Since the first bone marrow organ transplant went into clinical patterns in 1968, the application of root cell organ transplants were widely applied in the intervention of hematologic malignances for the last four decennaries. Autologous root cell deliverance after myeloablative therapy is a preferred option in disease intervention of haematological malignances including multiple myeloma ( MM ) , Non-Hodgkin ‘s lymphoma ( NHL ) and Hodgkin ‘s lymphoma ( HL ) . Mobilized peripheral blood root cell is replacing bone marrow as a major beginning of root cell in autologous organ transplant in the last two decennaries due to its rapid haematological reconstitution and turning away of general anesthesia in bone marrow aspiration. Furthermore, mobilized primogenitor cells are less tumour cell taint compared to cram marrow.
Infusion of important figure of root cells is paramount to reconstruct hematopoiesis in autologous root cell organ transplant. An infused dosage of 4 – 6 x 106/kg receiver organic structure weight is ideal for rapid hematopoietic recovery and 2 ten 106/kg is defined as minimal threshold for successful engraftment. Any infused dosage below this degree will sort as failure and will ensue in hazard of delayed or non-engraftment after myeloablative chemotherapy. However, root cells usually reside in bone marrow, merely a little figure of primogenitor cells are found in the blood circulation. After instilling leucocytes separated by blood centrifuge, these go arounding root cells enabled to reconstruct hematopoiesis after myelosuppressive chemotherapy. These findings postulated a new epoch of utilizing peripheral blood root cells in replacing bone marrow as a major root cell beginning in autologous and allogeneic organ transplant. However, multiple leukapheresis from non-mobilized steady-state peripheral blood is impossible to roll up sufficient root cells. In order to mobilise primogenitor cells by switching from bone marrow into peripheral blood that can be collected during leukapheresis process, mobilisation regimen is required. Richman et al. demonstrated that a combination of adriamycin and cyclophosphamide increased an about 20-folded above the baseline of go arounding root cells. These findings suggested that hematopoietic root cells could be mobilized by chemotherapy. Subsequently studied by Socinski et Al. and Duhrsen et Al. showed that go arounding colony-forming unit granulocyte macrophages ( CFU-GM ) and progenitor cells increased unusually by disposal of granulocyte-macrophage-colony stimulating factor ( GM-CSF ) and granulocytes-colony stimulating factor ( G-CSF ) . This hypothesized formed the foundation of utilizing hematopoietic growing factors ( HGFs ) in autologous root cell organ transplant.
Stem cell can be mobilized by chemotherapy, hematopoietic growing factor ( HGFs ) or moreover, combination of both. Chemotherapy was the first employed as mobilizating agent for clinical used. To et Al. found that the figure of CFU-GM increased by 25 times every bit compared to normal topics in acute non-lymphoblastic leukemia ( ANLL ) patients when thrombocyte started to bounce quickly after chemotherapy. Cyclophosphamide ( CY ) is the most normally chemtherapeutic agent that can be used entirely or combined with other agent. Lie et Al. found that output of CFU-GM every bit good as CD34+ output increased significantly by seting CY dosage from 4g/m2 to 7g/m2. Using chemothrapy in mobilisation regimen enjoyed benefit of tumour cytoreduction of mobilized tumor cells. However, high dosage of cyclophosphamide used as mobilising agent resulted in higher marrow toxicity. Morbidity and haemorrhagic cystitis were reported which was associated with utilizing high dose CY. Besides marrow toxicities, aphaeresis session was difficut to schdule because recovery from cytopenic stage after chemotherapy was unpredictable. Hence, utilizing chemotherapy entirely is less normally used presents.
Another mobilisation government is utilizing hematopoietic growing factors ( HGFs ) entirely, granulocyte-colony stimulating factor ( G-CSF, Filgrastim ) and granulocyte-macrophages settlement exciting factor ( GM-CSF, Sargarmostim ) are two normally used mobilising agents. Advantage of utilizing HGFs avoided cytotoxic side consequence caused by chemotherapy. Administraion of HGFs is besides applicabe to mobilising normal healthy givers in allogeneic root cell organ transplant. A 40- to 80-fold addition of go arounding CFU-GM over steady-state degree after 4-5 yearss of HGFs disposal had been observed. In a comparative surveies of mobilisation dynamicss between G-CSF and GM-CSF under same dosage ( 5Aµg/kg/day ) , the output 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 seldom use presents. However, for patients who were ill mobilized by G-CSF in their primary mobilisation, a combination of G-CSF with GM-CSF seems to be efficacious in re-mobilization.
However, the optimum dosage of G-CSF and GM-CSF are varied. The administrative dosage of G-CSF alone mobilisation regimen is runing from 10I?g/kg/day to high dosage ( 32I?g/kg/day ) . Normally, G-CSF was administrated at 10I?g/kg/day for 4 yearss before the start of aphaeresis and continued until the terminal of aphaeresis session. Kroger et Al. suggested that a important betterment in the figure of CD34+ collected if G-CSF was administrated subcutaneously twice daily in an interval of 12 hours at 5I?g/kg. For those patients who were failed in primary mobilisation, equal figure of root cells might be collected in re-mobilization by administrated at high dose G-CSF ( 32I?g/kg/day ) .
Around 20 % patients failed in G-CSF entirely root cell mobilisation. Based on the drawbacks of the above two mobilisation regimens, a combination of HGFs plus chemotherapy besides known as chemomobilization is used for root cell mobilisation. Chemomobilization serves several advantages over the above two regimens. It shortened the continuance of cytopenia and infirmary stay. Apheresis Sessionss scheduling was more predictable and manageable. Higher root cell outputs in combination therapy than HGFs entirely as a consequence of fewer aphaeresis Sessionss were needed. Most significantly, interactive consequence in chemomobilization suggested that low dose chemotherapy can be applied in the combination of HGFs in less marrow toxicity in add-on to roll up sufficient root cells. In chemomobilization regimen, chemotherapeutic agent was administrated followed by G-CSF. On contrary to HGFs mobilisation, leucocytes continue to lift after disposal. Even with day-to-day G-CSF support, blood leukocytes count will still go on to fall after chemotherapy for 7 to 10 yearss. Leukapheresis will get down when the circulating CD34+ cells exceed 20 ten 106/L when leukocytes count started to bounce from chemotherapy-induced low-water mark. Same as HGFs mobilisation regimen, the optimum dosage of CY in chemomobilization regimen is still unknown. The CY dosage in chemomobilization regimen depends on the experience in every individual Centre. Chemomobilization regimens encountered benefits and drawback. It benefited for better disease control by cytoreduction of tumor cell every bit good as cut downing tumour mass. High dose CY used in mobilisation regimen support better intervention result with faster root cell aggregation. However, high dose of CY damaged bone marrow microenvironment. Patients treated with high dosage of CY will ensue in higher incidence of pancytopenia and delayed in engraftment.
Numerous comparative surveies had been carried out to look into the impact of different dosage of CY in chemomobilization regimen. Administrative doses of CY from low dosage ( 1g/m2 ) to high dosage ( 7g/m2 ) had been reported. Goldschmidt et Al. showed that high dosage CY at 7g/m2 resulted in higher outputs of CD34+ cell collected than at the dosage of 4g/m2 and found that longer continuance of neutropenia and thrombopenia were associated with high dosage CY. In a survey of 63 myeloma patients, the per centum of holding neutropenic febrility that required hospitalization was lower in those who having 2 ten 1.2g/m2 CY than 7g/m2 CY during the preiod of root cell aggregation. Another surveies conducted by Fitoussi et Al. farther supported the above phenomenon and demonstrated that the figure of CD34+ cell collected ( a‰?2.5 x 106/kg receiver organic structure weight ) had no important difference between high ( 7g/m2 ) and intermediate dosage ( 4g/m2 ) CY whereas the continuance of neutropenia was shortened and fewer transfusion support were needed at intermediate dose CY. Jantunen et Al. compared the mobilisation efficiency between low dosage CY ( 1.2 – 2g/m2 ) and intermediate dosage CY ( 4g/m2 ) followed by G-CSF in 57 myeloma patients and illustrated that over 84 % efficaciously mobilized equal root cell ( a‰? 2 ten 106/kg CD34+ cells ) in individual aphaeresis under both chemomobilization regimen and fewer transfusion support every bit good as febrility taking to hospitalization under low dose chemomobilization. They besides found that the figure of CD34+ was usually peak before the first twenty-four hours of aphaeresis in low dose chemomobilization whereas the figure of CD34+ cell was usually continued to lift after the completion of first aphaeresis in intermediate chemomobilization.
For patients who were ill mobilized either G-CSF entirely or chemomobilization, the disposal of fresh agent MozobilA® ( Plerixafor, Genzyme Corporation, Cambridge, MA, USA ) showed a important betterment in root cell output. Randomized stage III test showed that 71.6 % myeloma patients achieved a‰? 6 ten 106 CD34+ cells/kg cells in a‰¤ 2 aphaeresis after disposal of MozobilA® together with G-CSF whereas merely 34.4 % under G-CSF entirely regimen making mark. In the same survey, the successful rate in roll uping a‰? 2 ten 106 CD34+ cells/kg in a‰¤ 4 aphaeresis Sessionss after add-on of Mozobil versus G-CSF entirely were 95.3 % and 88.3 % severally ( p = 0.031 ) . The figure of go arounding CD34+ cells increased 4.8-fold after add-on of Mozobil while merely increased 1.7-fold with G-CSF entirely. Patients usually received MozobilA® on Day 4 about 11 hours prior to apheresis on Day 5. However, extended survey of the impact on root cell mobilisation after add-on of Mozobil on Day 5 or onwards was deficiency of survey.
Gertz et Al. reported that by lengthening the period for over 30 yearss between completion of aphaeresis and autologous root cell organ transplant, no significance difference observed in term of hematopoietic reconstitution between chemomobilization and G-CSF entirely regimen. On contrary, Narayanasami et Al. reported that no significance difference in the continuance of neutrophil and platelet engraftment between G-CSF entirely and CY + G-CSF but they all agreed that more aphaeresis processs were required to roll up equal root cell under G-CSF entirely regimen. Dingli et Al. reasoning about the utility of intermediate dosage CY ( 3g/m2 ) chemomobilization in the relation to antimyeloma consequence in the betterment of disease result after autologous root cell deliverance and concluded that complete response rate did non better when compared to G-CSF entirely regimen.
Base on the above observations and research findings, mobilisation regimens varied among different Centres. The effectivity of the function of CY in mobilisation regimen is controversial. The impacts of different mobilisation regimens in relation to stem cell output every bit good as haematological reconstitution after autologous root cell organ transplant are seen to be necessary for farther surveies.
Correlation between CD34 to CFU-GM
In early old ages, in vitro cell civilization of myeloid primogenitor cells “ colony organizing unit granulocyte-macrophage ” ( CFU-GM ) clonogenic checks has been used to find the engraftment potency and quality of the transplant. Neither CFU-GM nor CD34+ genuinely represents the existent re-population capableness of haematopoietic cell. Infused dosage of CFU-GM and CD34+ were correlated to short-run haematological recovery. Siena et Al. and Ikematsu et Al. found that there was a strong positive correlativity between CFU-GM and CD34+ cells. Similar correlativity had been reported by Vogel et Al. and correlativity of R-value 0.63 ( P & lt ; 0.0001 ) was reported by Jansen et Al. . Enumeration of either CD34+ or CFU-GM can supply prognostic information about the engraftment potency in haematological reconstitution. Gianni et Al. observed that higher figure of transplanted CFU-GM related to early engraftment. However, CFU-GM clonogenic check required proficient accomplishments and consequences were non available instantly. Serke et Al. surveies the CFU-GM numeration among 6 European graft Centres found that the coefficient of fluctuation was approximately 30 % whereas the fluctuation of accessing CD34+ transplant by flow cytometry was merely 10 % . Impreciseness of CFU-GM numbering would take to wrong reading of infused dosage between Centres. Furthermore, Serke et Al. and Jansen et Al. observed that the clonogenicity of CFU-GM was negatively correlated by increasing figure of CD34+ cells. Two major accounts had been made to depict this scenario. First, grouping of many little CFU-GM settlements into a big settlement at high CD34+ cell concentration. Second, numbering of CD34+-expressing cells and CFU-GM clonogenic check were independently contributed to the anticipation of engraftment kinetic. Further survey by Fu et al. , suggested that there was no correlativity between CD34+ cells and CFU-GM ( p = 0.12 ) . Hence, the relationship between CD34+ cells and CFU-GM under different mobilisation regimens is another topographic point of involvement for farther probe.
Multiple Myeloma ( MM ) is one of the hematologic malignant upsets that comprises of about 10-15 % of haematological tumor and 1 % of all malignant neoplastic diseases. It is characterized by accretion of plasma cells in bone marrow and bring forthing Ig ( largely IgG or IgA ) or lone Ig visible radiation ironss ( Harousseau and Moreau, 2009 ) . Plasma cells are of import constituents in the immune system because of its ability in bring forthing specific antibodies to contend against infections. Under normal state of affairs, the population of plasma cells will extinguish to really low degree after infection has been eradicated. In myeloma, this self-controlled mechanism has been dysregulated but alternatively, plasma cell is uninterrupted bring forthing antibodies. This unnatural plasma cell is called myeloma cell. Clinical characteristics of multiple myeloma include bone lesion, anemia and nephritic failure and susceptible to infection ( Harousseau and Moreau, 2009 ) . Diagnosis of myeloma are based on the presence of monoclonal protein in blood serum and piss, unnatural population of myeloma cell in bone marrow and skeletal lesions.
Myeloma is normally found in aged patients with a average age group of 65 to 70 old ages. Mechanism of how multiple myeloma developed into plasma cell is still ill understood. It is believed that patterned advance of myeloma is originated from an symptomless premalignant growing of plasma cell in the blood marrow termed “ monoclonal gammopathy of unknown significance ” ( MGUS ) . The alteration of patterned advance to multiple myeloma is increasing by 1 % amongst the 3 % MGUS population. Another factor doing myeloma is change of familial stuff ( translocation, interpolation or omission ) within chromosomes ( Harousseau and Moreau, 2009 ) .
Choices of intervention rely on conventional chemotherapy by the usage of alkylator and steroid-based combination regimens or root cell organ transplant. The average endurance rate if disease untreated is 2 old ages. The life anticipation is about 3 old ages with conventional chemotherapy ( Blade and Rosinol, 2008, Harousseau and Moreau, 2009 ) . High dose chemotherapy ( HDC ) followed by root cell deliverance is considered outcome benefit as compared to conventional therapy entirely in protracting event free endurance, overall endurance and in higher complete response ( CR ) rate. Autologous haematopoietic root cell organ transplant after high dosage chemotherapy ( HDC ) is preferred because of faster haematopoietic recovery and easiness of aggregation than bone marrow. However, patients who are over 65 old ages of age or hindrances to hold intensive chemotherapy are non recommended for autologous or allogeneic root cell organ transplant ( Blade and Rosinol, 2008, Nakasone et al. , 2009, Mehta and Singhal, 2007 ) .
Surveies showed that high dosage chemotherapy ( HDC ) followed by autologous root cell deliverance for younger patient is better than conventional chemotherapy in bettering overall endurance and increase complete response rate, event-free endurance ( EFS ) are still controversial ( Blade and Rosinol, 2008 ) . Some of the research workers demonstrated that complete remittal ( CR ) and OS rate improved by dual organ transplant of multiple myeloma patients but these were merely benefited when the patients had hapless response in their first organ transplant ( Barlogie et al. , 1999 ) .
Autologous root cell mobilisation is an built-in portion of autologous root cell organ transplant. Patients will see different phases in the whole autologous root cell transplant process. That includes initiation intervention, haematopoietic root cell aggregation, high dosage chemotherapy followed by re-infusion of root cell that antecedently cryopreserved ( Harousseau and Moreau, 2009 ) .
Up to day of the month, the optimum mobilisation regimen in root cell mobilisation has non yet defined. In this survey, through a retrospective survey of autologous root cell organ transplant of multiple myeloma patients in one Centre. We would wish to look into the impacts of different mobilisation regimens on root cell output every bit good as haematological recovery after autologous root cell deliverance to happen out the best regimen to be used. Myeloma was selected of pick in this survey other than haematological malignances because of its singularity in intervention method. Therefore, the impact of mobilisation regimen on root cell dosage in respect to haematopoietic recovery and disease results would non be interfered by other intervention options and ease for comparings. Furthermore, through the survey of fresh agent MozobilA® , the effectivity in root cell mobilisation every bit good as haematological recovery could be investigated.
Purposes, aims and testable hypotheses
Overall Purposes:
The chief purpose of this survey is to look into the impact of different mobilisation regimens on root cell dosage and haematopoietic recovery in myeloma patients undergoing peripheral blood root cell crop and autologous root cell organ transplant.
Aim:
The most practical and effectual mobilisation regimen for root cell mobilisation.
The function of cyclophosphamide in root cell mobilisation in add-on to hematologic reconstitution
The function of infused dosage of CD34+ and CFU-GM in the anticipation of engraftment kinetic
The consequence of fresh agent ( MozobilA® ) for the betterment of root cell mobilisation
Hypothesis:
The root cell output, as enumerated by CD34+ cells, in peripheral blood root cell harvest depends on the dose of cyclophosphamide utilizing in mobilisation.
Hematopoietic recovery after root cell organ transplant is related to the dosage of CD34+ cells infused to the patients.
The per centum of CD34+ cells in the root cell crop is correlated with the frequence of settlement organizing unit- granulocytes macrophages ( CFU-GM ) .
Methods
Study design
We analyzed the root cell output and clinical result of patients undergoing root cell mobilisation and autologous organ transplant, with peculiar mention to the impact of different mobilisation regimens. Patients diagnosed with multiple myeloma from Jan-2000 to Oct-2010 who underwent root cell mobilisation followed by autologous root cell graft at Queen Mary Hospital were included in this retrospective survey. Personal specifics, clinical and laboratory parametric quantities that had an impact on root cell mobilisation and autologous root cell organ transplant were abstracted.
Stem cell aggregation
Informed consents have been obtained for patients undergoing root cell mobilisation, reaping and organ transplant harmonizing to the Hospital Authority ( HA ) ordinance. Patients were divided into two groups for analysis. One group received G-CSF ( 10Aµg/kg/day ) entirely 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 root cell reaping. The impact of root cell dosage and haematological recovery among different CY dose would be studied compared. Complete blood image ( CBP ) is monitored everyday after the beginning of mobilisation. Go arounding CD34+ count in peripheral blood will be enumerated by flow cytometry ( Beckman Coulter ) before root cell reaping harmonizing to ISHAGE protocol.
Patients having CY + G-CSF mobilisation regimen, CY was administrated at twenty-four hours 1 followed by G-CSF ( 2 x 5Aµg/kg/day ) on twenty-four hours 2 onwards until the terminal of LK. CD34+ count in peripheral blood would be monitored daily after chemotherapy from Day 8 harmonizing to ISHAGE protocol. Patients having G-CSF entirely were treated with G-CSF ( 2 x 5Aµg/kg/day ) daily for four yearss and go arounding CD34+ count in peripheral blood would be monitored on twenty-four hours 4 after G-CSF disposal. Go arounding CD34+ count in peripheral blood will be enumerated by flow cytometry ( Beckman Coulter ) before root cell reaping harmonizing to ISHAGE protocol.
Leukapheresis ( LK ) would be started when the go arounding peripheral blood CD34+ was greater than 20/Aµl otherwise hold. Continuous day-to-day G-CSF disposal would be received until the terminal of the LK process. LK was considered satisfactory when the CD34+ count reaches 2 ten 106/kg of patient organic structure weight. Leukapheresis was performed by Cobe Spectra centrifuge. Number of leukapheresis efforts would be recorded.
Cryopreservation of leukapheresis merchandise
Leukapheresis merchandise was cryopreserved harmonizing to our institutional in-house protocol. Final cryopreserved LK merchandise had adjusted to about 1-1.5 tens 108 cells/bag with 10 % DMSO ( dimethylsulfoxide ) and 5 % autologous plasma.
Autologous Stem cell graft conditioning regimen
Patients received high dose chemotherapy ( Melphalan 200 mg/m2 patient surface country ) before root cell graft harmonizing to the organ transplant protocol in the Bone Marrow Transplantation Unit, Queen Mary Hospital. Neutrophil engraftment was defined as the first twenty-four hours when absolute neutrophil count ( ANC ) a‰§ 0.5 ten 106/ml on three back-to-back yearss. Platelet engraftment was defined as the first twenty-four hours when thrombocyte count a‰§50 x 106/ml without transfusion.
Statistical analysis
Independent t-test was performed to mensurate the important of the above hypothesis. Any value within 95 % Confidence interval would describe in no significance. Data would be analyzed by statistical analysis plan SPSS version 13.
Timeline
