Since the first bone marrow transplantation went into clinical practices 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) . 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 . Furthermore, mobilized progenitor cells are less tumour cell contamination compared to bone marrow .
Infusion of significant number of stem cells is paramount to restore haematopoiesis in autologous stem cell transplantation. An infused dose of 4 - 6 x 106/kg recipient body weight is ideal for rapid haematopoietic recovery and 2 x 106/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 . However, stem cells normally reside in bone marrow, only a small number of progenitor cells are found in the blood circulation. After transfusing leukocytes separated by blood separator, these circulating stem cells enabled to restore haematopoiesis after myelosuppressive chemotherapy . 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 impossible to collect sufficient stem cells. In order to mobilize progenitor cells by shifting from bone marrow into peripheral blood that can be collected during leukapheresis procedure, mobilization regimen is required. Richman et al. demonstrated that a combination of adriamycin and cyclophosphamide increased an 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. and Duhrsen et al. 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 hypothesized formed the foundation of using hemopoietic growth factors (HGFs) in autologous stem cell transplantation.
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Stem cell can be mobilized by chemotherapy, hemopoietic growth factor (HGFs) or furthermore, combination of both. Chemotherapy was the first employed as mobilizating agent for clinical used. To et al. found that the number of CFU-GM increased by 25 times as compared to normal subjects in acute non-lymphoblastic leukaemia (ANLL) patients when platelet started to rebound rapidly after chemotherapy. Cyclophosphamide (CY) is the most commonly chemtherapeutic agent that can be used alone or combined with other agent. Lie et al. 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 of cyclophosphamide used as mobilizing agent resulted in higher marrow toxicity. Morbidity and hemorrhagic cystitis were reported which was associated with using high dose CY. Besides marrow toxicities, apheresis session was difficut to schdule because recovery from cytopenic phase after chemotherapy was unpredictable. Hence, using chemotherapy alone is less commonly used nowadays.
Another mobilization regime is using hemopoietic growth factors (HGFs) alone, granulocyte-colony stimulating factor (G-CSF, Filgrastim) and granulocyte-macrophages colony stimulating factor (GM-CSF, Sargarmostim) are two commonly used mobilizing agents. Advantage of using HGFs avoided 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 . Hence, the application of GM-CSF as mobilizating agent was 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 .
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 . Kroger et al. 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) .
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Around 20% patients failed in G-CSF alone stem cell mobilization . Based on the drawbacks of the above two mobilization regimens, a combination of HGFs plus chemotherapy also known as chemomobilization is 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 . 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 collect sufficient stem cells. In chemomobilization regimen, chemotherapeutic agent was administrated followed by G-CSF. On contrary to HGFs mobilization, leucocytes continue to rise after administration. Even with daily G-CSF support, blood leucocytes count will still 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. The CY dose in chemomobilization regimen depends on the experience in every single centre. Chemomobilization regimens encountered benefits and drawback. It benefited for better disease control by cytoreduction of tumour cell as well as reducing tumour mass. High dose CY used in mobilization regimen support better treatment outcome with faster stem cell collection . However, high dosage of CY damaged bone marrow microenvironment. Patients treated with high dose of CY will result in higher incidence of pancytopenia and delayed in engraftment .
Numerous comparative studies had been carried out to investigate the impact of different dose of CY in chemomobilization regimen. Administrative doses of CY from low dose (1g/m2) to high dose (7g/m2) had been reported . Goldschmidt et al. showed that high dose CY at 7g/m2 resulted in higher yields of CD34+ cell collected than at the dose of 4g/m2 and found that longer duration of neutropenia and thrombocytopenia were associated with high dose CY. In a study of 63 myeloma patients, the percentage of having neutropenic fever that required hospitalization was lower in those who receiving 2 x 1.2g/m2 CY than 7g/m2 CY during the preiod of stem cell collection . Another studies conducted by Fitoussi et al. further supported the above phenomenon and demonstrated that the number of CD34+ cell collected (â‰¥2.5 x 106/kg recipient body weight) had no significant difference between high (7g/m2) and intermediate dose (4g/m2) CY whereas the duration of neutropenia was shortened and fewer transfusion support were needed at intermediate dose CY. Jantunen et al. compared the mobilization efficiency between low dose CY (1.2 - 2g/m2) and intermediate dose CY (4g/m2) followed by G-CSF in 57 myeloma patients and illustrated that over 84% effectively mobilized adequate stem cell (â‰¥ 2 x 106/kg CD34+ cells) in single apheresis under both chemomobilization regimen and fewer transfusion support as well as fever leading to hospitalization under low dose chemomobilization. They also found that the number of CD34+ was normally peak before the first day of apheresis in low dose chemomobilization whereas the number of CD34+ cell was normally continued to rise after the completion of first apheresis in intermediate chemomobilization.
For patients who were poorly mobilized 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 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 Mozobil 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. However, extensive study of the impact on stem cell mobilization after addition of Mozobil on Day 5 or onwards was lack of study.
Gertz et al. reported that by lengthening the period for over 30 days between completion of apheresis and autologous stem cell transplantation, no significance difference observed in term of haemopoietic reconstitution between chemomobilization and G-CSF alone regimen. On contrary, Narayanasami et al. 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. arguing 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.
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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 seen to be necessary for further studies.
Correlation between CD34 to CFU-GM
In early years, in vitro cell culture of myeloid progenitor cells "colony forming unit granulocyte-macrophage" (CFU-GM) clonogenic assays has been used to determine the engraftment potential and quality of the graft . Neither CFU-GM nor CD34+ truly represents the real re-population capability of hematopoietic cell. Infused dose of CFU-GM and CD34+ were correlated to short-term hematologic recovery . Siena et al. and Ikematsu et al. found that there was a strong positive correlation between CFU-GM and CD34+ cells. Similar correlation had been reported by Vogel et al. and correlation of R-value 0.63 (p < 0.0001) was reported by Jansen et al. . Enumeration of either CD34+ or CFU-GM can provide predictive information about the engraftment potential in hematologic reconstitution . Gianni et al. observed that higher number of transplanted CFU-GM related to early engraftment. However, CFU-GM clonogenic assay required technical skills and results were not available immediately. Serke et al. studies the CFU-GM counting among 6 European transplant centres found that the coefficient of variation was about 30% whereas the variation of accessing CD34+ graft by flow cytometry was only 10%. Imprecision of CFU-GM counting would lead to incorrect interpretation of infused dose between centres. Furthermore, Serke et al. and Jansen et al. observed that the clonogenicity of CFU-GM was negatively correlated by increasing number of CD34+ cells. Two major explanations had been made to describe this scenario. First, grouping of many small CFU-GM colonies into a large colony at high CD34+ cell concentration. Second, enumeration of CD34+-expressing cells and CFU-GM clonogenic assay were independently contributed to the prediction of engraftment kinetic . Further study by Fu et al., suggested that there was no correlation between CD34+ cells and CFU-GM (p = 0.12). Hence, the relationship between CD34+ cells and CFU-GM under different mobilization regimens is another place of interest for further investigation.
Multiple Myeloma (MM) is one of the hematologic malignant disorders that comprises of about 10-15% of hematologic neoplasm and 1% of all cancers . 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 .
Myeloma is commonly found in elderly patients with a median age group of 65 to 70 years . 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 . Another factor causing myeloma is alteration of genetic material (translocation, insertion or deletion) within chromosomes (Harousseau and Moreau, 2009).
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 . 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).
Autologous stem cell mobilization is an integral part of autologous stem cell transplantation. Patients will experience different stages in the whole autologous stem cell transplant procedure. That includes induction treatment, hematopoietic stem cell collection, high dose chemotherapy followed by re-infusion of stem cell that previously cryopreserved (Harousseau and Moreau, 2009).
Up to date, the optimal mobilization regimen in stem cell mobilization has not yet defined. In this study, through a retrospective study of autologous stem cell transplantation of multiple myeloma patients in one centre. 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 to find out the best regimen to be used. Myeloma was selected of choice in this study other than hematological malignancies because of its uniqueness in treatment method. Therefore, 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. Furthermore, through the study of novel agent MozobilÂ®, the effectiveness in stem cell mobilization as well as hematologic recovery could be investigated.
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 mobilization
The stem cell yield, as enumerated by CD34+ cells, in peripheral blood stem cell harvest depends on the dosage of cyclophosphamide using 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. Patients diagnosed with multiple myeloma from Jan-2000 to Oct-2010 who underwent stem cell mobilization followed by autologous stem cell transplant at Queen Mary Hospital were included in this retrospective study. Personal particulars, clinical and laboratory parameters that had an impact on stem cell mobilization and autologous stem cell transplantation were abstracted.
Stem cell collection
Informed consents have been obtained for patients undergoing stem cell mobilization, harvesting and transplantation according to the Hospital Authority (HA) regulation. Patients were divided into two groups for analysis. One group received G-CSF (10Âµg/kg/day) alone and the other group treated with G-CSF plus cyclophosphamide at different doses (1.5g/m2, 2.5g/m2, 3g/m2 or 4g/m2) for stem cell harvesting. The impact of stem cell dose and hematologic recovery among different CY dosage would be studied compared. Complete blood picture (CBP) is monitored everyday after the commencement of mobilization. Circulating CD34+ count in peripheral blood will be enumerated by flow cytometry (Beckman Coulter) before stem cell harvesting according to ISHAGE protocol.
Patients receiving CY + G-CSF mobilization regimen, CY was administrated at 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 monitored 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 monitored on day 4 after G-CSF administration. Circulating CD34+ count in peripheral blood will be enumerated by flow cytometry (Beckman Coulter) before stem cell harvesting according to ISHAGE protocol.
Leukapheresis (LK) would be started when the circulating peripheral blood CD34+ was greater than 20/Âµl otherwise delay. Continuous daily G-CSF administration would be received 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 would be recorded.
Cryopreservation of leukapheresis product
Leukapheresis product was cryopreserved according to our institutional in-house protocol. Final cryopreserved LK product had 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 received 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.
Independent t-test was performed to measure the significant of the above hypothesis. Any value within 95% Confidence interval would report in no significance. Data would be analyzed by statistical analysis program SPSS version 13.