leukapheresis was performed by Cobe Spectra separator. Number of leukapheresis attempts were recorded.
Analysis of leukapheresis product
The content of CD34+ in leukapheresis product is determined by FACS analysis. Cells staining methods were according to the product inserts provided by the suppliers (Backman Coulter). Gating strategies and enumeration methods were following the ISHAGE guidelines.
Cryopreservation of leukapheresis product
Leukapheresis product was cryopreserved according to our institutional in-house protocol. Final cryopreserved LK product had been adjusted to approximately 1-1.5 x 108 cells/bag with 10% DMSO (dimethylsulfoxide) and 5% autologous plasma.
Autologous Stem cell transplant conditioning regimen
Patients were required to receive high dose chemotherapy (Melphalan 200 mg/m2 patient surface area) before stem cell transplant, according to the transplantation protocol in the Bone Marrow Transplantation Unit, Queen Mary Hospital. Neutrophil engraftment was defined as the first day when absolute neutrophil count (ANC) â‰§ 0.5 x 106/ml on three consecutive days. Platelet engraftment was defined as the first day when platelet count â‰§50 x 106/ml without transfusion.
CFU-GM clonogenic assay
An aliquot of LK product suspension was diluted and adjusted to 0.5 x 106/ml WBC with Hanks' balanced salt solution (Gibco). 0.4ml at 0.5 x 106/ml cells suspension were dispensed into 4ml methylcellulose based media containing colony-stimulating factors (MethoCult H4434, Stem cell technologies, Vancouver British Columbia, Canada). Mixture of cell-methylcellulose media was dispensed triplicate into petri dish at 1ml each. The final concentration of each petri dish contained 0.5 x 105 WBC cells/ml. After 14-days of humidified (>95%) incubation at 37oC with 5% CO2, CFU-GM colonies were counted under inverted microscope. The number of CFU-GM colonies were averaged and the total CFU-GM was calculated based on total WBCs in the LK product.
Results are expressed as mean ï‚± standard error of mean (SEM). Comparison between groups of data will be evaluated by Mann-Whitney U and Kruskal-Wallis Tests (SPSS, Version 13). A p-value of less than 0.05 is considered statistical significant.