Impact Different Mobilization Regimens Stem Cell Dose Biology Essay

High dosage chemotherapy followed by autologous haematopoietic root cell organ transplant ( HSCT ) is the pillar intervention for multiple myeloma and has been shown to better patient endurance. During the intervention, aggregation of sufficient HSC is of import for successful engraftment and haematopoietic recovery. The optimum scheme for root cell mobilisation has yet been determined and regimens integrating chemotherapy and granulocyte-colony stimulating factor ( G-CSF ) or G-CSF entirely are widely used. More late, fresh mobilisation agent, viz. plerixafor that antagonizes the CXCR4 ( CXC chemokine receptor 4 ) and SDF1 ( stromal-cell-derived factor-1I± ) interaction, has been approved by the FDA as salvage agent after G-CSF mobilisation failure. In this retrospective survey, the mobilisation and HSCT results of 28 myeloma patients who underwent different mobilisation regimens were compared: cyclophosphamide ( CY ) plus G-CSF ( CY group ) , G-CSF entirely ( G-CSF group ) and G-CSF + plerixafor ( P group ) . Mobilization in the CY group resulted in significantly higher HSC yield ( 9.46A±1.33 x 106/kg ) and few aphaeresis Sessionss ( 1.17A±0.09 ) compared with those in the G-CSF ( 2.68A±0.28 x 106/kg, P & lt ; 0.001 ; 2A±0.00 Sessionss, P & lt ; 0.001 ) and P groups ( 2.57A±0.24 x 106/kg, p=0.001 ; 2A±0.32 Sessionss, p=0.002 ) . However, the CY group resulted in neutropenia and thrombopenia at low-water mark ( 0.15A±0.05 x 109/L and 76.4A±9.23 tens 109/L ) and a demand for infirmary stay for 13A±0.26 yearss during mobilisation. These were non present in the G-CSF and P groups. Despite the difference in HSC output, there was no difference in absolute neutrophil ( CY: 10A±0.19 vs G-CSF: 10.8A±0.2 V P: 10, p=0.062 ) and platelet engraftment ( CY 15.1A±1.41 vs G-CSF 18.8A±4.57 V P 14.5A±0.65, p=0.319 ) every bit good as infirmary stay ( CY: 22.72A±1.71 vs G-CSF: 20.2A±1.77 V P: 24.5A±1.5, p=0.209 ) between the three groups. The consequences suggested that G-CSF entirely could be used for mobilisation in patients with multiple myeloma if there could be back up by plerixafor to accomplish equal HSC crop. The hazard and morbidity associated with CY mobilisation would hold to be balanced with the drug cost of plerixafor. The long-run difference in disease control in these three groups needs to be determined.

Chapter 1

Literature reappraisal

1.1 Introduction

High dosage chemotherapy and autologous haematopoietic root cell organ transplant ( HSCT ) is the pillar of intervention for multiple myeloma ( MM ) and relapsed instances of Non-Hodgkins ‘ Lymphoma ( NHL ) and Hodgkin ‘s lymphoma ( HL ) ( Pusic et Al. 2008 ) . Direct crop of bone marrow ( BM ) used to be the major beginning of HSC for organ transplant as it is the site where HSC usually reside. In the last two decennaries, peripheral blood root cell ( PBSC ) is emerging as an of import root cell beginning in autologous organ transplant and it avoids the hazard of general anesthesia and the injury associated with bone marrow aspiration ( Leung & A ; Kwong, 2010 ) . Furthermore, PBSCT is associated with a more rapid haematological reconstitution and mobilized primogenitor cells are less contaminated with tumour cells ( Alegre et al. 1997 ) . In fact, autologous PBSCT has now become the mainstay intervention of multiple myeloma, taking to better patient endurance and minimum toxicity. In the undermentioned subdivisions, an overview of the construct of root cells, the assorted mobilisation regimens, myeloma biological science and the foretelling factors of PBSC output will be described.

1.2 Concept of haematopoietic root cells and primogenitors

In human grownup, blood formation ( haematopoiesis ) occurs in the bone marrow and it arises from a little population of cells, known as the hematopioetic root cells ( HSC ) that are replicatively quiescent but are capable of uninterrupted self-renewal and distinction into primogenitors and functional blood cells of different line of descents. Most of import, HSC are capable of reconstructing haematopoiesis when transplanted into hosts ( patients ) who have received high dose chemotherapy and irradiation that would hold caused fatal bone marrow aplasia without the graft. The features of human HSC have non been to the full defined. Early surveies in Primatess demonstrated that HSC expresses CD34+ ( Berenson et al. 1988 ) . However, CD34+ cells are heterogeneous and the true HSC may be enriched in a more immunophenotypically restricted population i.e. CD34+CD38-Lin- based on xenogeneic organ transplant theoretical accounts. For most clinical organ transplant protocols, CD34+ remained to the standard phenotypical markers for the numbering of HSC. Unlike HSC, the haematopoietic primogenitors are defined by their proliferative potency ( clonogenic potency ) in semi-solid civilization medium in which single primogenitors ( settlement organizing unit, CFU ) signifier offsprings that would be clustered as a settlement. Progenitors that are restricted to myeloid distinction will organize settlements of granulocytes ( G ) and macrophage ( M ) are known as CFU-GM. ( fig. 1 ) .

Fig. 1 Human Hematopoiesis

1.3 Mobilization regimen

1.3.1 Chemotherapy and HGFs

The construct of PBSC mobilisation arose from the phenomenal observations by McCredie et Al. ( 1971 ) and Richman et Al. ( 1976 ) that high dose chemotherapy significantly increased go arounding root cells at a clip that coincides with the early neutrophil recovery from the low-water mark. Since so, the usage of chemotherapeutic agents, peculiarly cyclophosphamide together with haematopoietic growing factors ( HGF ) , has been widely used in mobilisation protocol. The output of PBSC could be farther enhanced by increasing the dosage of chemotherapy ( Lie et al. 1996 ) . Using chemotherapy as a mobilisation agent has a theoretical benefit of in-vivo tumour purging prior to PBSC aggregation and resulted in higher cell output and few aphaeresis Sessionss ( Gertz et al. 2009 ) . However, it is associated with neutropenia and thrombopenia, ensuing in morbidity and occasional mortality. Generally, PBSC can be collected when the circulating CD34+ cells exceed 20 ten 106/L as neutrophil counts begin to lift from low-water mark. However, the optimum clip of PBSC mobilisation could be variable because recovery from cytopenic stage after chemotherapy was unpredictable ( To et Al. 1997 ) .

1.3.2 Hematopoietic growing factors ( HGFs )

Another mobilisation government is by 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 use mobilising agents. Socinski et Al. ( 1988 ) and Duhrsen et Al. ( 1988 ) showed that go arounding CFU-GM increased unusually by disposal of GM-CSF and G-CSF. This formed the footing of utilizing hematopoietic growing factors ( HGFs ) entirely in root cell mobilisation.

Advantage of utilizing HGFs avoid cytotoxic side consequence caused by chemotherapy. Administraion of HGFs is besides applicabe to mobilising normal healthy givers in allogeneic HSCT. 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 in same dosage ( 5Aµg/kg/day ) , the output of CD34+ cells in GM-CSF arm was 3-folded lower than G-CSF and more side effects had been observed in GM-CSF ( Bensinger et al. 2009 ; Fischmeister et Al. 1999 ) . Hence, GM-CSF is seldom used presents. However, for patients who failed mobilisation by G-CSF, a combination of G-CSF with GM-CSF might be efficacious for re-mobilization ( Cottler-Fox et al. 2003 ) .

However, the optimum dosage of G-CSF and GM-CSF have non been deteremined. The dosage of G-CSF scopes from 10I?g/kg/day to high dosage every bit high as 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 ( Bensinger et al. 2009 ) . Kroger et Al. ( 2000 ) suggested that a important betterment in CD34+ cell output if G-CSF was administrated subcutaneously twice daily in an interval of 12 hours at 5I?g/kg. For those patients who failed primary mobilisation, equal figure of root cells might be collected in re-mobilization by administrated at high dose G-CSF ( 32I?g/kg/day ) ( Gazitt et al, 1999 ) .

1.3.4 Plerixafor

Plerixafor ( MozobilA® ; Genzyme Corp. , Cambridge, MA, USA ) was licensed in 2008 by the United States Food and Drug Administration for usage with G-CSF to mobilise HSC for autologous organ transplant in patients with NHL and MM. For patients who failed mobilisation by conventional regimens, Plerixafor showed a important betterment in root cell output. Plerixafor is a fresh agent that binds specifically to CXCR4 receptor in bone marrow stroma and interferes with the formation of SDF-1I±/CXCR4 composite between BM stromal cells and HSC, taking to the release of the latter into the blood watercourse. Phase III clinical test ( DiPersio et al. 2009 ) showed that 71.6 % myeloma patients achieved a‰? 6 ten 106 CD34+ cells/kg cells in less than 2 aphaeresis after disposal of plerixafor with G-CSF whereas merely 34.4 % achieved under G-CSF entirely. 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 plerixafor versus G-CSF entirely were 95.3 % and 88.3 % severally. The figure of go arounding CD34+ cells increased 4.8-fold after plerixafor while merely increased 1.7-fold with G-CSF entirely. Patients usually received plerixafor on Day 4 12 hours prior to apheresis on Day 5. Fowler et Al. ( 2009 ) showed that 90 % patients having combination of G-CSF plus plerixafor gathered sufficient root cells ( scope 2.5 – 6.2 x 106/kg ) for autologous root cell organ transplant in their 2nd efforts who have failed mobilisation antecedently.

The optimum mobilisation regimens for PBSC aggregation has non been determined. Previous surveies have demonstrated that the deficiency of haematopoietic reconstitutions by PBSC transplants collected by either chemomobilization or G-CSF entirely ( Gertz et al. 2009 ; Narayanasami et Al. 2001 ) . Dingli et Al. ( 2006 ) besides argued about the in vivo purging effects of intermediate dosage CY ( 3g/m2 ) chemomobilization and demonstrated a deficiency of difference in complete response rate of PBSCT when compared to G-CSF entirely regimen.

1.4 Multiple Myeloma ( MM )

Multiple Myeloma ( MM ) comprises about 10-15 % of haematological tumor and 1 % of all malignant neoplastic diseases ( Kyle & A ; Rajkumar, 2008 ) . It is characterized by the presence of unnatural plasma cell in the bone marrow and the skeleton, ensuing in secernment of monoclonal Igs or visible radiation ironss. MM is by and large classified by the specific monoclonal proteins secreted from the plasma cells. It is a group heterogenous diseases with overall endurances changing from few old ages to decennaries. The bulk of patients are aged with a average age of oncoming of about 60-70 old ages old. Clinically, the patients typically present with anemia and bone hurting ensuing from infiltration of bone marrow and skeleton by neoplastic plasma cells. Patients with terrible diseases can besides show with nephritic failure and hyperviscosity. A figure of presenting systems are available for omen, including the Durie Salmon ( DS ) and the International Staging System ( ISS ) , taking into consideration of unnatural biochemical trials, the grade of anemia, skeletal devastation and nephritic disfunction.

The optimum intervention of MM has non been defined. In the yesteryear, aged patients were treated with steroid and alkylating agents like Alkeran and less than 5 % patients can accomplish complete remittal. Occasional immature patients were treated with chemotherapy followed by allogeneic bone marrow organ transplant with the possibility of a remedy. However, this attack was associated with important transplant-related mortality ( TRM ) of every bit high as 30-40 % . As a consequence, the intervention has been unsatisfactory ( Barlogie et al. 2004, Kyle and Rajkumar, 2009 ) . Recent progresss in the pathogenesis of MM have led to novel therapy for this disease. This includes immunomodulatory agents like thalidomide and lenolidomide and proteosome inhibitor bortezomib ( VelcadeA® ) . These agents are used entirely or in combination with steroid and chemotherapy, ensuing in higher complete remittal rate ( Table 1 ) . Despite these interventions, the diseases often backslidings and farther consolidation with autologous root cell organ transplant is now considered as the criterion intervention, with TRM of less than 5 % and a possibility of protracting endurance in this group of patients. Collection of equal autologous PBSC for organ transplant becomes a requirement for successful MM intervention. The impact of these fresh curative agents on PBSC output is soon ill-defined.

Table 1. Summary of consequences associated with CR ( Complete response ) and OS ( overall endurance ) rate utilizing fresh curative agent for initiation therapy of multiple myeloma

Mention

Palumbo et Al. 2008.

Facon et Al. 2007.

Harousseau et Al. 2010.

Cavo et Al. 2010.

Luwig et Al. 2011.

Abbreviaions: MPT, Alkeran, Orasone, and thalidomide ; MP, Alkeran and prenisone ; VD, Velcade and Decadron ; VAD, Oncovin, doxorubicine, and Decadron ; VTD, bortezomib, thalidomide, and Decadron ; TD, thalidomide, Decadron ; PAD, bortezomib, doxorubicin, and Decadron.

Median Overall endurance ( OS )

45 months vs 47.6 months ( p = 0.79 )

51.6 months vs 33.2 months ( p = 0.027 )

3 old ages OS, 81.4 % vs 77.4 % ( p = 0.572 )

3 old ages OS, 86 % vs 84 % ( p = 0.3 )

Superior in PAD arm

Complete response ( CR ) ( % )

15.6 % vs 3.7 %

13 % vs 2 %

39.5 % vs 22.5 %

58 % vs 41 %

49 % vs 34 %

Number of patients

129 V 126

125 V 196

240 V 242

236 V 238

371 V 373

Regimens

MPT V MP

VD V VAD

VTD V TD

PAD V VAD

1.5 Determination of hematopoietic recovery

Successful hematologic reconstitution after autologous PBSCT depends on the dosage of root cells infused. A minimal figure of CD34+ cell dosage at 2 x 106 cells/kg receiver organic structure weight is needed to guarantee rapid hematologic reconstitution. In the pre-CD34+ epoch, CFU-GM clonogenic check of the transplant facilitates appraisal of granulopoiesis reconstitution after PBSCT. An appraisal of 10-50 ten 104 CFU-GM/kg are considered optimum for hematologic reconstitution ( To et Al. 1997 ) . However, CFU-GM check is labour intensive and can merely supply information about myeloid primogenitors but non true HSC. On the other manus, numbering of CD34+ by flow cytometry provides quantitative measuring of root cell in the transplant. Research findings demonstrated that there is a important correlativity between CD34+ cells and CFU-GM ( Gianni et al, 1989 ; Jansen et Al, 2007 ; To et Al, 1986 ) .

1.6 Research inquiry and hypothesis

In this present survey, I attempted to turn to the inquiry if G-CSF entirely can practically replace chemomobilization for patients with MM during PBSC mobilisation in a cohort of patients who have been treated with thalidomide and bortezomib containing regimens. The usage of plerixafor in patients who might neglect G-CSF mobilisation will besides be evaluated. The pros and cons of these mobilisation regimens will be compared.

Chapter 2

Patients and Methods

2.1 Patients

Back-to-back MM patients who underwent autologous root cell organ transplant in Queen Mary Hospital, Hong Kong, between 2009 – 2010 were studied. Clinical informations including the infirmary stay for mobilisation and PBSCT, CD34+ cells, CFU-GM output every bit good as haematological recovery after PBSCT were analyzed. Autologous PBSC were cryopreserved and re-infused to patients harmonizing to the criterion processs in the Bone Marrow Transplantation Centre, Queen Mary Hospital. Absolute neutrophil engraftment was defined when neutrophil count reaches systematically above 0.5 tens 109/L for at least 3 yearss. Platelet engraftment is defined when thrombocyte count rises to above 50 ten 109/L unsupported by transfusion.

2.2 Mobilization and aggregation of PBSC

Patients were divided into 3 cohorts. In the chemomobilization ( CY-G-CSF ) group, patients received a dosage of cyclophosphamide ( 3g/m2 ) on twenty-four hours 1 of mobilisation followed by G-CSF ( 5Aµg/kg G-CSF administrated at every 12 hours ) disposal on twenty-four hours 2 onwards. PB CD34+ cells were monitored mundane after twenty-four hours 10. In the G-CSF group, patients were given G-CSF entirely ( 5Aµg/kg G-CSF administrated every 12 hours ) for four yearss. Plerixafor ( 0.24 mg/kg ) was given subcutaneously on twenty-four hours 4 eventide in patients ( G-CSF-P ) in whom the treating doctors considered probably to neglect mobilisation based on low PB CD34+ cells or deficient PBSC harvested with G-CSF entirely. CD34+ count in peripheral blood and PBSC merchandise were enumerated by flow cytometry ( Cytomics FC500, Beckman Coulter, USA ) under ISHAGE guideline. Go arounding CD34+ cells was enumerated on twenty-four hours 8 in the CY-G-CSF, and twenty-four hours 5 in the G-CSF and G-CSF-P group. PBSC aggregation by aphaeresis will get down when peripheral blood CD34+ rose above 10/Aµl and G-CSF would be continued until completion of PBSC aggregation. Aphaeresis was performed by continuous-flow blood cell centrifuge ( COBE Spectra, Gambro BCT, Lakewood, CO, USA ) . Optimum CD34+ dosage was defined as 4 x 106/kg and minimum dosage was 2 ten 106/kg of receiver organic structure weight.

Multiple Myeloma Patient underwent autologous root cell organ transplant

Mobilization regimen

G-CSF+P group

G-CSF group

Figure 2. Study algorithm

Continuous G-CSF support and administered plerixafor ( 10 hours before approaching aphaeresis ) until a‰? 2 x 106/kg CD34+ cells were collected

2 ten 5Aµg/kg/day G-CSF

( 5 yearss )

Sign of diminishing in

PBSC CD34+ cells

Sign of diminishing in

PB go arounding CD34+ cells

PB CD34+ count

~ 10 cells/Aµl

Leukapheresis

Enumeration of CD34+ cell in root cell merchandise

PB CD34+ count

~ 10 cells /Aµl

Leukapheresis

Enumeration of CD34+ cell in root cell merchandise

Continuous G-CSF support until a‰? 2 x 106/kg CD34+ cells were collected

Daily WBC monitoring

Daily WBC monitoring

Monitoring yearss to neutrophil engraftment after autologous root cell organ transplant

CFU-GM clonogenic check

3g/m2 Cyclophosphamide ( Day 1 )

plus

2 ten 5Aµg/kg/day G-CSF

( Day 2-9 )

CY+G-CSF group

2.3 Cryopreservation

The aphaeresis merchandise incorporating PBSC was transferred to stem cell transplant research lab for cryopreservation on the same twenty-four hours. Fresh stop deading medium was prepared day-to-day as stock solution for each aphaeresis merchandise incorporating 20 % dimethyl sulfoxide ( DMSO ) ( Cat. WAK-DMSO-10, WAK-Chemie Medical GmbH, Germany ) , 70 % Hanks ‘ balanced salt solution ( HBSS ) ( Cat. 14170-112, Gibco, USA ) and 10 % autologous plasma. Freezing medium was kept in 4oC until usage. Volume of the harvested root cell merchandise was measured by aspiration syringe and diluted by HBSS to want volume. Equal volume of stock stop deading medium was easy added to the aphaeresis merchandise which was pre-cooled at 4oC for 30 proceedingss with soft agitation. The nucleated cell concentration of each bag was adjusted to 1 – 1.5 ten 108/ml. Concluding concentration of DMSO in the cryopreserved root cell merchandise would be 10 % , incorporating 5 % autologous plasma. Mixture of root cell merchandise was so divided into cryocyte-freezing bags ( Nexell, Irvine, CA, USA ) or CryoMACS freeze bags ( Miltenyi Biotec, GmbH, Germany ) together with three cryogenic phials each incorporating 0.5ml of root cells constituent as quality control. The cell merchandises were placed into a controlled-rate deep-freeze ( Kryo-10, Planer, England ) and cooled under a stepwise chilling plan ( Ramp 1: 10oC to 4oC at 5oC/min. , Ramp 2: clasp at 4oC for 10 min. , Ramp 3: 4oC to -5oC at 2oC/min. , Ramp 4: -5oC to -40oC at 1oC/min. , Ramp 5: -40oC to -160oC at 5oC/min. ) . The cryopreserved bags together with the phials were stored in a liquid N storage armored combat vehicle until ready for re-infusion.

2.4 CFU-GM clonogenic check

An aliquot of aphaeresis merchandise was diluted and adjusted to 0.5 tens 106/ml WBC with Hanks ‘ balanced salt solution ( Cat. 14170-112, Gibco, USA ) . 0.4ml at 0.5 x 106/ml cells suspension was dispensed into 4ml methylcellulose based media incorporating colony-stimulating factors ( MethoCult H4534, Stem cell engineerings, Vancouver, British Columbia, Canada ) . 1 ml aliquot of cell-methylcellulose media was plated in triplicate in 35-mm civilization dishes. The concluding concentration of each civilization dish contained 0.5 ten 105 nucleated cells. After 14-days of humidified ( & gt ; 95 % ) incubation at 37oC with 5 % CO2, CFU-GM settlements were counted utilizing an upside-down microscope. A bunch of 30 cells formed together would be classified as one settlement ( fig 3a, 3b ) . The frequence of CFU-GM settlements was averaged and entire CFU-GM was calculated based on entire nucleated cells in the aphaeresis merchandise.

Entire figure of CFU-GM was calculated by the undermentioned equation:

Entire WBC in the PBSC merchandise

0.5 x 105

Average CFU-GM settlements

ten

Entire CFU-GM =

Entire CFU-GM/kg was determined by spliting the entire CFU-GM by organic structure weight ( kilogram ) of receiver.

Entire CFU-GM

organic structure weight of receiver ( kilogram )

Entire CFU-GM/kg =

Fig. 3a. CFU-GM, 40x

Fig. 3b. CFU-GM, 100x

2.5 Enumeration of CD34+ cell and gating scheme

The CD34+ flow cytometric analysis in peripheral blood and root cell merchandise was carried out by Flow Cytometry Laboratory, Department of Haematology, Queen Mary Hospital, Hong Kong. Analysis of absolute count of CD34+ cells was performed by commercial analytical kit Stem-Kita„? . Cells staining was performed harmonizing to the maker ( ref. IM3630, Beckman Coulter, USA ) . CD34+ cells finding was harmonizing to ISHAGE guidelines in which the Stem-Count flourospheres enabled absolute numeration of CD34+ cells. Briefly, two tubings labelled CD45/CD34/7-AAD each incorporating 20Aµl anti-CD45-FITC/anti-CD34-PE were prepared for CD34+ cell numbering. Another tubing labelled CD45/Ctrl/7-AAD incorporating 20Aµl anti-CD45-FITC/IsoClonic Control-PE was prepared for negative control. 100Aµl of fresh blood/apheresis sample in WBC concentration a‰¤ 15 ten 109/ml ( if necessary, sample needed to be diluted in DPBS to make the coveted concentration ) was added into each tubing. The tubings were instantly vortex smartly for 5 seconds and incubated at room temperature for 15 proceedingss in the dark. Two milliliters ( 2ml ) of ammonium chloride lysing solution was added to each of the three tubings after incubation to lyses ruddy cells with gently whirls. Then, all tubings were incubated at room temperature for another 15 proceedingss in the dark. After ammonium chloride lysis, 100Aµl of Stem-Count flourospheres was added to each tubing with instantly whirl. Samples were kept in dark on runing ice and ready for flow cytometric analysis within 1 hr. The sample was good vortexed before informations acquisition.

2.5.1 Gating Scheme

Enumeration of feasible CD34+ cells in blood/apheresis specimen was performed by single-platform protocol with consecutive gating method harmonizing to ISHAGE ( International society of hematotherapy and transplant technology ) guidelines. Beckman Coulter CytoMic FC500 ( USA ) flow cytometer equipped with CXP system package was employed to execute samples analysis. As shown in fig. 4, histogram 1 displayed side-scatter versus 7-AAD where part J ( 0.3 % ) was set to include all the non-viable events. Histogram 2 displayed all feasible events on histogram 1. Region A on histogram 2 was positioned to include all leucocytes except cell dust, thrombocyte sums and CD45- events. Feasible CD34+ cells ( ruddy bunch with low side spread and low/immediate CD45+ staining ) and lymphocytes part ( bright CD45+ , low side spread ) are besides displayed within part A. A entire figure of 75,000 CD45+ events were collected on histogram 2. Appropriate scene of part is of import to function as a denominator for the computation of per centum of CD34+ cells. Histogram 3 was gated on part A. A distinct bunch from dim to bright feasible CD34+ events with low side spread was displayed in part B. Region C on histogram 4 which was set on part A and B to except thrombocyte sums and cell dust to qualify true CD34+ events. Region D was gated by part A, B and C whether other events with weak CD34+ and CD45+ staining will be eliminated. Same process was done in negative control. After gating processs established, absolute figure of feasible CD34+ cells per Aµl was automatically calculated by the system package.

Histogram 1

Histogram 2

Histogram 3

Histogram 4 ( CD34+ cells )

Histogram 6 ( Negative control )

Histogram 5

Fig. 4. Gating scheme of CD34+ numbering by utilizing Stem-Kita„? . Methodology of informations acquisition was described in patients and methods. ( Section 2.6.1 )

2.6 Autologous Stem cell graft conditioning regimen

Patients received high dose chemotherapy ( Melphalan 200 mg/m2 patient surface country ) 2 yearss before root cell graft, harmonizing to the organ transplant protocol in the Bone Marrow Transplantation Unit, Queen Mary Hospital. Cryopreserved PBSC was thawed by submergence in a 37oC to 40oC waterbath with uninterrupted shaking and gently massage until seeable ice was wholly melted. Thawed merchandises were transfused instantly to the patients.

2.7 Statistical analysis

Consequences are expressed as mean i‚± criterion mistake of mean ( SEM ) . Comparison between groups of informations was evaluated by 2-sided t-test. Comparison of neutrophil and thrombocyte engraftments between groups was evaluated by Kruskal-Wallis H Tests. Pearson ‘s correlativity coefficient was used to analyze the correlativity between CD34+ and CFU-GM. Statistical computations were performed with computing machine package IBM SPSS, Version 13. A p-value of less than 0.05 is considered statistical important.

Chapter 3

Consequences

3.1 Patient population

From Jan-2009 to Oct-2010, a entire figure of 28 multiple myeloma patients were enrolled in this survey. Eighteen patients received CY-G-CSF mobilisation. Ten patients were mobilized by G-CSF entirely of whom five of them have required plerixafor at the discretion of handling hematologists. The baseline features of the three groups were tabulated in Table 2. One patient whose PBSC was mobilized have non underwent autologous PBSCT.

Table 2 Patients features

Mobilization regimen

CY+G-CSF

( CY group )

G-CSF entirely

( G-CSF group )

G-CSF + Plerixafor

( P group )

Gender

Male

9

3

4

Female

9

2

1

Age ( old ages )

Mean

54.11

50.6

55.2

Median ( scope )

53 ( 46-63 )

54 ( 50-60 )

57 ( 46-63 )

Body Weight ( kilogram )

Mean

64.8

54.6

60.1

Median ( scope )

62 ( 49-89 )

54 ( 50-60 )

61.7 ( 44-77 )

Myeloma type harmonizing to type of paraprotein

Immunoglobulin g

( kappa )

9

2

( lambda )

1

Immunoglobulin a

( kappa )

2

1

1

( lambda )

2

Immunoglobulin d

( lambda )

1

Non-secretory

1

Light concatenation

( kappa )

2

1

( lambda )

1

2

2

Induction therapy #

Thal/Dex

3

1

Pad

10

2

VTD

2

5

2

VCMD + PAD

1

VAD + VTD + TD

1

VTD + Thal/Dex

1

Conditioning Regimens

Melphalan ( 200mg/m2 )

18

5

4

# Thal/Dex: Thalidomide/Dexamethasone ; PAD: Bortezomib, Adriamycin, Dexamethasone ; VTD: Bortezomib, Thalidomide, Dexamethasone ; VCMD: Vincristine, Cyclophosphamide, Melphalan, Dexamethasone ; VAD: Vincristine, Adriamycin, Dexamethasone

3.2 Clinical features during root cell mobilisation

The average infirmary stay in the CY+G-CSF group was 13 yearss ( scope: 11-15 yearss ) . The average low-water mark neutrophil and thrombocyte count were 0.095 ten 106/ml ( scope: 0 – 0.9 x 106/ml ) and 68.5 tens 106/ml ( scope: 13 – 175 x 106/ml ) despite uninterrupted G-CSF disposal. Patients in the G-CSF or G-CSF-P groups did non necessitate hospitalization and there was no lessening in neutrophil or thrombocyte counts during mobilisation ( Table 3 ) .

Table 3. Clinical features during root cell mobilisation

Mobilization regimen

CY+G-CSF

G-CSF

G-CSF+P

Hospitalization yearss

Mean

13

4.8

4.2

Median

13 ( 11-15 )

5 ( 3-6 )

4 ( 3-6 )

Nadir neutrophil counts ( x 106 /ml )

0.095 ( 0-0.9 )

n/a

n/a

Nadir thrombocyte counts ( x 106 /ml )

68.5 ( 13-175 )

n/a

n/a

3.3 Impact of CD34+ cell dosage in different mobilisation regimen

The entire figure of CD34+ cells/kg collected was shown in fig. 2. The average figure of entire CD34+ cells collected in CY+G-CSF, G-CSF and G-CSF+P groups were 8.265 ten 106/kg ( scope: 3.1 – 24.5 x 106/kg ) , 2.8 ten 106/kg ( scope: 2.10 – 3.56 x 106/kg ) and 2.38 tens 106/kg ( scope: 2.06 – 3.33 x 106/kg ) severally.

Fifteen out of 18 ( 15/18 ) patients in the CY+G-CSF group achieved the minimal figure of 2 ten 106/kg CD34+ root cell in one aphaeresis session. Three patients achieved sufficient PBSC in two Sessionss. All patients in the G-CSF and 4 patients in G-CSF+P group have required at least two aphaeresis Sessionss. Plerixafor significantly increased PBSC output compared with patients who received G-CSF merely ( Figure 4 and Table 5 ) .

Table 5. Apheresis output of CD34+ cells/kg ( ten 106/kg ) in each group

CY+G-CSF

G-CSF

G-CSF+P

No. of patients

18

5

5

Median values and ( scope )

Aphaeresis 1

8.265 ( 1.64 – 24.4 )

1.81 ( 1.34 – 2.01 )

0.84 ( 0.31 – 2.89 )

Aphaeresis 2

1.46 ( 0.91 – 1.72 )

0.92 ( 0.31 – 1.55 )

1.43 ( 0.64 – 2.07 )

Aphaeresis 3

n/a

n/a

1.51 ( 1.51 )

Entire CD34/kg

8.265 ( 3.1-24.5 )

2.8 ( 2.01 – 3.56 )

2.38 ( 2.06 – 3.33 )

Mean

9.46

2.68

2.57

S.E. Mean

1.33

0.28

0.24

p-value

0.001

0.001

Fig. 4. Entire CD34+ cells /kg collected in each group

3.4 Impact of CFU-GM clonogenic check

The average doses of infused CFU-GM in the CY-G-CSF, G-CSF and G-CSF-P groups were 119 ten 104/kg ( scope: 44.7 – 311 x 104/kg ) , 67.76 ten 104/kg ( scope: 20.6 – 107.2 x 104/kg ) and 102.84 tens 104/kg ( scope: 41 – 251.6 x 104/kg ) severally.

Table 5. Clonogenicity of CFU-GM ( x 104/kg ) infused in PBSC merchandise

CY+G-CSF

G-CSF

G-CSF+P

No. of patients

18

5

5

Median values and ( scope )

Aphaeresis 1

119 ( 25.1 – 311 )

35 ( 5.3 – 66.5 )

25.2 ( 10.3 – 73.6 )

Aphaeresis 2

28.1 ( 19.6 – 35.9 )

30 ( 15.3 – 52.4 )

42.05 ( 30 – 57.9 )

Aphaeresis 3

n/a

n/a

166 ( 166 )

Entire CFU-GM/kg

119 ( 44.7 – 311 )

65 ( 20.6 – 107.2 )

73.6 ( 41 – 251.6 )

Mean

132.28

67.76

102.84

S.E. Mean

16.93

16.69

37.84

p-value

0.052

0.371

Fig. 6. Entire CFU-GM/kg ( x 104 ) infused under different mobilisation regimen

3.5 Correlation between CD34+ cell and CFU-GM

The correlativity coefficient between CD34+ and CFU-GM in CY-G-CSF and G-CSF groups were shown in Figure 6. There was statistical correlativity in CD34+ cells collected and transplanted CFU-GM in CY group ( R = 0.822, P & lt ; 0.001 ) . However, we found that there was a weak correlativity and no important correlativity between collected CD34+ cells and transplanted CFU-GM in G-CSF group ( R = 0.377, P = 0.282 ) .

Fig. 7a. CY group

Fig. 7b. G-CSF and G-CSF+P group

Fig. 7 Correlation graph associating entire CD34+/kg collected and entire CFU-GM/kg infused. a ) . correlativity between collected CD34+ cells ( x 106/kg ) and CFU-GM infused ( ten 104/kg ) in CY group ; r = 0.822, P & lt ; 0.0001. B ) . correlativity between collected CD34+ cells ( x 106/kg ) and CFU-GM infused ( ten 104/kg ) in G-CSF and G-CSF+P group ; r = 0.377, P = 0.282.

3.6 Dynamicss of go arounding CD34+ cells after Plerixafor disposal

There were 5 patients in the G-CSF-P group. Their PB CD34+ cell counts and outputs were listed in Table X. Plerixafor disposal resulted in important addition in PB CD34+ counts 12 hours after disposal ( before: 14.75 i‚± 2.14/Aµl V after: 41.75 i‚± 4.85/Aµl, p=0.02 ) . The entire CD34+ cell output was besides increased ( before: 0.88 i‚± 0.33 x 106/kg V after: 2.49 i‚± 0.29 x 106/kg, p=0.021 ) .

Table 6. Fold addition of peripheral blood CD34+ cell count after plerixafor disposal

Patient

Pre-Plerixafor

Post-Plerixafor

Fold addition ( x )

( cells/Aµl )

PB CD34+ ( cell/Aµl )

CD34/kg

( x 106 )

Collected

PB CD34+ ( cell/Aµl )

CD34/kg

( x 106 )

Collected

1

14

0.571

33

1.64

2.36x

2

12

0.635

45

1.51

3.75x

3

21

0.841

35

1.21

1.67x

4

12

0.31

54

2.07

4.5x

5

6

n/a

n/a

2.89

n/a

3.7 Impact on figure of aphaeresis processs

Merely three patients under CY+G-CSF groups needed for 2nd aphaeresis Sessionss while all the patients in G-CSF groups and 4 patients in the G+CSF-P were required at least 2 aphaeresis Sessionss to roll up sufficient root cells.

Table 7. Number of aphaeresis processs

CY+G-CSF

G-CSF

G+CSF-P

No. of patients

18

5

5

Aphaeresis 1

15

0

1

Aphaeresis 2

3

5

3

Aphaeresis 3

n/a

n/a

1

Mean

1.17

2

2

S.E.M.

0.09

0

0.32

p-value

& lt ; 0.001

0.002

3.8 Impact on engraftment yearss

In the entire 28 patients that underwent root cell mobilisation, one patient in the G-CSF-P group did non continue to autologous PBSCT. Neutrophil engraftments were similar in the three groups ( Kruskal-Wallis H Test, P = 0.062 ) . Furthermore, no significance difference were observed in platelet engraftment among the three groups ( Kruskal-Wallis H Test, P = 0.319 ) . In fact, there was no correlativity between neutrophil and thrombocyte engraftments and the CD34+ or CFU-GM doses in each graft ( Figure XX )

Table 8. Dayss of neutrophil and platelet engraftment in different group

CY+G-CSF

G-CSF

G-CSF+P

No. of patient

18

5

4

Engraftment Days* :

ANC

Plt

ANC

Plt

ANC

Plt

Mean

10.06

15.1

10.8

18.8

10

14.5

S.E.M.

0.19

1.41

0.2

4.57

0

0.65

Median

10

13

11

15

10

14.5

Scope

8-11

9-31

10-11

13-37

0

13-16

*Neutrophil engraftment: ANC a‰? 0.5 ten 106/ml for 3 back-to-back yearss

Platelet engraftment: Plt & gt ; 50 x 106/ml

R = -0.366, P = 0.135

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to neutrophil engraftment in CY+G-CSF group

R = -0.4, P = 0.1

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to platelet engraftment in CY+G-CSF group

R = -0.395, P = 0.105

R = -0.224, P = 0.371

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to neutrophil engraftment in G-CSF group

R = 0.706, P = 0.182

R = 0.494, P = 0.397

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to platelet engraftment in G-CSF group

R = -0.423, P = 0.478

R = 0.152, P = 0.807

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to neutrophil engraftment in G-CSF + P group

Fig. Xx Correlation of entire CD34+ cells/kg collected and entire CFU-GM/kg infused to platelet engraftment in G-CSF + P group

R = -0.825, P = 0.175

R = -0.466, P = 0.534

Chapter 4

Discussions

4. Discussion

High dosage chemotherapy followed by autologous PBSCT is presently the criterion intervention for patients with multiple myeloma but the optimum mobilisation regimens have non been determined ( Lie & A ; To, 1997 ) . In this survey, we demonstrated that Cy+G-CSF mobilisation resulted in higher CD34+ cell output and fewer figure of aphaeresis Sessionss compared with those who received GCSF with or without plerixafor. Similar observations have been extensively reported. A figure of retrospective surveies have demonstrated a 1 to 5 fold addition in CD34+ cell output in CY+G-CSF compared with G-CSF entirely ( Gertz et al. 2009 ; Alegre et Al. 1997 and Mark et Al. 2008 ) . CY may damage the ability of HSC to adhere to the BM microenvironment, thereby heightening their mobilisation into the circulation Cesana et Al. ( 1998 ) . The difference in CD34+ cell output between chemomobilization and G-CSF entirely might be overcome by a higher dosage of G-CSF ( 24Aµl/kg/day ) of the latter as demonstrated by Kroger et Al. ( 1998 ) . The evident high quality of chemomobilization over standard dosage of G-CSF has led to the usage of former regimen in Queen Mary Hospital, Hong Kong. However, as new curative agents for myeloma intervention have emerged that are much less myelotoxic than conventional chemotherapy, and fresh mobilisation agent plerixafor has become available, it is possible that HGF entirely may do as mobilisation agent. This issue was tested in the present survey in which we addressed its feasibleness in a individual establishment and there were a figure of issues which might hold an impact on patient direction.

First, we demonstrated that a higher CD34+ cell output in the CY+G-CSF group had no clinical advantage into footings of engraftment or infirmary stay during organ transplant. A cell dosage of 2 x106 cells/kg was considered minimum for successful engraftment. Our consequences suggested that a higher cell dose had no advantage and corroborated with those of Stiff et Al. ( 2010 ) and Narayanasami et Al. ( 2001 ) who showed that neutrophil and thrombocyte engraftment were non cell dose dependant. However, this was different from those of Ketterer et Al. ( 1998 ) , Shpall et Al. ( 1998 ) and Statkute et Al. ( 2007 ) who demonstrated a more rapid haematopoietic recovery with higher cell dosage. The consequences were besides in contrast to those of umbilical cord blood organ transplant in which engraftment and organ transplant result were critically dependent on the cell dosage in the transplant ( Wagner et al. 2002, Avery et Al. 2010 ) . Therefore, a lower cell output in the G-CSF groups with or without plerixafor had no negative impact on patient result. However, the long-run result in these patients would hold to be carefully evaluated in prospective survey.

Second, we demonstrated that the usage of cyclophosphamide during mobilisation necessitate hospitalization and resulted in profound neutropenia and thrombopenia during mobilisation. This was associated with possible hazard of potentially fatal infection and hemorrhage. All 18 patients undergoing CY+G-CSF mobilisation recovered uneventfully from cytopenia. However, mortality during chemomobilization has been reported ( Fitoussi et al. 2001 ) ( Ref, allow me cognize if you ca n’t happen it ) .

Third, despite the fact that our patients have received minimum myelotoxic agents in their initial intervention, five out of 10 patients failed to accomplish the minimum cell dosage for organ transplant and have required the add-on of plerixafor. An norm of 2.57-fold addition in go arounding CD34+ cells was observed and all patients achieved sufficient cell output after plerixafor. Plerixafor was usually given on twenty-four hours 4 eventide in a 5 yearss G-CSF mobilisation protocol ( Calandra et al. 2008 ; Fowler et Al. 2009 ; Basak et Al. 2010 ) . However, in the present survey, plerixafor was merely given to patients in whom the treating hematologists considered likely to neglect G-CSF mobilisation based on low PB CD34+ counts or CD34+ cell output in the initial crops. This “ risk-adapted ” scheme might restrict the usage of this expensive agent to patients who truly necessitate it and may cut down the drug cost ( HKD $ 50,000 per dosage ) incurred to the establishment.

Finally, we have besides examined CFU-GM in the PBSC transplant. Before the broad handiness to flux cytometry, To et Al. ( 1986 ) suggested that CFU-GM in the transplant could be used to foretell the hematologic recovery after organ transplant. Later, a strong correlativity between go arounding CD34+ cells and CFU-GM were demonstrated ( Siena et al. 1991 ) . Similar correlativity was observed in the present survey. In the present survey, we demonstrated a close relationship between CFU-GM/kg and CD34+ cells/kg in CY+G-CSF group with R value of 0.822, ( P & lt ; 0.0001 ) but non in G-CSF group ( R = 0.377, P = 0.282 ) . The correlativity coefficient in CY+G-CSF group of this survey was higher than that reported by Jansen et Al. ( 2007 ) . On the other manus, the deficiency of important correlativity in the G-CSF could be explained by the smaller patient figure compared with the CY+G-CSF group. Whether there was a echt functional difference in HSC or primogenitors mobilized by different mobilisation regimens would hold to be farther investigated. At present, CD34+ cell numbering remained to be the criterion in most organ transplant Centre although CFU-GM may be helpful in confidence of quality of the cryopreserved transplant after dissolving.

Our survey is limited in sample size and its prospective nature. In the hereafter, prospective and carefully controlled survey should be performed to compare the PBSC output and clinical result of the three regimens. Furthermore, the indicant for the usage of plerixafor should be standardized so that the consequences could be translated into practical guidelines based on the “ risk-adapted ” attack in this survey. The impact of cyclophosphamide as in vivo purging on long term disease control would hold to be prospectively evaluated.

Chapter 5

Decision

5.1 Decision

The handiness of newer mobilisation agent plerixafor has enabled us to measure the feasibleness of G-CSF mobilisation in our Centre in which CY+G-CSF has been the criterion. The CY+G-CSF regimen was superior in PBSC output and few aphaeresis session needed but these benefits were offset by the demand of hospitalization and profound cytopenia at low-water mark during mobilisation and the deficiency of advantages in footings of haematopoietic recovery. The long-run result of patients mobilized with these protocols would hold to be evaluated prospectively.