Post on 29-Jul-2022
BIOLOGICAL DETERMINANTS OF THERAPEUTIC RESPONSE TO L-
ASPARAGINASE IN CHILDREN WITH ACUTE LYMPHOBLASTIC
LEUKAEMIA.
A thesis submitted to the University of Manchester for the degree of Doctor in Philosophy
In the Faculty of Medical and Human Sciences
2012
Dr. Ashish Narayan Masurekar
Supervisor
Professor Vaskar Saha
School of Cancer and Enabling Sciences Division of Cancer
Contents
2
Contents
Pages
Title 1 Contents 2 Figures and Tables 6 Abbreviations 9 Abstract 12 Declaration 13 Copyright Statement 14 Dedicated to 15 Gratitude 16 Acknowledgements 17 Chapter 1 Introduction 19 1.1 Background of childhood ALL 19 1.1.1 Clinical aspects 19 1.1.2 Aetiology 20 1.1.3 Therapy of ALL 24 1.1.4 Current outcome in childhood ALL 25 1.1.5 Heterogeneity observed in the clinical response to therapy 26 1.1.6 Current issues in treatment of childhood ALL 27 1.1.7 Relapse in childhood ALL 28 1.2 L-Asparaginase 29 1.2.1 Rationale for studying the determinants of response to ASNase 29 1.2.2 Structure of ASNase 30 1.2.3 Mechanism of action of ASNase 31 1.2.4 ASNase- source and products 31 1.2.5 Problems with ASNase therapy 32 1.2.6 Determinants of therapeutic response to ASNase 33 1.2.7 Preliminary data 35 1.3 Asparaginase Study 35 1.3.1 Rationale of this study 35 1.3.2 Hypothesis 37 1.3.3 Eligibility 37 1.3.4 Aims of the study 37 1.3.5 Objectives of the study 37 1.3.6 Study design 37 Chapter 2 Material and Methods 39 2.1 Reagents 39 2.2 Asparaginase study 39 2.2.1 Patient sample collection 39 2.2.2 Sample processing 39
Contents
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2.3 Techniques 40 2.3.1 Tissue culture 40 2.3.1.1 Cell lines 40 2.3.1.2 Cell culture, passage and collection of cell pellets and
supernatants 40
2.3.2 Molecular methods 41 2.3.2.1 RNA extraction 41 2.3.2.2 cDNA synthesis 42 2.3.2.3 Real time TaqMan gene expression assay for AEP 43 2.3.2.4 MicroRNA (miRNA) array 44 2.3.3 Biochemical methods 46 2.3.3.1 Protein extraction 46 2.3.3.2 Protein quantification 46 2.3.3.3 L-Asparaginase activity assay 46 2.3.4 Immunological methods 48 2.3.4.1 Chemiluminescent AEP ELISA 48 2.3.4.2 Immunoblotting 50 2.3.4.3 Immunoprecipitation assay 54 2.3.5 Other techniques 55 2.3.5.1 Capture of microvesicles 55 2.3.5.2 Immunoblotting of microvesicle lysate 58 2.3.5.3 In-vivo labelling of cells for AEP by fluorescent probe 58 2.3.5.4 Statistical methods 58 Chapter 3 Development of assays 60 3.1 Plasma Asparaginase activity assay-Indoxine method 60 3.1.1 Background for the assay 60 3.1.2 Principle of the assay 60 3.1.3 Performance of the assay 61 3.1.4 Pre-analytical effect of sample transport on Asparaginase activity 64 3.1.5 Summary 64 3.2 Expression of AEP by RQ-RTPCR in cell lines and patient
samples 66
3.2.1 Quality of RNA obtained from patient samples 66 3.2.2 Dynamic range of the assay 68 3.2.3 Consistency of the step of reverse transcription 68 3.2.4 Amplification efficiency of the assay 68 3.2.5 Selection of control gene 68 3.2.6 Expression of AEP by RQ-RTPCR in cell lines 72 3.2.7 Expression of AEP by RQ-RTPCR in patient samples 72 3.3. AEP ELISA 73 3.3.1 Development history of the assay 74 3.3.1.1 1st generation AEP ELISA 74
3.3.1.2 2nd generation AEP ELISA 77
3.3.1.3 3rd generation AEP ELISA 80
3.4 Quantifying active AEP 82 3.5 Summary of assays tested for AEP expression 89 Chapter 4 Asparaginase Study: L-Asparaginase activity 90 4.1 Background 90 4.1.1 Aims of the Asparaginase Study 94 4.1.2 Objectives of the Asparaginase Study 94 4.1.3 Design of the Asparaginase Study 95 4.1.4 Recruitment in the Asparaginase Study 97
Contents
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4.2 Results 98 4.2.1 Recruitment in the UKALL2003 arm of the Asparaginase Study 98 4.2.1.1 Patient characteristics 99 4.2.1.2 ASNase activity at individual time points 100 4.2.1.3 Serial ASNase activity results 102 4.2.1.4 Correlation of induction ASNase activity with biological factors and
therapeutic outcome 104
4.2.1.4 Time to event analysis 106 4.2.2 Results of patiens recruited in ALLR3 arm of the Asparaginase
Study 109
4.2.2.1 ALLR3 ASNase activity results 109 4.2.2.2 Outcome analysis of patients in ALLR3 who did not get PEG-
ASNase 110
4.3 Discussion 112 Chapter 5 Asparaginase Study: Determinants of ASNase activity 115 5.1 Background 115 5.2 Results 115 5.2.1 Role of cysteine proteases as predictive biomarkers to ASNase
therapy 115
5.2.2 Impact of Anti L-Asparaginase antibodies 121 5.2.2.1 Anti L-Asparaginase antibodies: Patients enrolled in the
UKALL2003 trial 121
5.2.2.1.1 The correlation between serial ASNase activity and antibodies to L-Asparaginase
121
5.2.2.1.2 Correlation between anti L-Asparaginase antibodies and clinical hypersensitivity
124
5.2.2.2 Anti L-Asparaginase antibodies: Patients enrolled in the ALLR3 study
124
5.2.3 Toxicity after PEG-ASNase 124 5.3 Discussion 128 Salient points of chapters 4 and 5 131 Chapter 6 Role of Bone Marrow Stromal Exosomes in Conferring Chemoprotection to Leukaemic Cells
133
6.1 Background 133 6.1.1 Drug resistance in ALL is multi-factorial 133 6.2 Results 137 6.2.1 Bone marrow stromal cell derived conditioned medium (BMSC-
CM) confers chemoprotection to SUPB15 cell line 137
6.2.2 Generation of SUPB15 MR cells 138
6.2.3 Exosomes in the BMSC-CM contributed to the BMSC-CM mediated chemoprotection
139
6.2.4 HS5 derived exosomes are taken up by SUPB15 and primary ALL cells
140
6.2.5 BMSC derived exosomes conferred broad spectrum chemoprotection to SUPB15 cells
141
6.2.6 Horizontal transfer of Micro-RNS (miRNA) from BMSC to leukaemic cells- a mechanism by which BMSC derived exosomes could confer broad spectrum chemoprotection to SUPB15MR cells
142
6.2.7 BMSC derived exosomal miRNAs target ROS pathway in leukaemic cells
150
Contents
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6.2.8 Further characterisation of SUPB15MR cells 151
6.3 Discussion 154 Chapter 7 Work in progress 158 7.1 Background 158 7.1.1 Nomenclature of membrane-limited vesicles 158 7.1.2 Functional role of vesicles 161 7.2 Preliminary results- Harvesting B cell derived MV 163 7.2.1 Pellet obtained after stage one of the method was enriched in
microvesicle markers and contained CD19 164
7.2.2 Immobilisation of CD19 expressing SD1 microvesicles on UltraLink A/G resin
166
7.2.3 Quantifying PKH67 labelled cell line MV by flow cytometry 168 7.2.4 Immobilised vesicles expressed microvesicle markers 170 7.2.5 MV from patient plasma 170 7.3 Discussion 172 Chapter 8 Concluding remarks 174 References 189 Achievements 213 Appendix Appendix 1 216 Appendix 2 219 Appendix 3 223 Appendix 4 235 Appendix 5 236
Total word count, including reference and appendix: 49,284
Tables and Figures
6
Figures and tables Pages
Chapter 1
Figure 1.1 Cytogenetic abnormalities in childhood ALL 22
Figure 1.2 Overview of standard chemotherapy treatment in childhood ALL
24
Figure 1.3 Outcome of ALL in successive MRC chilhood ALL trials 25
Figure 1.4 Event free survival according to cytogenetics 27
Figure 1.5 Schedule of 1st two weeks of therapy in UKALL2003 30
Figure 1.6 Quartenary structure of ASNase 31
Table 1.1 Genetic alterations in childhood ALL 23
Table 1.2 ASNase products in clinical use 33
Table 1.3 Predicted pattern of response to PEG-ASNase 36
Chapter 2
Figure 2.1 Composition of mix 1 and 2 for cDNA synthesis 42
Figure 2.2 Schematic diagramm of AEP gene and the region of binding of TaqMan probe
44
Figure 2.3 Composition of the reverse transcription mix 45
Figure 2.4 Capture of microvesicles 57
Table 2.1 Reagents used for infra red and chemiluminescent immunoblotting
53
Table 2.2 Concentrations and source of antibodies 58
Chapter 3
Figure 3.1 MAAT assay 61
Figure 3.2 Linear range of the Indoxine assay 62
Figure 3.3 Performance of the Indoxine assay 63
Figure 3.4 Effect of sample transport on ASNase activity 65
Figure 3.5 Quality of patient RNA 67
Figure 3.6 Dynamic range of the assay 69
Figure 3.7 Consistency of the RT step 70
Figure 3.8 Performance of the control gene 71
Figure 3.9 Expression of AEP by RQ-RTPCR in cell lines 72
Figure 3.10 Expression of AEP by RQ-RTPCR in clinical samples 73
Figure 3.11 Performance of the 1st generation AEP ELISA 75
Figure 3.12 Quantification of AEP in SD1 cells using a 1st generation AEP ELISA
76
Figure 3.13 Optimisation of capture antibody of the 2nd generation AEP ELISA
78
Figure 3.14 Linear range and intra-assay variation of the 2nd generation AEP ELISA
79
Figure 3.15 Quantification of AEP in SD1 cells using a 2nd generation AEP ELISA
80
Figure 3.16 3rd Generation AEP ELISA 81
Figure 3.17 Basis of action of LP1 probe 83
Figure 3.18 Localisation of active AEP in SD1 cells 84
Tables and Figures
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Figure 3.19 Expression of active AEP by flow cytometry/Image stream 85
Figure 3.20 Suitability of LP 1 probe to measure active AEP 87
Figure 3.21 Correlation of expression of AEP at transcrip and protein levels
88
Chapter 4
Figure 4.1 Schedule of L-Asparaginase and sample time points 96
Figure 4.2 Overview of the statistical methods used to analyse the results
97
Figure 4.3 Recruitment in the Asparaginase Study-UKALL2003 arm 98
Figure 4.4 ASNase activity at individual time points 100
Figure 4.5 ASNase activity levles at TP1 and TP2 in NCI SR, precursor B ALL patients with good risk cytogenetics
106
Figure 4.6 Time to event analysis of patients in UKALL2003 with respect to response to PEG-ASNase
108
Figure 4.7 ASNase activity in ALLR3 patients 109
Figure 4.8 Contribution of PEG-ASNase at relapse 110
Table 4.1 Use of L-Asparaginase in chilhood ALL 91
Table 4.2 Patient characteristics 99
Table 4.3 Median ASNase activity levels 101
Table 4.4 Serial ASNase activity 103
Table 4.5 Correlation between induction ASNase and baseline characteristics
104
Table 4.6 Correlation between induction ASNase levels and outcome 105
Chapter 5
Figure 5.1 Correlation between AEP expression and baseline characteristics
117
Figure 5.2 Correlation between AEP expression, response to PEG-ASNase and outcome (SER/MRD)
118
Figure 5.3 Correlation between CTSB expression and baseline characteristics
119
Figure 5.4 Correlation between CTSB expression, response to PEG-ASNase and outcome (SER/MRD)
120
Figure 5.5 Correlation between hypersensitivity and ASNase activity 123
Table 5.1 Correlation between ASNase activity and anti L-Asparaginase antibodies
121
Table 5.2 Incidence of toxicity to PEG-ASNase 125
Table 5.3 Reported adverse reactions to ASNase 126
Chapter 6
Figure 6.1 Comparision between 2D and organotypic 3D culture systems
137
Figure 6.2 BMSC-CM confers non selective chemoprotection to leukaemic cells
138
Figure 6.3 Protective ability of <3kd fraction of BMSC 139
Tables and Figures
8
Figure 6.4 Transmission electron microscopy of <3kd fraction of BMSC pelleted by ultracentrifugation
140
Figure 6.5 Cellular uptake of BMSC exosomes 140
Figure 6.6 BMSC derived exosomes confer chemoprotection. 141
Figure 6.7 Exosomes contain small RNA 143
Figure 6.8 miRNA expression profiles in host and tumour- drug sensitive and drug resistant
148
Figure 6.9 Connection between miRNA and ROS 151
Figure 6.10 Gene expression in SUPB15MR cells 152
Figure 6.11 ROS levels in SUPB15 compared with SUPB15MR 153
Figure 6.12 Origin of exosomes 155
Table 6.1 Pharmacological heterogeneity in childhood ALL 135
Table 6.2 Expression of miRNAa in HS5, HS5 exosomes, SUPB15 and SUPB15MR cells
145
Table 6.3 Previously described role of miRNA in chemoprotection and or cell survival or cell cycle or cell metabolism
149
Chapter 7
Figure 7.1 Characteristics of pellet obtained after stage one of MV isolation method
165
Figure 7.2 Immobilisation of SD1 microvesicles on to protein A/G Ultralink resin
167
Figure 7.3 Quantification of MV in cell supernatants 169
Figure 7.4 Protein content of immobilised MV 170
Figure 7.5 Isolation of MV from patient plasma 171
Table 7.1 Key features of membrane limited vesicles 160
Table 7.2 Vesicles in health and disease 162
Table 7.3 Microvesicle cargo and cancer 163
Chapter 8
Figure 8.1 Proposed central role of BMSC derived soluble factors in conferring chemoprotection to leukaemic cell
178
Figure 8.2 Concise summary of normal processes involved in regulating coagulation during homeostasis
185
Figure 8.3 Mechanism involved in clot formation in response to vessel injury
187
Table 8.1 MV participate in coagulation 184
Abbreviations
9
Abbreviations
AEP Asparaginyl Endopeptidase
AIEOP Associazione Italiana Ematologia Oncologia Pediatrica
ALL Acute Lymphoblastic Leukaemia
Ara C Cytosine Arabinoside
ARID AT-rich interaction domain
Asn Asparagine
ASNase Asparaginase
ASNS Asparagine Synthetase
Asp Aspartic acid
BCR B cell receptor
BFM Berlin Frankfurt Münster
BM Bone marrow
BMSC Bone marrow stromal cells
BMSC-CM Bone marrow stromal cell-conditioned medium
BSA Bovine serum albumin
CD Cluster of differentiation
cDNA Complementary deoxyribonucleic acid
CI Confidence interval
CIGMR Centre of Integrated and Genomic Medical Research
CNS Central nervous system
CR Complete remission
CSF Cerebrospinal fluid
CS Citrate Synthetase
CT Cycle threshold
CTSB Cathepsin B
CV Coefficient of variation
D/D Double-distilled (deionised)
DAPI 4',6-diamidino-2-pheylindole
DEPC Diethylpyrocarbonate
DMSO Dimethylsuplhoxide
DNA Deoxyribonucleic acid
DTT Dithiotreitol
E.coli Escherichia coli
EBV Ebstein Barr Virus
ECL Enhanced chemiluminescence
EDTA Ethylenediamine tetra acetic acid
ELISA Enzyme Linked Immunosorbitant Assay
FAB French American British
FAM 6-carboxy-fluorescein phosphoramidite
FBS Fetal bovine serum
FISH Fluorescent in situ hybridisation
g Relative centrifugal force
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GLS Glutaminase
HCl Hydrochloric acid
HH High hyperdiploid
HR High risk
HRP horseradish peroxidise
ICAM1 Inter-cellular adhesion molecule 1
IDH Isocitrate dehydrogenase
IKZF1 Ikaros family zinc finger protein 1
Im Intramuscular
Abbreviations
10
IR Intermediate risk
IT Intrathecal
Iv Intravenous
JAK Janus Kinase
JmjC Jumonji domain-containing
kDa Kilodalton
LFA Leukocyte funtion-associated antigen-1
LIMS Laboratory information management system
MFI Mean fluorescent intensity
MGG May-Grünwald Giemsa
MHC Major Histocompatibility
miRNA micro RNA
MMP Matrix metalloproteinase
MNC Mononuclear cells
MRC Medical Research Council
MRD Minimal residual disease
MREC Multi Regional Ethical Committee
MTx Methotrexate
MV Microvesicles
MW Molecular weight
NAD Nicotinamide adenine dinucleotide
NADH Nicotinamide adenine dinucleotide, reduced form
NADP Nicotinamide adenine dinucleotide phosphate
NADPH Nicotinamide adenine dinucleotide phosphate, reduced form
NOPHO Nordic Society for Pediatric Hematology
OAA Oxaloacetate
OD Optical density
PAGE Polyacrylamide gel electrophoresis
pBALL Precursor B acute lymphoblastic leukaemia
PBS Phosphase buffered saline
PCR Polymerase chain reaction
PEG Polyethyleneglycol
PEG-ASNase Pegalyted E coli L-Asparaginase
PEITC Phenethyl isothiocyanate
Ph+ALL Philadelphia chromosome positive ALL
PI Protease inhibitor
PICR Paterson Institute for Cancer Research
PVDF Polyvinylidene fluoride
r2
Pearson coefficient of determination
RNA Ribose nucleic acid
ROC Receiver operating characteristic curve
ROS Reactive oxygen species
RPMI Roswell Park Memorial Institute
RQ-RTPCR Real time reverse transcriptase polymerase chain reaction
RT Room temperature
SD Standard deviation
SDS Sodium dodecyl sulphate
SE Standard error
SER Slow early response
SIL SCL(stem cell leukaemia) interrupting locus
SR Standard risk
TAL1 T cell acute lymphoblast leukaemia 1
TAMRA 6-carboxy-tetramethyl-rhodamine
Abbreviations
11
TCCSG Tokyo Children's Cancer Study Group
TCF3-PBX Transcription factor 3- pre b cell leukaemia homeobox 1
TCR T-cell receptor
TEMED Tetramethylethylenediamine
TET2 tet methylcytosine dioxygenase 2
TGF-β Transforming growth factor beta
Tris Tris(hydroxymethyl)aminomethane
UNG Uracil-N-Glucosidase
v/v Volume/volume
VAMP3 Vesicle associate membrane protein 3
w/v Weight/volume
WCC White cell count
α KG Alpha ketoglutarate
β2M Beta-2 microglobulin
2-HG 2 hydroxyglutarate
Abstract
12
Institution: The University of Manchester Name: Dr Ashish Narayan Masurekar Degree Title: Doctor of Philosophy (PhD) Thesis title: Biological Determinants of Therapeutic Response to L-Asparaginase in Children with Acute Lymphoblastic Leukaemia. Introduction: Intensification of chemotherapeutic agents in use since the 1970’s has led to over 80% survival in childhood ALL. The price of cure is treatment related morbidity and mortality that now approaches the incidence of relapse. Further improvement in outcome need better understanding the biological basis behind disease heterogeneity and identifying new targets and therapeutic strategies. Patients and Methods: We monitored trough ASNase activity at one or more time points in 451 patients treated in UKALL2003 and ALLR3 trials. The activity was correlated with prognostic determinants using assays mostly developed in house and data collated from the national trials. Two in-vitro models were created, one to test microenvironment mediated drug resistance and the second to test tumour related thrombosis. Results: Over 85% of patients [n=451; 427 (UKALL2003) and 24 (ALLR3)] had adequate ASNase activity levels. For UKALL2003 patients, the incidence silent neutralising antibodies (4.7%); clinical hypersensitivity (3.7%); thrombosis (3.1%) and pancreatitis (1.5%) was lower than previously reported. Hypersensitivity was mostly (n=16/17) seen in regimen C. There was a significant association between inadequate activity in induction and MRD levels in regimen A (p=0.03), especially so if they additionally had good risk cytogenetics (p=0.006). Older patients had higher incidence of inadequate ASNase activity during induction (p=0.0097). Patients in regimen C were more likely to inactivate ASNase in post induction phase (p=<0.01); less likely to recover from inadequate response to ASNase during induction (p=<0.01) more likely to experience toxicity to ASNase. ALLR3 patients: Silent antibodies were not observed in 16/24 patients that were tested. ASNase did not appear to influence outcome of patients in ALLR3. A model of microenvironment mediated multi-drug resistance showed a role of exosomal miRNA, of bone marrow stromal cell origin, in altering the redox state and chromatin pattern of leukaemic cell. In the second model, tumour associated microvesicles were shown to have a thrombogenic potential. Conclusion: Patients with good risk features depend on ASNase for disease clearance in the early phase. Routine testing would identify those with inadequate ASNase levels and improve the resolution of the current prognostic system used to decide post induction therapy. High risk patients have higher incidence of toxicity to ASNase and show a multidrug resistant phenotype where response to ASNase is not sufficient. A better understanding of disease biology is needed to design new treatment strategies in these patients.
Declaration
13
DECLARATION: I hereby declare that no portion of the work referred to in the thesis has been
submitted in support of an application for another degree or qualification of
this or any other university or other institute of learning.
Dr Ashish Narayan Masurekar
Copyright statement
14
COPYRIGHT STATEMENT:
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to this thesis) owns any copyright in it (the “Copyright”) and s/he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and/or teaching purposes.
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iv. Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available from the Head of School of (insert name of school) (or the Vice-President) and the Dean of the Faculty of Life Sciences, for Faculty of Life Sciences’ candidates.
Dedicated To
15
This thesis is dedicated to my parents Narayan Achyutrao
Masurekar and Ranjan Narayan Masurekar and to all my
teachers.
Gratitude
16
Gratitude
I am most grateful to Professor Vaskar Saha for providing me the opportunity
to pursue a PhD and for his constant help, encouragement and guidance as a
mentor. Under his leadership I experienced a sense of abundance of
resources and enjoyed total freedom to pursue lateral ideas.
I am thankful to Dr Jizhong Liu, for supervising me in the laboratory and for
being a friend, philosopher and guide during my journey.
Dr Mark Holland and Dr David Gilham for their input and help in optimising
AEP ELISA.
Members of the Children’s Cancer Group (CCG) both past and present for
their support.
Paterson Institute for Cancer Research for providing an academic and
scientific environment.
Housemates at 33 Morningside Drive for their constant support and
encouragement and for putting up with my unsocial hours.
Cancer Research UK for giving me the fellowship and Leukaemia and
Lymphoma Research Fund for funding the project
Parents of children, young adult patients and clinical colleagues who supported the project.
Acknowledgement
17
Acknowledgements
I am grateful to the following people for their advice, help and support
Sample tracking Charlie O' Horo, Catriona Parker, Parisa Mahjoob Afag, Neelofar Charfer
Sample Processing Centre of Integrated and Genomic Medical Research: Rebecca Cole, Genevieve Pridham,
Late John-Paul Allen, Debbie Payne & Hazel Platt
Maintaining of Database Adiba Hussain, Catriona Parker AEP ELISA Jizhong Liu, Mark Holland, David
Gilham, John Bridgeman AEP RQ-RTPCR Stuart Pepper. Molecular biology
core facility, PICR Providing baseline patient characteristics & data on toxicity
Professor Ajay Vora, Sue Richards
Anti Asparaginase Antibodies Hans-Jürgen Kühnel, Monica
Essink; Medac GmbH, Wedel, Germany
Native E.coli Asparaginase Lorna Livingstone, Medac GmbH
(UK), Stirling, UK Ewinase Jonathan Morgan; EUSA
Pharma, Stevenage, UK Cytogenetics Professor Anthony Moorman MRD Jeremy Hancock Statistical analysis Sue Richards, Rachel Wade, Ric
Swindell Helping hand in generating results Asparaginase activity assay
(Adiba Hussain, Caroline Fong);
Acknowledgement
18
Protein quantification (Adiba Hussain, Caroline Fong); CTSB
ELISA (Caroline Fong); RNA extraction (Adiba
Hussain). Imaging Achille Dunne, Steve Bagley Flow cytometry Morgan Blaylock, Geoff Barry dRVVT assay Lynne Keighley, Central
Manchester Foundation Trust Hospitals
Informal advice Dave Gilham, Geoff Margison,
Karim Labib, Nullin Divecha, Iman van Debout, Mandy
Watson, Christopher Morrow
Introduction
19
Chapter 1 INTRODUCTION
______________________________________________________________
1.1 BACKGROUND OF CHILDHOOD ALL:
The normal development of B and T lymphocytes is characterised by an
orderly and progressive differentiation of haematopoietic stem cells into
common lymphoid progenitor cells followed by lineage restricted B and T
precursor cells and then the development of mature lymphocytes. Acute
lymphoblastic leukaemia (ALL) is a neoplasm affecting lymphocytes at any
stage during their development in the bone marrow (B cells) or in thymus (T
cells). It is classified into two categories: precursor B lymphoblastic leukaemia
and T lymphoblastic leukaemia according to the 2008 World Health
Organisation classification of acute leukaemia (1). In the UK approximately
400 children are diagnosed to have ALL each year. It accounts for
approximately 80% of all cases of childhood leukaemia and a quarter of all
childhood cancers making it the most common paediatric malignancy.
Amongst children with ALL, approximately 80% of the cases are children have
precursor B ALL (pBALL).
1.1.1 Clinical aspects: ALL has a peak incidence between 2 and 5 years of
age. pBALL originates in the bone marrow, while T-cell ALL on occasion
originate in the thymus. Children present with symptoms and signs of bone
marrow failure, with or without additional involvement of extra medullary sites
such as the liver, spleen, lymph nodes, thymus, meninges and gonads. The
presenting blood white cell count (WCC) can vary from being undetectable to
greater than 100 x 109/L (hyperleukocytosis) [Normal WCC: 4 -11 x 109/L].
The ensuing bone marrow failure leads to multi-organ failure and death of a
child in the absence of prompt treatment.
The diagnosis of ALL is suspected on the basis of a combination of clinical
findings, abnormal blood counts and presence of lymphoblasts in blood
smears. Tests done to confirm the diagnosis of ALL include a bone marrow
Introduction
20
aspirate with or without trephine biopsy, and immunophenotyping of blast cells
in bone marrow or peripheral blood.
ALL follows a heterogeneous clinical course. With therapy, >85% of children
will be cured (2, 3). The most important adverse event in the treatment of ALL
is disease recurrence. The current relapse rate is ~ 10% (4-6), which is
associated with a high incidence of CNS involvement ~ 40%, compared to ~
2% at presentation (5, 7, 8). The outcome of children who relapse is poor
with survival rates between 15 to 69% (9-11).
1.1.2 Aetiology of ALL: The aetiology of ALL is unknown. Only in <5% of
cases do we find an underlying inherited predisposing condition such as
Down syndrome, Bloom syndrome, Ataxia-Telangiectasia or Nijmegen
breakage syndrome. Ionising radiation (2), prior therapy involving DNA
topoisomerase II inhibitors (12) and germline polymorphisms of IKZF, a gene
encoding for transcription factor IKAROS, as well as the ARID5 genes (13, 14)
have been implicated in ALL as well. The role of a host of other potential
environmental factors predisposing to ALL such as parental occupation,
maternal reproductive history, parental tobacco or alcohol use maternal diet,
exposure to pesticides and magnetic fields have been studied. The evidence
for these factors is conflicting and their roles have not been established (15).
Clustering of cases of ALL between 2-5 years of age has fuelled two
hypotheses suggesting a role of abnormal response to infection: population
mixing hypothesis and the delayed infection hypothesis. Population mixing
hypotheses suggests that the peak in ALL observed in this age group is due
to exposure of non immune children to infections after population mixing with
carriers. The delayed infection hypothesis in addition suggests a mechanism
whereby a pre existing pre-leukaemic clone transforms to leukaemia following
an abnormal response to infection (16).
Conventional cytogenetics and FISH detects genetic abnormalities in greater
than 75% of cases of ALL(3). Newer high resolution techniques such as a)
microarray based gene expression profiling, comparative genomic
Introduction
21
hybridization arrays and single nucleotide polymorphism analysis; b) DNA
methylation arrays and c) whole genome sequencing techniques can identify
changes in virtually all cases of ALL. ALL is biologically a heterogeneous
disease. Broadly the lesions in ALL can be classified into recurrent
chromosomal aberrations including balanced or reciprocal translocations,
aneuploidies (hyperdiploidy or >50 chromosomes; hypodiploidy or <45
chromosomes and trisomies of chromosome 4, 10, 17 and 21) and gene-
specific alterations [Figure 1.1 and Table 1.1]. The genes most commonly
altered are those that are involved in regulation of lymphoid differentiation, cell
cycle, apoptosis and cell signalling (3).
Whilst the above mentioned techniques have identified a number of somatic
genetic abnormalities, they do not in all cases separate mutations that drive
the leukaemia (driver mutations) from passenger mutations. Of the genetic
abnormalities identified, chromosomal translocations are the most well
described. The exchange of material between two chromosomes leads to
either i) a promoter substitution mutation where a normally quiescent
oncogene becomes constitutively active under the influence of the new
promoter or ii) creation of an in-frame chimeric or fusion gene which is
oncogenic. Promoter substitution mutations are more common in T
lymphoblastic leukaemia whereas fusion genes are more common in B
lymphoblastic leukaemia. These translocations involve either transcription
factor genes that are crucial in B or T cell differentiation or genes that are
involved in key signalling pathways such as protein kinases and they disrupt
key pathways of cell differentiation, proliferation and survival.
Chromosomal translocations are important in the initiation of leukaemia and in
most instances arises in-utero (16-21). They induce, expand and sustain a
pre-leukaemic clone. For example the ETV6-RUNX1 fusion is thought to
sustain the pre-leukaemic clone by a reduced sensitivity of the pre-leukaemic
clone to TGF-β mediated inhibition of cell proliferation (22).
Introduction
22
Chromosomal translocations differ in their capacity to generate a full
leukaemic phenotype (3, 23, 24). ETV6-RUNX1 fusions arise in B lineage
progenitor/stem cells and are seen in 1% of normal prenatal haematopoiesis
but only 1% of those cases go on to develop ALL (21). ETV-RUNX1 ALL is
associated with on average six copy number variations (CNV). This indicates
that the malignant transformation of lymphocytes bearing the ETV6-RUNX1
fusion is a multistep process that requires additional gene specific ‘co-
operating oncogenic lesions (23). Contrary to this, MLL translocations have a
greater leukaemogenic potential as evidenced by their ability to cause ALL
during infancy, show concordance rates that are close to 100% in identical
twins and be associated with less than one CNV per case(2, 3).
Currently, chromosomal translocations are considered to be chance events
(25) that occur following DNA double strand breaks coupled with illegitimate
recombination by non homologus end joining processes (17, 26). At the DNA
level, most gene fusions remain poorly characterized and why only a select
few genes, mostly encoding transcription factors or tyrosine kinases, are
involved in chromosomal translocations is unknown (25). Potentially, these
genetic recombination events produce aberrant expression of genes that
provide a mechanism of survival.
Hyperdipliody, 25%
Hypodiploidy, 1%
ETV6-RUNX1, 25%
ERG deletion, 7%
CRLF2 over expression, 6%
E2A-PBX1, 5%
iAMP21, 2%
Others, 7%
T cell, 12%
MLL rearrangements, 8%
BCR-ABL, 3%
Hyperdipliody, 25%
Hypodiploidy, 1%
ETV6-RUNX1, 25%
ERG deletion, 7%
CRLF2 over expression, 6%
E2A-PBX1, 5%
iAMP21, 2%
Others, 7%
T cell, 12%
MLL rearrangements, 8%
BCR-ABL, 3%
Introduction
23
Figure 1.1: Cytogenetic abnormalities of childhood ALL. The lesions illustrated
in the dark pink colour are observed in exclusively in B lymphoblastic
leukaemia. Lesions illustrated in light pink (not characterised) are observed
exclusively in T lymphoblastic leukaemia and includes rearrangements
involving HOX11L2, LYL1, TAL1, HOX11 genes. MLL rearrangements shown
in grey are more common in B cell than T cell lymphoblastic leukaemia.
Lesions found in the ‘others’ category in dark blue are more common in T cell
than B cell lymphoblastic leukaemia and include normal karyotype which is
observed in ~50% of cases of T lymphoblastic leukaemia. Adapted from (3,
27).
Table 1.1: Genetic alterations in ALL
ALL is biologically a heterogeneous disease involving a number of different
mutations ranging from gross cytogenetic abnormalities to gene specific point
mutations identified only by single nucleotide polymorphism (SNP) arrays.
MLL-ENL fusion* is observed in both T cell and B cell acute lymphoblastic
leukaemia. CALM-AF10 is observed in acute myeloid leukaemia as well as in
both T and B ALL. Adapted from (17).
Subtype of ALL molecular lesion Functional product
B cell acute lymphoblastic leukaemia
i) translocations
t(12;21) ETV6-RUNX1 fusion Chimeric transcription factor
t(1;19) TCF3-PBX1 fusion Chimeric transcription factor
t(9;22) BCR-ABL fusion Activated kinase
t(17;19) TCF3-HLF fusion Chimeric transcription factor
11q23 translocations MLL rearrangements Chimeric transcription factor
ii) aneuploidy
hyperdiploidy Altered gene dose
hypodiploidy Altered gene dose
trisomsy 4, 10, 17 & 21 Altered gene dose
iii) gene specific abnormalities
>50
T cell acute lymphoblastic leukaemia
i) translocations
7q34 translocations multiple TCRB & TCRG rearrangements promotor substitution involving transcription factors
14q11 translocations multiple TCRA & TCRD rearrangements promotor substitution involving transcription factors
t(10;11) CALM-AF10 fusion Chimeric transcription factor
11q23 translocation MLL-ENL fusion* Chimeric transcription factor;
t(4:11) translocation NUP98-HOXA9 Chimeric transcription factor
iii) extrachromosomal amplification
NUP214-ABL1 fusion Activated kinase
iv) gene duplication
MYB MYB over-expression
v) gene mutations
NOTCH1, FLT3, NRAS, FBW7 Activating mutations
vi) chromosomal deletions
9p21 P15, P16 cell cycle deregulation
6q Unknown Unknown
1p SIL-TAL1 fusion Chimeric transcription factor
Introduction
24
1.1.3 Therapy of ALL: Standard treatment consists of combination
chemotherapy involving often more than 10 drugs. Additionally for some
children central nervous system (CNS) radiotherapy or a bone marrow
transplant is required for cure. The treatment spans over a minimum of 24
months and is divided into following phases: induction, CNS directed therapy,
post induction intensification and continuation [Figure 1.2]. The absolute
minimum goal of induction phase is to reduce the population of lymphoblasts
(leukaemic cells) present at diagnosis in the bone marrow to less than 5%
along with the restoration of normal bone marrow function. This is defined as
a complete morphological remission (CR). The incidence of CR at the end of
induction phase is greater than 95% with current protocols. The goal of
induction therapy is recently directed towards achievements of a molecular
remission. This is defined as minimal residual disease (MRD) of <1 leukaemic
cell/10,000 cells in bone marrow on assessment by techniques such as flow
cytometry or quantitative polymerase chain reaction (RQ-PCR) (28).
Subsequent therapy is still needed in order to aim for a cure. CNS directed
therapy and post induction intensification are essential in order to reduce the
incidence of CNS relapse (29-32) and to consolidate further upon the initial
therapeutic response (33-37). The exact mechanism underlying the protective
effect of continuation therapy is unclear (38). Attempts to taper continuation
therapy have led to inferior results (39).
Figure 1.2: Overview of standard chemotherapy treatment in childhood ALL.
Some children require cranial radiotherapy ± bone marrow transplant. Total
therapy lasts well over 2 years and involves at least 10 drugs delivered in 4
Induction 5 weeks
ContinuationCNS directed therapy/
Consolidation Post inductionintensification
5 weeks 3-9 weeksDivided into 2-4 different
blocks each over 6-7 weeks
125-140 weeks
5-6 drugs 2-6 drugs 8-9 drugs 5 drugs
•Dexamethasone•L-Asparaginase•Vincristine•Daunorubicin*•IT MTx•Mercaptopurine
•IT MTx•Mercaptopurine•Ara C•L-Asparaginase•Vincristine•Cyclophosphamide
•IT MTx•IV MTx•L-Asparaginase•Vincristine•Dexamethasone•Cyclophosphamide•Cytarabine•Doxorubicin•Mercaptopurine
•IT MTx•Oral MTx•Dexamethasone•Vincristine•Mercaptopurine
Introduction
25
main blocks. The intensity of treatment in each phase and the number and
design of each phase especially in the post intensification period depends on
the risk stratification. MTx= Methotrexate; Ara C= Cytosine Arabinoside; iv=
intravenous; IT= intrathecal
1.1.4 Current outcome in childhood ALL: During the last three decades,
the outcome of paediatric ALL has steadily improved from around 50% to over
80% across the developed world (2, 3), including the UK (40), as shown in
Figure 1.3. No new drugs have been incorporated into therapeutic regimens
during this time. This achievement is a result of: i) carefully planned large
scale multicentre trials across the world which have optimised the delivery of
drugs given in the above mentioned phases. ii) adoption of risk stratification
strategies that segregate patients into standard, intermediate and high risk
groups based on their relapse risk and tailors treatment accordingly (30) and
iii) the availability of better supportive therapy.
Figure 1.3: Outcome of ALL in successive Medical Research Council
Childhood ALL trials. UKALL VIII: 1980-1985, UKALL XI: 1985-1997 & ALL
97/99/01: 1997-2002. (Courtesy Professor Ajay Vora).
0 1 2 3 4 5 6 7 8 9 100
25
50
75
100
Per
cen
t ev
ent
free
su
rviv
al
Time in years
UKALL VIII (1980-1985) – 54%
UKALL X & XI (1985-1997) – 60%
UKALL 97/99/01 (1997-2002) - 74%
UKALL 2003 ~85%
Introduction
26
1.1.5 Heterogeneity observed in the clinical response to therapy: There
are a number of risk factors that are predictive of relapse such as age ≥ 10
years and/or presenting white cell count ≥ 50 x109/L (41), adverse
cytogenetics [Hypodiploidy, t(9;22), iAMP21, E2A-HLF and MLL gene
rearrangements] (42), figure 1.3, slow response to early therapy (SER),
defined as presence of ≥ 25 leukaemic cells/100 cells on morphological
assessment of bone marrow after 1-2 weeks of starting therapy, failure to
achieve complete morphological remission at the end of induction phase (2)
and persistence of disease at a molecular level after the end of induction
phase (43). The importance of SER and persistence of disease at molecular
level is expanded below. The advent of new techniques mentioned above
have led to the identification of new lesions associated with poor outcome
such as the deletion of IKZF1(24, 44, 45); over expression of CRLF2 in some
but not all studies (46-49) and presence of JAK mutations (in case of
precursor B ALL) (50); and a distinct gene expression profile associated with
HOX11L2 (51)(in case of T ALL).
The current UKALL 2003 protocol has the following 3 risk groups:
a) Standard risk: all children > 1 & < 10 years with a highest white cell
count before starting treatment of < 50x109/L and who do not have
adverse cytogenetics
b) Intermediate risk: all children ≥10 years old and/or with a diagnostic
white cell count ≥50x109/L(41) and who do not have adverse
cytogenetics
c) High risk: all children, irrespective of initial white cell count or age who
have a SER and or adverse cytogenetics [Hypodiploidy, t(9;22),
iAMP21, E2A-HLF and MLL gene rearrangements] (42) (Figure 1.4).
Children with standard, intermediate and high risk are treated respectively on
regimen A, B and C of the UKALL 2003 protocol. The greater the clinical risk,
the more intense the therapy is in terms of the total number of drugs used,
Introduction
27
their maximum dose, the frequency of administration and the duration of
overall therapy.
Figure 1.4: Event free survival according to their cytogenetics at diagnosis in
childhood ALL-MRC ALL 97 trial. Courtesy Dr. Anthony Moorman, LRF,
cytogenetic group, Newcastle.
1.1.6 Current issues in the Treatment of Childhood ALL: There are two
main issues in the treatment of childhood ALL. Firstly, the improvement in the
outcome of ALL has come at a price. Current treatment protocols have
progressively intensified therapy over the last four decades; as a result a
significant number of children are over-treated on current protocols compared
to the ones used in the 1980’s. This is true even when we take the current risk
stratification strategy into account and compare children on Regimen ‘A’ of
UKALL 2003, who are less likely to relapse and receive least therapy, with all
those treated in UKALL VIII (1980-1985). Current protocols are complex,
expensive and associated with long term toxicities including cardiotoxicity,
secondary neoplasms, osteonecrosis and radiation induced endocrinopathies
or decline in neurocognitive function. Children who have had a bone marrow
transplant are at risk of additional long term side effects in the form of chronic
graft vs host disease of the skin, gut and lungs. Secondly, even on current
treatment protocols the disease relapses in 10-20% of children (4-6) and
EFS According to Cytogenetics - ALL
t(12;21)
High Hyperdiploid
Other
<Hypodiploid
t(9;22)
iAMP21
0.0
00
.20
0.4
00
.60
0.8
01.
00
Eve
nt F
ree
Sur
viva
l
0 1 2 3 4 5 6 7 8 9
Time from diagnosis (years)
Introduction
28
relapse is the most important adverse event in childhood ALL. The clinical
variation in the treatment response is most likely to be due to the underlying
biological heterogeneity of the disease. The two main challenges in childhood
ALL are i) to accurately identify at diagnosis all children who are at a high risk
of disease relapse and separate them from the subgroup of children who can
be cured with the less intensive treatment given in the protocols of the 1980’s
and ii) to understand the mechanisms behind disease relapse in-order to
optimise therapy and improve outcome in this subgroup.
1.1.7 Relapse in childhood ALL: Within all risk factors, slow early response
(SER) (34, 52, 53) and persistent MRD either at the end of induction phase
(43, 54-56) or at day 15 (57) are the most important determinants of poor
outcome (58). While SER is an indicator of speed of response to therapy,
persistent MRD is an indicator of the depth of response. Children with SER
have a worse outcome in all risk categories defined by clinical and biological
features (59, 60). SER is also linked with adverse cytogenetics as over 50%
of children with t(9;22) ALL have a SER (7).
Analysis of genomic copy number abnormalities (CNAs) in samples at
diagnosis and relapse suggests that in >90% of cases disease recurrence is
due to a subpopulation of cells present at diagnosis (61, 62). This suggests
that the clone responsible for relapse is able to survive front line therapy and
therefore undergoes further selection and expansion. Protection from
chemotherapy could be either due to intrinsic drug resistance to one or more
chemotherapeutic agents or extrinsic where the leukaemic blasts are
protected by the host or a combination of the two reasons.
In this thesis I chiefly investigated the incidence of intrinsic resistance to L-
Asparaginase in childhood ALL, it’s possible causes and it’s contribution to
treatment failure (Chapters 4 and 5). In collaboration with Dr. Liu, I looked at
the role of soluble factors secreted by bone marrow mesenchymal cells in
conferring chemoprotection to ALL cells (Chapter 6). My primary task was to
investigate the biological determinants of the response to L-Asparaginase
Introduction
29
(ASNase) (Chapter 4). I shall therefore give an introduction to ASNase in this
chapter.
1.2 L-ASPARAGINASE
ASNase is the only example of a bacterial enzyme used in cancer therapy.
1.2.1 Rationale for studying the determinants of response to ASNase: As
relapse is linked to adverse outcome variables such as SER and or high MRD,
it is plausible that the clone responsible for relapse is resistant to one or more
drugs used during the first 1-2 weeks of therapy. The drugs used during this
phase are steroid, vincristine, ASNase, intrathecal methotrexate and
additionally an anthracycline in patients who are at a higher risk of relapse
[Figure 1.5]. These drugs have different mechanisms of actions and in theory
will tackle the problem of clones with differing chemosensitivity. Clinical trials
over three decades show that ASNase is probably the single most effective
agent used in the induction phase and it has further benefits when repeated in
the post induction period (32, 63-70). It is the only drug used in the first 1-2
weeks of therapy where we have not reached dose limiting toxicities, making
it possible to further optimise its dose (71). There is evidence to suggest that
optimal ASNase activity potentiates the efficacy of dexamethasone which is
another key drug used in ALL induction therapy (72). Thus, ineffective
ASNase therapy could be a major determinant of the response to induction
therapy.
Introduction
30
Days 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Dexamethasone
Vincristine
PEG-ASNase
Daunorubicin*
IT MTx
Figure 1.5 Schedule of first 2 weeks of therapy in UKALL 2003.
Dexamethasone (6 mg/m2) is given as a daily oral dose, PEG-ASNase (1000
U/m2) is given as a single intramuscular injection and Daunorubicin (25 mg/m2)
and vincritsine (1.5 mg/m2) are given intravenously. Daunorubicin * is given
only to high risk patients. IT MTx= intrathecal methotrexate.
1.2.2 Structure of ASNase: The enzyme forms a tetramer in a soluble state
that consists of 4 identical subunits: A, B, C and D arranged in 2-2-2
symmetry [Figure 1.6]. Amongst these subunits the interaction between A and
B and between C and D are the most extensive, resulting in a tetramer which
is in fact a dimer of identical intimate dimers (73). ASNase is active only as a
tetramer. Residues in the N-terminal domain of subunit A interact with
residues of the C-terminal domain of subunit B to create one half of the active
substrate binding pocket. Identical interactions between subunits C and D
contribute to the other half of the binding pocket.
Introduction
31
Figure 1.6: Quaternary structure of E.coli-ASNase. Each monomer (subunit)-
A(pink), B(blue), C(green) & D(coffee) is identical and has a molecular mass
of 35.6 kDa. The green spheres in the centre of the tetramer represent
asparagine molecule sitting inside the active catalytic site that is made by
contributions from all four monomers. The orange regions in each monomer
represent the known B cell epitopes [Paul Bates, London Research Institute].
1.2.3 Mechanism of action of ASNase: ASNase has been used in ALL since
the 1970’s. It converts asparagine and glutamine to aspartate and glutamate
respectively resulting in their depletion from the extracellular fluid
compartment. This is thought to lead to impaired protein synthesis and
apoptosis of leukaemic blasts which are selectively susceptible to ASNase
owing to low/absent Asparaginase Synthetase (ASNS) activity (74).
1.2.4 ASNase- source and products: ASNase used in clinical practice is
either derived from either E.coli (E.coli-ASNase) or from Erwinia (Erwinase).
E.coli-ASNase includes Elspar (Merck Sharp & Dohme, USA) and Medac
(Medac GmbH, Germany) amongst other products (Table 1.2). E.coli-ASNase
and Erwinase have identical mechanisms of action and side effects, although
they do differ in potency. E.coli-ASNase is more potent than Erwinase (75)
A=pink
B=blue C=green
D=coffee
Introduction
32
1.2.5 Problems with ASNase therapy: Apart from drug related toxicities,
there are two major issues with the use of ASNase in clinical practice. Firstly it
is a bacterial protein. Frequent administration leads to the development of
silent neutralising antibodies and/or clinical hypersensitivity to ASNase (76,
77). Secondly, ASNase degradation is probably primarily via the reticulo-
endothelial system (78) leading to wide inter-patient variability in levels after
administration. In small cohort studies using E.coli ASNase, up to 35% of
patients developed neutralising antibodies and 15% developed clinically
significant hypersensitivity reactions (79, 80). The immune response to
ASNase appears to be more prevalent in high-risk ALL patients of whom up to
60% may develop antibodies (79).
The pharmacokinetics of the drug varies between different products; E.coli
ASNase has a longer half life compared to Erwinase. Not all patients achieve
adequate therapeutic levels of ASNase (81), considered to be ≥ 100 IU/Litre
of serum (76, 82, 83). This has clinical implications. Not all children show an
adequate response to ASNase. An adequate therapeutic response is
correlated with both a rapid early response (76) and an improved outcome in
de novo and relapsed ALL (84, 85).
To improve the pharmacokinetic profile of E.coli ASNase, Elspar and Medac
Asparaginases are now available conjugated with polyethylene glycol (PEG-
ASNase). PEG-ASNase has a longer half-life (5.7 days for PEG-ASNase
compared to 1-1.5 days with Erwinase and E.coli-ASNase) (75, 76, 86) and
produces similar ASNase activity with a lower dose. This permits the use of
PEG-ASNase at a lower and less frequent dosage interval when compared to
ASNase (76). PEG-ASNase is also associated with a reduced incidence of
immune response (76, 87), reflecting the lower antigen load. Several studies
have shown the efficacy of PEG-ASNase during induction and post induction
phases when used in newly diagnosed as well as in relapsed patients (32, 76,
84, 88). As a result, PEG-ASNase has replaced ASNase in the frontline and
relapse ALL protocols in the UK. Patients who develop clinical hypersensitivity
to PEG-ASNase can be give Erwinase as antibodies to E. coli ASNase are
Introduction
33
not cross reactive. More recently recombinant PEG-ASNase has been tested
in the setting of a randomised phase 2 clinical trial. This showed that the
recombinant product is comparable to PEG-ASNase in terms of its efficacy,
pharmacokinetics and toxicity (89).
Table 1.2: ASNase products in clinical use.
In the United Kingdom we use PEG-Asparaginase (PEG-ASNase) in front line
treatment protocols. The native product is Medac Asparaginase which is
pegylated by Enzon and marketed by Medac as Oncaspar™ (Recently,
Enzon was bought by Sigma-Tau which will now market PEG-ASNase
directly). Erwinase is used in case of clinical hypersensitivity to PEG-ASNase.
It is manufactured by the health protection agency (HPA) and marketed by
EUSA pharmaceuticals.
1.2.6 Determinants of the therapeutic response to ASNase: A number of
studies have investigated whether resistance to ASNase is due to the
production of asparagine synthetase (ASNS) by lymphoblasts. Some such
studies have demonstrated a correlation between cellular levels of ASNS and
in vitro resistance to ASNase in cell lines and experimental models of
leukaemia (90-92). A correlation between ASNS expression, in vitro
resistance to ASNase and inferior treatment outcome has also been reported
in some but not all genetic subtypes of ALL (93, 94). However, other studies
ASNase Manufacturer Marketed by
E.coli -ASNase
Elspar MSD (Ireland) MSD
Kidrolase Kyowa-Hakka (Japan) EUSA
Colaspase Kyowa-Hakka (Japan)
Medac Asparaginase Kyowa-Hakka (Japan) Medac
E.coli PEG-Asparaginase
Oncospar (UK) Enzon/Kyowa-Hakka (Japan) Sigma-Tau
Oncospar (US) Enzon/MSD Sigma-Tau
Erwinia -ASNase
Erwinase HPA (UK) EUSA
Introduction
34
have failed to find a correlation between the expression of ASNS and in vivo
sensitivity to ASNase or response to therapy in ALL (93, 95). For example,
ALL with t(12;21) is characterised by up-regulation of ASNS in response to
ASNase and yet it is exquisitely sensitive to ASNase with a good clinical
outcome (96) (Figure 1.4).
Despite systemic asparagine depletion lymphoblasts have been shown to
have high levels of intracellular asparagine (97). Recently we have shown that
the glutaminase activity of ASNase is critical to cytotoxicity, i.e the asparagine
depletion alone is insufficient for killing the cancer cell (98). Cancer cells are
dependent on glutamine for many cellular functions, including
chemoresistance. Thus glutamine depletion by ASNase may also sensitise
lymphoblasts to cytotoxicity by other agents. These data cast doubt on the
role of asparagine depletion alone as the main mechanism of cytotoxicity and
the expression of ASNS as the main mechanism behind drug resistance.
Furthermore, in response to systemic asparagine depletion, mesenchymal
cells up-regulate the synthesis of asparagine and protect leukaemic cells by
preventing apoptosis in response to ASNase (99).
We have recently identified that two lysosomal cysteine proteases, Cathepsin
B (CTSB) and Asparaginyl Endopeptidase (AEP) which degrade ASNase
(100). CTSB is ubiquitously expressed by most cells including lymphoblasts
and may play a crucial role in the pharmacokinetic clearance of the drug. AEP
is normally expressed by renal cells. It is aberrantly expressed primarily by
pre-B lymphoblasts of high risk cytogenetic subtypes (101). While CTSB
degrades both Erwinase and E.coli ASNase, AEP is specific for E.coli
ASNase. AEP cleaves at distinct sites carboxy-terminus to either an
asparagine or aspartate (102), in contrast CTSB cleavage sites are non-
specific. In the case of E.coli ASNase AEP was shown to cleave ASNase at
N24, D124 & N143 (100). The AEP induced cleavage fragments retain intact
known antigenic epitopes of E.coli-ASNase. AEP has been previously shown
to accelerate the presentation of tetanus toxin fragment to MHC class II
molecules (103). Thus our observations suggest that the co-expression AEP
Introduction
35
and CTSB by lymphoblasts may hasten the degradation of ASNase leading to
inadequate levels of ASNase during induction therapy. As ASNase modulates
the dexamethasone response, this process may significantly contribute to the
SER in high risk cytogenetic subtypes. Additionally AEP may induce
development of allergic reactions such as hypersensitivity or the development
of neutralising antibodies. The latter would lead to inadequate drug levels
during the delayed intensification phase.
1.2.7 Preliminary data: In a pilot study (n=86, collaboration with Prof. Tim
Eden), we have noted 4 different patterns of response to PEG-ASNase.
Around 60% of patients had good ASNase activity (>100U/L) throughout all
phases of treatment. Twenty percent show good activity during induction but
not subsequently, suggesting the development of neutralising antibodies
during subsequent therapy. 15% show inadequate activity during induction but
satisfactory activity later on as disease burden starts to reduce on therapy.
This suggested that the inhibition of ASNase activity could be due to the
leukaemic state, possibly mediated by cysteine proteases in the leukaemic
cells. About 5% never develop adequate ASNase activity. These patients are
all in the high risk category where a combination of above reasons would lead
to inadequate ASNase activity.
1.3 ASPARAGINASE STUDY
1.3.1 Rationale of this study: The current trial data (UKALL 2003) shows a
major improvement in outcome compared to the previous trial. This can to be
attributed in part to the introduction of PEG-ASNase for all risk groups
(personal communication Professor Vora, Sheffield). Thus optimal usage of
PEG-ASNase is likely to underpin all future clinical trials in childhood ALL in
the UK. There are no studies done to date that link pharmacokinetic response
of PEG-ASNase with early response to therapy and outcome in ALL. As
observed in the pilot study, patients can be classified into four categories
(Table 1.3). Table 1.3 also suggest possible mechanisms for the variations in
the therapeutic response. If these hypothesis and preliminary observations
Introduction
36
are correct, then routine pharmacokinetic monitoring of PEG-ASNase could
become mandatory for all patients. If patients with AEP over-expression show
a poor initial response to PEG-ASNase, they could benefit from the primary
use of Erwinase. It is also possible to inhibit the action of the proteases using
AEP specific inhibitors or develop a cleavage resistant ASNase. Any of these
strategies would optimise ASNase therapy and potentially improve the
outcome of high risk patients with ALL. In patients who show immunological
response, one could further undertake T cell activation studies to identify
allergic epitopes of PEG-ASNase. This will permit the development of
modified PEG-ASNase that is likely to be less immunogenic and equipotent to
the parent drug.
Table 1.3 Predicted pattern of response to PEG-ASNase
Patient groups
I
II
III
IV
Expected resultsAsp activity (induction)
Good
Poor
Good
Poor
Asp activity (post induction)
Good
Good
Poor
Poor
Likely explanation
Good response
Biological factors (blasts or mesenchymalcells) at diagnosis
Immune related
Combination offactors in II & III
Anticipated therapeutic response
RER, followed by CR; MRD week 5 neg and week 11 neg
RER/SER, followed by CR; MRD week 5 neg/pos and week 11 neg
SER/RER, followed by poor(no CR) or inadequate(MRD pos) response
SER and a poor response or inadequate (MRD pos) response.
The major determinants of poor response to the drug are likely to be
neutralising biological factors (such as AEP and/or CTSB) in the initial phase
and major mechanism of destruction in the subsequent phase would be due
to immune response to ASNase. In either case a poor response to ASNase
will result in an inadequate therapeutic response. RER= rapid early response
(≤25% lymphoblasts on morphology of bone marrow aspirate done 1-2 weeks
after starting therapy); SER= slow early response (>25% lymphoblasts on
morphology of bone marrow aspirate done 1-2 weeks after starting therapy);
CR= complete remission (<5 lymphoblasts/100 cells on morphology of bone
marrow aspirate at the end of induction therapy); MRD= minimal residual
disease (>1 lymphoblasts/10,000 cells on assessment by techniques including
Introduction
37
flow cytometry and quantitative polymerase chain reaction (RQ-PCR) at
weeks 5 & 11 of therapy); pos= positive; neg=(negative)
1.3.2 Hypothesis:
1) PEG-ASNase at 1000 U/m2 given i.m. fortnightly during induction will
produce adequate activity levels throughout induction for most children
with ALL
2) Variations in ASNase pharmacokinetics will influence the early
response to therapy and outcome
3) Lymphoblast proteases regulate inadequate ASNase activity levels and
the allergic response to ASNase.
1.3.3 Eligibility: Following approval by a Multi Regional Ethical committee,
this study was incorporated into two current national trials for childhood ALL -
UKALL 2003 (de novo patients) and ALL R3 (relapsed cases) in 2008.
1.3.4 Aims of the study (discussed further in Chapter 4):
1) To provide recommendations on whether routine pharmacokinetic
monitoring of ASNase is required in clinical practice
2) To further optimise PEG-ASNase therapy in childhood ALL.
3) To investigate whether AEP and/or CTSB emerged as prognostic
biomarkers in childhood ALL.
1.3.5 Objectives of the study (discussed further in chapter 4): At
diagnosis we measured the expression of AEP and CTSB. We serially
measured ASNase activity and determined the incidence of anti-ASNase
antibodies in children who either had clinical hypersensitivity to ASNase or
who showed inadequate activity. We determined if initial inactivation of
ASNase correlated with SER and or high MRD and if this could be predicted
by expression of AEP and/or CTSB and lastly if late inactivation of ASNase is
indeed due to an immune response that is facilitated by high expression of
AEP.
Introduction
38
1.3.6 Study design (discussed further in chapter 4): Asparaginase activity
was serially measured during induction and post induction phases. Induction
ASNase activity results were correlated with expression of AEP and CTSB at
diagnosis, cytogenetic subtype, therapeutic response (SER, MRD at week 5
and 11 for de novo ALL and week 5 and 13 for children with relapsed ALL)
and eventual outcome. The expression of AEP and CTSB was correlated with
PEG-ASNase activity and with the occurrence of clinical hypersensitivity.
Samples with inadequate activity were assayed for antibody development
using risk matched controls from the study population.
Material and Methods
39
Chapter 2 MATERIALS AND METHODS
______________________________________________________________
2.1 REAGENTS: Details of the reagents and buffers used in this thesis are
shown in appendix 1 and appendix 2 respectively.
2.2 ASPARAGINASE STUDY
The study recruited patients from two national trials: the frontline UKALL2003
and the relapsed ALLR3 trials. Details of the biological samples collected,
their time points, along with the laboratory assays employed in this study are
described in detail in chapter 4.
2.2.1 Patient Sample Collection: Consent for enrolment in the Asparaginase
Study was obtained by treating physicians. Diagnostic samples were sent by
dedicated courier and subsequent samples by a post box drop service.
Samples were received and processed by the Centre for Integrated Genomic
Medical Research (CIGMR) at the University of Manchester. They were bar-
coded and tracked on a dedicated LIMS system. Samples were subsequently
collected from CIGMR and assayed at the Paterson Institute for Cancer
Research, Manchester.
The Children’s Cancer Group has a trial office based at the Christie Hospital.
This unit was notified electronically about all new registrations and also about
the occurrence of SAE (serious adverse events) related to ASNase in both
above mentioned trials. Dr. Catriona Parker and Charlotte O’Horo based in
the trial office then tracked the flow of diagnostic and followed up samples for
each patient. This was done by electronically reminding individual treatment
centre each time before the next sample time point and also by liaising with
CIGMR to confirm whether the sample was received or not.
2.2.2 Sample Processing: All stages of sample processing and storage have
been optimised and standard operating procedures (SOP’s) established,
Appendix 3.
Material and Methods
40
2.3 TECHNIQUES
2.3.1 Tissue Culture
2.3.1.1 Cell Lines: ALL cell lines; SD1, SUPB15 and REH (appendix 4) were
used to validate the TaqMan gene expression (Applied Biosystems, Europe
BV, Warrington, UK) and ELISA assays. SD1 is an EBV-immortalised
polyploid human precursor B-lymphoblastic leukaemia cell line with t(9;22).
REH is a human diploid precursor B-lymphoblastic leukaemia cell line with
t(12;21). SUP B15 is a human precursor B-lymphoblastic cell line with a
t(9;22). SD1 expresses AEP at the protein level whereas REH and SUP B15
don’t (100).
2.3.1.2 Cell culture, passage and collection of cell pellets & supernatants:
Cell lines were cultured for 48-96 hours at 37°C/5%CO2 in BioWhitaker®
RPMI-1640 Ultraglutamine (Lonza, Wokingham, UK) supplemented with 10%
foetal calf serum (Sera Laboratories Ltd, Haywards Heath, UK). Cells were
grown in 162 cm2 cell culture flasks with 0.2 um Vent cap (Corning
Incorporated, N.Y, USA). The initial concentration of cells was 2 x105/ml. At
the end of 48-96 hours the cells were either sub cultured or collected for
downstream applications. Decision to sub culture cells was influenced by the
cell count (>1x 107/ml), colour of media and confluency. Excess cells at the
end of a given experiment were cryo-preserved using 90% foetal calf serum
and 10% DMSO (Sigma-Aldrich, Dorset, UK). The number of cells preserved
was 10-30 x 107/vial and the volume of the cryo-protectant was 0.8- 1.5µl/vial.
Cells were also cultured for 48 hours in 6 well plates in low serum medium
(RPMI- 1640 glutamax supplemented with 0.75% foetal calf serum) and
serum free medium (RPMI-1640 glutamax) under identical conditions
described above. The starting concentration of the cells in these cases was 1
x107/ml. Cell viability was determined using 0.4% Trypan blue (Sigma-Aldrich,
Dorset, UK) staining and the cell count were performed using a
haemocytometer. Cells were checked daily by inverted phase contrast
microscopy to determine their morphology, the degree of confluency and also
to rule out presence of sediments or obvious microbial contamination. Testing
Material and Methods
41
for Mycoplasma was done routinely once a month. After doing an initial cell
count, cells in medium were centrifuged at 200g for 7 minutes at R.T.
Resultant supernatants were collected and split into either 1ml aliquots in
1.5ml microfuge tubes containing 20µl of P.I. cocktail (Roche, Welwyn, UK)
for future protein work or 300µl aliquots along with 1ml of TRIzol in 1.5ml
microfuge tubes for RNA isolation. Aliquots were snap frozen on dry ice and
stored in freezer at -80°C. Cell pellets were generated only from cells grown in
conditioned medium. They were re-suspended in appropriate volume of ice
cold PBS so that the final concentration of these cells was 1x107/ml. Cells
were washed twice with ice cold PBS in a micro centrifuge at 400g for 7
minutes at 4° C. After discarding the supernatant, cell pellets were lysed
either in 1.0ml of TRIzol L.S. for RNA extraction or in lysis buffer for
proteomics.
2.3.2 Molecular Methods
2.3.2.1 RNA extraction: The entire procedure was performed in a fume
cabinet with strict aseptic precautions and use of pipette tips and microfuge
tubes dedicated for this procedure. The work surfaces were pre-treated with
RNAase Zap (Sigma-Aldrich, Dorset, UK). Frozen samples in TRIzol reagent
(Invitrogen, Paisley, UK) were defrosted and incubated at R.T. for 5 minutes
to allow complete dissociation of nucleoprotein complexes. Next, 200μl of
chloroform (VWR International Ltd. Poole, UK) was added per 1ml of initial
TRIzol reagent. Samples were shaken vigorously for 15 seconds and
incubated at R.T. for 5 minutes. They were then centrifuged at 12,000 x g for
10 minutes at 2 to 8°C. 400μl of upper aqueous phase containing RNA was
isolated and transferred to a fresh 1.5ml microfuge tube. 500μl of propan-2-ol
(Fisher Scientific, Loughborough, UK) was added to this aqueous phase to
precipitate the RNA. The samples were mixed and incubated at R.T. for 10
minutes and then centrifuged at 10,000 x g for 10 minutes at 2 to 8°C. The
supernatants were removed and RNA pellets washed by adding 1ml of 75%
ethanol (Fisher Scientific, Loughborough, UK). The samples were mixed by
vortexing and centrifuged at 7500 x g for 5 minutes at 2 to 8°C. Ethanol was
Material and Methods
42
discarded and the RNA pellets were allowed to air dry by inverting the
microfuge tubes for 10-15 minutes. RNA was re-suspended in 50μl of DEPC
(Ambion, Warrington, UK) treated water. 2μl aliquots were taken in order to
determine the chemical purity of RNA by measuring the A260/280 and A260/230
ratios on a spectrophotometer (Nanodrop ND-100 Labtech International,
Ringmor, UK). The remaining samples were stored in 10-20 μl aliquots in a
freezer at -80°C. The integrity of RNA was determined by measuring the RIN
(RNA integration number) on 2001 Bio-analyser (Agilent Technologies UK Ltd.
Stockport, UK).
2.3.2.2 cDNA synthesis: cDNA synthesis was done using random hexamers
(Promega, Southampton, UK) and SuperScript™ II Reverse Transcriptase
(Invitrogen, Paisley, UK). This enzyme is an engineered version of M-MLV RT
(Moloney Murine Leukemia Virus Reverse Transcriptase) with reduced RNase
H activity and increased thermal stability. Higher temperatures increases the
specificity of the enzyme and reduced RNase H activity results in higher yields
of cDNA compared to conventional M-MLV RT. As shown in figure 2.1 below,
Mix 1 and Mix 2 were prepared separately in PCR tubes. Mix 1 was vortexed
and centrifuged briefly to ensure that the components collected to the bottom
of the PCR tubes and then incubated at 65°C for 5 minutes. It was then
quickly chilled on ice for 5 minutes. Mix 2 was made up just before use and
7μl of Mix 2 was added to each Mix 1 sample
Mix1
Total RNA sample 1000ng of RNA in DEPC H2O; max 9l
Random Hexamer 500 ng/µl 2l
dNTP mix (10mM each) 1l
DEPC H2O Volume necessary to adjust to 12 μl in total
Figure 2.1 Composition of Mix 1 and Mix 2 prepared during cDNA synthesis.
5X first strand buffer 4l
DTT (0.1M) 2l
RNaseOUT 1l
Mix 2
Material and Methods
43
The contents were mixed by pipetting gently up and down several times.
Samples were then incubated at 25°C for 10 minutes (to maximize primer-
RNA template binding), and then at 42°C for 2 minutes. Following this 1l of
SuperScript™ II Reverse Transcriptase was added and mixed by pipetting
gently up and down several times followed by brief centrifugation. Samples
were then incubated at 42°C for 50 minutes followed by 70°C for 15 minute to
inactive the reaction. Samples were stored at -80°C.
2.3.2.3 Real time TaqMan gene expression assay for AEP (Legumain;
LGMN): Real time TaqMan gene expression assay for AEP (gene of interest)
and β2 microglobulin (control gene) were performed using inventoried (on-
demand) reagents from Applied Biosystems (Foster City, CA, USA). The AEP
probe hs00271599_ml spans boundaries of exon 5 and 6 in case of transcript
NM_005606.6 and exon 6 and 7 in case of transcript NM_001008530.1 In
each case the resultant amplicon is identical and has a length of 79 base
pairs, Figure 2.2. The probe for β2 microglobulin gene hs99999907_m1 spans
exon 2 and 3. The reaction was performed in a 384 well plate on a 7900HT
Fast Real-Time PCR System (Applied Biosystems). The total volume of
reaction per well was 10μl and the proportion of reagents in each well was as
follows: 0.4μl of cDNA, 4.1μl of DEPC treated water, 0.5μl of probe and
primers and 5.5μl of mastermix. The reagents were pipetted in each well with
the help of a automated robotic system (Eppendorf). The reaction was carried
on for 40 cycles each consisting of the following steps: i) UNG (uracil N
glycosylase) incubation at 50°C for 2 minutes, ii) AmpliTaq Gold Activation at
95°C for 10 minutes, iii) Denaturation at 92°C for 15 seconds and finally iv)
Annealing/extension at 60°C for 1min. In this assay the 5’reporter dye was
FAM and the 3’quencher dye was TAMRA. The CT value for was obtained
using SDS2.1 software and the threshold was selected automatically by the
software. Analysis of AEP expression was done by relative quantification
using the 2 -∆∆CT method (104).
Material and Methods
44
Figure 2.2 Schematic diagram of AEP (Legumain/LGMN) gene on
chromosome 14q32.1 showing the position of exon spanning TaqMan probe.
The gene has two transcript variants NM_001008530.1 & NM_005906.5. The
former has an additional exon in the 5’UTR of the gene. Both variants encode
for the same isoform. Modified from Entrez Gene, NCBI.
2.3.2.4 MicroRNA (miRNA) array: Expression of 754 human miRNAs per
sample was performed using a two card set consisting of TaqMan human
MicroRNA A+B cards Set v3.0 (Applied Biosystems, part number 4444913).
Included on each card were three TaqMan® MicroRNA assay endogenous
controls to aid in data normalisation and one TaqMan® MicroRNA assay not
related to human to function as a negative a control.
RNA was extracted as described above. Reverse transcription was performed
in fume cabinet with all the precautions described under RNA extraction.
Reverse transcription mix was made in a 1.5ml microcentrifuge tube, figure
2.3 A. Primers for reverse transcription were specific for each card (Megaplex
™ RT primers: Human pool A v2.1 part number 4399966 for card A and
human pool B v3.0 part number 4444281 for card B). Rest of the components
of reverse transcription mix were common for cards A and B. For each
reverse transcription reaction, 3μl of RNA (at least 350ng) was added to 4.5μl
of the above mix. The 1.5ml microcentrifuge tube was incubated on ice for 5
minutes and went through the thermal cycling conditions on DNA Engine
Dyad ® Peltier Thermal Cycler (BioRAD) as described in figure 2.3B. For a
NM_005606.6
NM_001008530.2
AEP Ch 14q32.193,170,152 93,215,047
5 ’ 3 ’
TaqMan Probe
Untranslated region
Coding region
NM_005606.6
NM_001008530.2
AEP Ch 14q32.193,170,152 93,215,047
5 ’ 3 ’
TaqMan Probe
Untranslated region
Coding region
Material and Methods
45
real time polymerase chain reaction reagents were mixed as shown in figure
2.3C. Samples were then dispensed into each probe of the TaqMan
MicroRNA array cards and PCR reactions were done as per manufacturer’s
instructions on the 7900HT Fast Real-Time PCR System (Applied
Biosystems).
Figure 2.3 Composition of reverse transcription mix (A), the thermal cycling
condition for reverse transcription (B) and the components of the polymerase
chain reaction (C).
Stage Temp Time
Hold
50°C
85°C 5min
1 sec
1 min
2 min
42°C
16°C
Cycles (40 cycles)
RT Reaction Mix Components Volume for ten samples (μl)‡
Megaplex™ RT Primers (x10)†
9.00
dNTPs with dTTP (100mM) 2.25
MultiScribe Reverse Transcriptase (50U/μl) 16.88
x10 RT Buffer 9.00MgCl2 (25mM) 10.12
Rnase Inhibitor(20U/μl) 1.12
DEPC nuclease free water 2.25
Total 50.62†
Primers were specific for Cards A and B‡
Includes 12.5% excess for volume loss from pippetting
Component Volume for one Array‡ (μl)
Total
444
900
6
450TaqMan® Universal PCR Master Mix, no
AmpErase® UNG,2X
Megaplex™ RT product
Nuclease-free water
A
B
C
Material and Methods
46
2.3.3 Biochemical Methods
2.3.3.1 Protein extraction: Cell pellets were incubated with 150μl mixture
made up of lysis buffer and PI cocktail solution (125ul of lysis buffer per 1x107
cells and 25ul of PI) for 35 minutes on ice. Initially cell pellets were lysed
using CelLytic M (Sigma-Aldrich, Dorset, UK) lysis buffer. From March 2009
onwards, CelLytic M buffer was replaced by modified NP-40 lysis buffer (in
house) as initial testing shows that in some patients CelLytic M interferes with
the quantification of AEP by ELISA. Following the incubation step, samples
were centrifuged at 16,000 x g for 35min at 4°C. The resultant supernatant
containing cytosolic proteins was collected and split into 15-150μl aliquots and
stored at -80°C.
2.3.3.2 Protein quantification: This was done using a protein assay method
(Bio-Rad, Hemel Hempstead, UK) based on the Lowry assay (105). In order
to generate a standard curve, bovine serum albumin (Sigma-Aldrich, Dorset,
UK) was used as a reference protein and serially diluted in PBS to achieve
concentrations between 1.44 and 0.08875 mg/ml. Cell lysates were diluted in
PBS at 1:5 and 1:10. For each concentration, 5μl of standards and samples
were added in duplicates into wells of the 96 well microplate. 25µl of freshly
made Reagent A‘ was added into each well followed by 200μl of reagent B.
After 15 minutes, absorbance was read at 650 nm in FLUOstar Omega
microplate reader (BMG Labtech, Aylesbury, UK). The amount of protein in
each well was quantified by reading the OD value off the standard curve.
2.3.3.3 L-Asparaginase activity assay:
Reagents:
1) Native E. coli Asparaginase, Medac GmbH, Germany: A vial containing
10000 Units of E. coli Asparaginase was reconstituted with 10 ml of normal
saline. The drug is unstable for long term storage in aqueous solution. Hence
2994μl of volunteer fresh frozen plasma (FFP) was spiked with 6μl of drug
solution to give a concentration of 2000 Units/L of plasma and stored at -80°C
in 80μl aliquots.
Material and Methods
47
2) 10mM L-aspartic β-hydroxamate (AHA, Sigma-Aldrich, A6508) in 0.015
MTris buffer, pH 7.3, supplemented by 0.015% (w/v) Bovine Serum Albumin
(BSA):Dissolve 1.82 gms of Trizma® base (Sigma-Aldrich, T1503) in 80 ml of
DD H2O. Adjust pH to 7.3 with help of HCl, bring volume to 1000 ml by adding
required volume of DD H2O. Add 150 mg of lyophilized BSA (Sigma-Aldrich,
A9647). Pass through 0.2 micron filter. Take 67.5 ml of this buffer to re-
suspend 1 vial of lyophilized AHA (100 mg) to make a 10mM solution. Store
the solution in 5 to 10 ml aliquots at -80° C.
3) 24.5% Trichloro Acetic Acid (Sigma-Aldrich, T9159): 24.5 gm of Trichloro
Acetic Acid in 100 ml of DD H2O. Stored at room temperature.
4) 8 hydroxyquinoline solution: Made freshly each time just before use by
mixing one volume of 2% 8-hydroxyquinoline (Sigma-Aldrich, 252565) in
absolute ethanol to three volumes of 1M sodium carbonate solution. Both the
above reagents were stored separately at room temperature.
5) Non reagent components:
Finnpipette ®F2 (30-300μl) Multi-channel pipette (Thermo Scientific)
MULTIWELL ™ 24 well flat bottom tissue culture polystyrene non pyrogenic
plates (Becton Dickinson, Oxford, UK).
Sterile 1.5ml polypropylene microcentrifuge (Eppendorf) tubes
Sterile flat bottomed polystyrene 96 well plate (BD)
Heating block
Incubator or water bath at 37°C
BMG FLUOstar Omega microplate reader (BMG LABTECH, Aylesbury, UK)
for absorbance spectrometry.
Method:
1) ASNase standards were freshly made each time by serially diluting the
stored drug at concentration of 2000 Units/L with volunteer FFP to give a
range between 0-800 Units/L. Unspiked volunteer FFP was used as blank
concentration.
Material and Methods
48
2) Patient plasma samples were thawed at room temperature.
3) Then, 20μl of standards and patient plasma were added in duplicate into
each well of the MULTIWELL ™ 24 well flat bottom tissue culture polystyrene
non pyrogenic plates (Becton Dickinson, Oxford, UK). The positions of the
standards and patient plasma were accurately recorded.
4) With the help of Finnpipette®F2 (30-300μl) multi-channel pipette, 180μl of
10mM AHA solution is next added to each well of the plate. The sequence of
step 3 and 4 was always preserved to ensure uniform incubation times with
AHA for all samples.
5) Plate was then incubated in an oven at 37°C for exactly 15 minutes.
6) Next, with the help of Finnpipette®F2 (30-300μl) multi-channel pipette,
250μl of 24.5% TCA was added to each well of the plate and it was
centrifuged at 1000g for 10 minutes at 37° C.
7) 2mls of freshly prepared 8 hydroxyquinoline solution was added to each
well of the plate following which the plate was put into the oven at 95°C for
exactly 12 minutes.
8) Plate was then allowed to cool at room temperature for 10 minuter.
9) 100μl of supernatant from each well of the 24 well flat bottom plate was
carefully aspirated and transferred into a 96 well flat bottom plate. The
absorbance from each well was measured at 710nm on a BMG FLUOstar
Omega microplate reader.
10) Plasma ASNase activity in patient samples was read off linear standard
curve obtained by joining the OD values of ASNase standards.
2.3.4 Immunological methods
2.3.4.1 Chemiluminescent AEP ELISA:
Material and Reagents:
1) White opaque 96 well microplate- Lumitrac 600, catalogue number 15042,
ThermoFisher Scientific.
2) Capture Antibody: Monoclonal mouse anti human AEP antibody, clone
322109, catalogue number MAB21992, R&D Systems. Lyophilized powder
Material and Methods
49
of capture antibody was reconstituted in 1ml of sterile PBS to give a
concentration of 500 μg/ml. Antibody is stored at -80°C in 50μl aliquots.
3) Reagent diluent (0.5% Bovine Serum Albumin PBS): 19 parts of D/D water
mixed to 1 part of Reagent Diluent Concentrate.
4) Calibrator Solution: 9 parts of Reagent Diluent and 1 part of Lysis
buffer/Protease inhibitor solution.
5) Standard: Recombinant human AEP protein (R&D systems, Catalogue
Number 2199-D Y-100 accession #Q99538). The recombinant protein is
available as 20μl of 0.2 micron filtered solution in 20mM Tris and 150mM
NaCl, pH 7.5 at a concentration of 0.5mg/ml. Protein was diluted in
Tris/NaCl buffer to give a concentration of 5ng/μl and stored in 5μl aliquots
at -80°C.
6) Detection Antibody: Biotinylated polyclonal goat anti human AEP antibody,
catalogue number BAF2199. Lyophilized powder of detection antibody
was reconstituted in 1ml of Tris-buffered saline pH 7.3 (20 mM Trizma
base, 150 mM NaCl) containing 0.1% BSA to give a concentration of
50ng/μl. Antibody was stored at -80°C in 90μl aliquots.
7) Streptavidin-Horse Radish Peroxidase (HRP), R&D systems Minneapolis,
US. Catalogue no DY998 (1/2500 solution): Add 4μl of Streptavidin-HRP
to 10 ml of reagent diluent just before use.
8) Chemiluminescent Substrate (Reagent A & B): Super Signal West Pico,
37070, Thermo Scientific. The two reagents were stored at room
temperature in dark and mixed in 1:1 proportion just before use.
9) FLUOstar OMEGA plate reader, BMG Labtech.
Each well of the white opaque 96 well microplate was coated with 100μl of
capture antibody that was diluted in coating buffer at a concentration of
2μg/ml. Plate was sealed and kept at 4°C overnight. The following morning
unbound antibody was aspirated and each well was washed 3 times with
400μl of washing buffer. This aspiration/washing step was repeated each time
before successive steps. Blocking was achieved by adding 400μl of blocking
buffer per well and incubating the plate for 2 hours at room temperature.
Standard, controls and samples were all diluted in calibrator solution and
Material and Methods
50
loaded in duplicates in volumes of 100μl per well. Using 2-fold serial dilutions
in calibrator solution, 7 concentrations of standards were generated ranging
from 50ng/ml (highest) to 75pg/ml (lowest). Samples consisted of cell lysates
at 1: 9 dilutions and plasma at 1:99 dilutions. Cell lysates of AEP+REH cell
lines were used as positive control and volunteer plasma was used as a
negative control. Next, plate was sealed and kept at 4°C overnight. Detection
of bound AEP protein used sequential incubation at room temperature in dark
conditions of 100μl detection antibody at a concentration of 400ng/ml
incubated for 2 hours, followed by 100μl of Streptavidin/HRP solution for 1
hour and lastly 100μl of chemiluminescent substrate for 5 minutes. The
luminescence was measured in relative light units using a luminescent probe
on the FLUOstar OMEGA plate reader.
2.3.4.2 Immunoblotting:
a) Chemiluminescent immunoblotting
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
Glass plates, spacers and combs of the mini-PROTEAN II electrophoresis cell
(Biorad) were first cleaned with 20% ethanol to remove any contaminating
proteins and assembled as per instructions from the manufacturer. The
discontinuous SDS-PAGE gel electrophoresis system was achieved by first
pouring resolving gel between the two layers of glass and overlaying it with
isopropanol. The gel was left to polymerise. Isopropanol ensures a smooth
top surface of the polymerised gel. Following polymerisation, isopropanol
layer was removed by gently tilting the assembly and the glass plate washed
with D/D water to remove any trace of alcohol. The stacking gel was next
poured on top of the polymerised resolving gel. Combs were placed to create
sample loading well. Following polymerisation, the combs were removed and
the wells were washed with 1x running buffer. Cell lysates were diluted
appropriately with x 5 SDS reducing buffer to ensure at least 10μg of protein
per 20μl volume. Samples were boiled for 5 minutes at 95° C and 20 μl of
reduced sample lysates were loaded into the wells using Gel loading tips (2-
20μl, Anachem). To reference the molecular weight of protein of interest, each
run had a protein standard (Precision Plus Dual Colour, BioRad) to estimate
Material and Methods
51
the protein size. Electrophoresis was performed at 55 V whilst the samples
ran though the stacking gel and 100 V through the resolving gel.
Semi-dry protein transfer:
Polyvinylidene fluoride (PVDF) microporous membrane (Immobilon-P
Transfer Membrane; Milipore) was first activated by 1 minute exposure to
methanol. After washing off methanol from the activated membrane, it; along
with the extra think blot paper were pre soaked in transfer buffer. Proteins
separated at the end of SDS-PAGE electrophoresis were transferred on to a
methanol exposed activated by applying 25mV of current for 34 minutes
across the gel nitrocellulose sandwich. The sandwich had the following
sequence from top to bottom consisting of thick filter paper, resolving gel,
activated PVDF membrane and finally another thick filter paper.
Western blotting
Following transfer, the membrane was rinsed in wash buffer and blocked by
incubating it with non fat milk blocking buffer on a rocking platform unit for 1
hour at R.T. Membranes were extensively washed with wash buffer and then
incubated overnight on a rocking platform unit with a primary antibody solution
against protein of interest. The following day, the membranes were washed x5
with wash buffer to remove primary antibody and were incubated with
appropriate species specific horseradish-peroxidase conjugatged secondary
antibody for one hour. Following the one hour incubation, the membranes
were washed x 5 with wash buffer to remove any free (unbound) secondary
antibody.
Detection
Membranes were then placed on a clean Cling-Film (Saran™ ClingPlus®
Wrap) sheet and layered with 10 ml of 1:1 mix of Luminol/Ehancer and
Peroxide buffer solutions contained in the SuperSignal® West Dura Extended
Duration Enhanced Chemiluminescent Substrate Kit. Following its incubation
for 5 minutes, the excess solution was drained off and the membranes were
placed in the film cassette and exposed on X-ray films (Kodak Medical X-ray
films) for varying intervals and developed using a Film Processor to visualise
protein band.
Loading control
Material and Methods
52
In order to show uniform loading the sample proteins in each lane,
membranes were stripped using ‘Restore’ proprietary membrane stripping
buffer and re-blotted with GAPDH.
b) Infrared Fluorescent immunoblotting
In the Infrared fluorescent immunoblotting detection system, the secondary
antibodies are conjugated with infra red dyes that show emission at 680 and
800nm. Employing a combination of primary antibodies from two distinct
species and secondary antibodies from one species having two distinct
fluoropores corresponding to two distinct primary antibodies allows two-colour
multiplexed detection and quantification of proteins on the Odyssey® CLx
Infrared Imaging System (LI-COR). Table 2.1 summarises the steps that are
distinct to the chemiluminescent immunoblotting. Aside from the distinct
detection system, table 2.1 shows the specifics of primary and secondary
antibodies for AEP and CTSB and the key differences, highlighted in bold,
between the chemiluminescent and infra immunoblotting techniques.
Material and Methods
53
Table 2.1 Reagents used for infra-red and chemiluminescent immunoblotting
Steps Infra-red immunoblotting (LICOR) Chemiluminescent immunoblotting
a) Membrane Millipore FL Millipore P
b) Blocking 5% BSA (w/v) PBS (no tween) PBST (0.1%) + Marvel
c) Primary Antibody AEP R & D Systems, AF2199 R & D Systems, AF2199 Type Goat, polyclonal Goat, polyclonal Dilution 1 in 2500 5% BSA (w/v) PBST (0.1%) 1 in 2300 in 5% Marvel PBST (0.1%) CTSB Biomol SA-361, Enzo Life Sciences) Biomol SA-361, Enzo Life Sciences) Type Rabbit, poly clonal Rabbit, poly clonal Dilution 1 in 20000 5% BSA (w/v) PBST (0.1%) 1 in 20000 5% Marvel (w/v) PBST (0.1%) GAPDH Abcam ab8245 Abcam ab8245 Type Monse, monoclonal Monse, monoclonal Dilution 1 in 20000 5% BSA (w/v) PBST (0.1%) 1 in 20000 5% Marvel (w/v) PBST (0.1%)
d) Secondary Antibody AEP LICOR 926-32224 Santa Cruz, sc-2020 Type IRDye 680LT donkey anti goat HRP conjugated Donkey anti goat Dilution 1:25000 5% BSA (w/v) PBST (0.1%) 1:10000 5% Marvel (w/v) PBST (0.1%) CTSB LICOR 926-32213 Pierce Type IRDye 800CW donkey anti rabbit HRP conjugated goat anti rabbit Dilution 1:25000 5% BSA (w/v) PBST (0.1%) 1:4000 5% Marvel (w/v) PBST (0.1%) GAPDH LICOR 926-68022 Pierce 31430 Type IRDye 660LT donkey anti mouse HRP conjugated goat anti mouse Dilution 1:25000 5% BSA (w/v) PBST (0.1%) 1:10000 5% Marvel (w/v) PBST (0.1%) *Highlighted text refers to the key differences between the two techniques
Material and Methods
54
2.3.4.3 AEP Immunoprecipitation assay:
The protocol was obtained from Dr Nullin Divecha’s group based at the
Paterson Institute for Cancer Research
Day 1
a) Pre clear step:
Fifty micro-litres Protein A/G UltraLink resin (Thermo Scientific, catalogue
number PN53132) was added to three 1.5ml eppendorf tube containing 250μg
of SD1 lysates in 200μl of lysis buffer. The samples were incubated on a
rotator at 10RPM for 1 hour at 4°C and then centrifuged at 3000g for 3
minutes at 4°C. Clear supernatants free of non specific immunoglobulin and
containing the protein of interest were collected.
b) Pull down:
Two micrograms each of 1) monoclonal mouse anti-human AEP antibody:
MAB21992, R&D Systems-pull down antibody; 2) Polyclonal goat antihuman
AEP antibody: AF2199, R&D System-positive control; and 3) mouse IgG
antibody: negative control; were added to the clear supernatants and the
samples were incubated at 10RPM on the rotator overnight at 4°C. AF2199
was previously optimised for AEP immunoblotting in the lab served.
Day 2
Fifty micro-litres of Protein A/G UltraLink resin was added to each tube and
the samples were incubated on a rotator at 10RPM for 2 hours at RT.
Samples were then centrifuged at 3000g for 3 minutes at 4°C, clear
supernatant was discarded and the resins re-suspended in 500μl of lysis
buffer. This centrifugation step was repeated three times. A mixture of 10μl x2
loading buffer, 0.5μl 1 M DTT and 7.5 μl of DD H2O was added to resins in
each tube and the samples were heated to 96°C for 10 minutes do denature
and detach the protein(s) captured onto the resins. Finally samples were
incubated in dark with 2μl of 500mM iodoacetamide for 20 minutes at R.T. to
break apart the pull down antibody into heavy and light chains. The proteins
Material and Methods
55
were separated by SDS-Polyacrylamide gel electrophoresis. The recognising
antibody was a polyclonal goat anti-human AEP antibody at conditions
optimised in the AEP immunoblotting protocol and the detecting antibody was
a true blot anti-goat antibody at 1:10,000 dilution that was incubated for 1 hour
at R.T.
2.3.5 Other techniques
2.3.5.1 Capture of microvesicles: Microvesicles (MV) were isolated from the
cell suspensions and plasma samples by a combination of centrifugation
protocols described in the literature and using an antibody based approach
that adopted the principle of the immunoprecipitation (IP) assay, figure 2.4.
The novelty in the technique consisted of capturing microvesicles instead of
capturing proteins in a standard IP assay. The “pull down antibody” was a
mouse anti human anti CD19 antibody, clone HIB19, BD Pharmingen™;
catalogue number 555410.
Technique: On day 1, debris free SD1 supernatant was obtained by
subjecting 5ml of SD1 cell suspension to 2 successive spins consisting of 750
g for 5 minutes (to pellet the SD1 cells), 1500 g for 15 minutes (to pellet
debris). Microvesicles were pelleted by subjecting the debris free SD1
supernatant to 3rd spin consisting of 15,000 g for 45 minutes at 4 C.
Microvesicles were labelled with PKH-67 (SigmaAldrich, Dorset, UK;
Catalogue number PKH67GL-1KT) as per manufacturer’s instructions. PKH-
67 incorporates into cell membrane lipid bilayer and contains a green
fluorochrome that has an excitation and emission spectrum of 490nm and
504nm respectively. Labelled MVs were re-suspended in 800 μl of PBS and
incubated with either anti-CD19 antibody (BD Biosciences, clone HIB19,
catalogue number 555410) at 4 C at 5 RPM (revolutions per minute). The
anti-CD3 antibody (Thermo Scientific, clone RIV9, catalogue number MA1-
7639)) and Protein A/G UltraLink resin were used as negative controls for
anti-CD19 antibody.
Material and Methods
56
On day 2, 50 μl of Protein A/G UltraLink resin was added MV suspensions
and samples were incubated at room temperature for 2 hours under dark
condition to immobilise the anti-CD19 antibody/labelled MV complex.
Material and Methods
57
Figure 2.4 Capture of MV. Schematic description of method developed to
isolate CD19 expressing MV. For labelling of MV with green fluorescent
Isolating CD19 expressing micro vesicles from extracellular compartmentDay 1
Collect supernatant
1st spin 750g x 5min; pellet cells
2nd
spin 1500g x 15min; pellet debris
3rd
spin 15000g x 45min, 4°C;
Stop the staining reaction +
4thspin 15000g x 45min 4 °C
Re-suspend MV 800 μl of PBS
5mls SD1 suspension
Collect supernatant
Label MV with PKH67 dye*
Pellet microvesicles (MV)
Re suspend MV in 1ml Diluent C
Add 50μl of Ultralink A/G
incubate RT, 2hrs, 10RPM
Day 2
Spin 3000g with PBS, 4°C for 5min
Collect 1st supernatant for time lapse microscopy
Wash beads x3
Collect beads for time lapse microscopy
Material and Methods
58
PKH67 dye, both the initial incubation step (ϯ) and the subsequent stopping of
the reaction by adding bovine albumin (*) was done as per manufacturer’s
instructions.
2.3.5.2 Immunoblotting of microvesicle lysates: Concentrations of primary
antibody used in the immunoblotting experiments is shown in table 2.2
Table 2.2 Concentration and source of antibodies:
2.3.5.3 In-Vivo labelling of cells for AEP by Fluorescent probe:
Ten million SD1, REH, SD1kd, REH+AEP cells were suspended in 2 ml of
culture medium described above and incubated at 37°C for 1 hour with a
fluorescent labelled, cell permeable AEP probe (LP-1) (106) at a
concentration of 1μmolar.. Unbound probe was removed by x2 PBS washes
at 200g for 5 minutes and cell pellets were re- suspended in 10 ml of excess
media and left in the incubator at 37°C for 1 hour. Finally media was removed
by x2 PBS washes at 200g for 5 minutes and expression of AEP was
analysed by flow cytometry on the BDTM LSR II flow cytometer (Becton
Dickinson Biosciences, Oxford, UK) and on Amnis ImageStreamX flow
cytometer (Amnis Corporation, Ipswich, UK)
2.3.5.4 Statistical methods: Categorical variables were compared with the
Chi-squared test and continuous variables with Mann-Whitney or a Kruksal-
Walis test depending upon the number of groups being compared. SPSS-16
Table
Target Protein Host species Clone Company Catalogue number Concentration of Antibody
MMP2 Mouse F14P4D3 Biolegend 303602 1 in 500
MMP9 Mouse F11P2C3 Biolegend 635002 1 in 500
Actin Mouse AC-15 Sigma-Aldrich A5441 1 in 5000
Cathepsin B Rabbit Polyclonal BioMol, Alexis BML-SA361 1 in 10000
CD11a Rabbit EP1285Y Abcam ab52895 1 in 1000
VAMP3 Rabbit Polyclonal Fisher Scientific PA1-767A 1 in 1000
CD19 Goat Polyclonal Santa Cruz SC-8498 1 in 400
CD63 Rabbit MEM-259 Abcam ab8319 1 in 1000
LAMP1 Rabbit Polyclonal Abcam ab24170 1 in 1000
TSG101 Mouse 4A10 Abcam ab8319 1 in 1000
CD81 Mouse 5A6 Biolegend 349501 1 in 1000
Table
Target Protein Host species Clone Company Catalogue number Concentration of Antibody
MMP2 Mouse F14P4D3 Biolegend 303602 1 in 500
MMP9 Mouse F11P2C3 Biolegend 635002 1 in 500
Actin Mouse AC-15 Sigma-Aldrich A5441 1 in 5000
Cathepsin B Rabbit Polyclonal BioMol, Alexis BML-SA361 1 in 10000
CD11a Rabbit EP1285Y Abcam ab52895 1 in 1000
VAMP3 Rabbit Polyclonal Fisher Scientific PA1-767A 1 in 1000
CD19 Goat Polyclonal Santa Cruz SC-8498 1 in 400
CD63 Rabbit MEM-259 Abcam ab8319 1 in 1000
LAMP1 Rabbit Polyclonal Abcam ab24170 1 in 1000
TSG101 Mouse 4A10 Abcam ab8319 1 in 1000
CD81 Mouse 5A6 Biolegend 349501 1 in 1000
Material and Methods
59
software was used for statistical analysis. For time to event outcome, Kaplan-
Meier curves were calculated and compared with the log-rank test. Event free
survival was defined as time to relapse, secondary tumour or death, counting
only the first event. Overall survival was defined as time to death. Relapse
free survival was defined as time to relapse, excluding those who never
achieved remission.
.
Development of assays
60
Chapter 3 DEVELOPMENT OF ASSAYS ______________________________________________________________
Most of the assays used in this thesis were developed in house and therefore
I have devoted a chapter to describe them in detail.
3.1 PLASMA ASPARAGINASE ACTIVITY ASSAY- Indoxine Method:
3.1.1 Background for the assay: Plasma ASNase activity is expressed in
Units/L. One unit of activity is defined as the amount of enzyme which
releases 1μmol of ammonia and L-Aspartic acid from 1μmol of L-Asparagine
per minute at 37°C. The liberated ammonia can be measured
spectophotometrically after nesslerization. Alternatively, assays use an L-
Asparagine analogue substrate that is cleaved by ASNase in plasma to
produce an intermediate product. This intermediate product in turn reacts with
a chromogen to produce a colour that is measured at a specific wavelength in
a plate reader.
Initially we used a commercially available substrate assay, MAAT (Medac
Asparaginase Aktivitäts-Test) kit from Medac GmbH Wedel, Germany was
used. Optimisation of the MAAT assay identified one difficulty. The standard
curve generated using the reagents provided in the kit was linear only when
the ASNase was reconstituted in a proprietary calibrator solution. This was
not the case (Figure 3.1) when the standard curve was generated using
volunteer plasma, which is a better comparator for measuring plasma ASNse
activity in clinical samples. Hence the “indoxine method”(107) was adopted
and standardised.
3.1.2 Principle of the assay: Plasma containing ASNase is incubated for 15
minutes at 37°C with an excess amount of L-Aspartic β Hydroxamate (AHA).
Plasma ASNase cleaves AHA to generate proportionate amount of
hydroxylamine. The reaction is stopped by adding Trichloroacetic acid (TCA).
TCA is then neutralised by adding sodium carbonate (Na2CO3).
Development of assays
61
Hydroxylamine when condensed with 8-hydroxyquinoline at 95°C undergoes
oxidation to produce green coloured indoxine that is read at 710nm.
Figure 3.1: MAAT assay (Medac, GMBH, Wedel Germany). X axis is the
concentration ASNase in U/L, Y axis is the optical density value obtained by
measuring absorbance at 700nm.
3.1.3 Performance of the assay: Figure 3.2 shows the dynamic range, intra-
assay variation and limit of detection of the assay and figure 3.3 shows the
inter-assay and inter-person variation of the assay. The assay is linear for
plasma ASNase concentrations of up to 800U/L. The limit of detection (LOD)
of the assay is the lowest concentration of analyte that can be detected and
reliably differentiated from the background values. It can be calculated by two
methods. In the first method, LOD is expressed in Units of ASNase/Litre of
plasma and is calculated as the lowest concentration of ASNase standard that
had a value that was greater than (3.3 x σ of background value)/Slope of the
calibration curve (108). The second method first calculates limit of blank (LOB)
which is mean background OD value + 1.645 σ of background value. LOD is
then expressed as a raw OD value obtained by adding LOB to 1.645 x σ low
concentration sample (109). Limit of detection for this assay was 33.8 Units of
ASNase/Litre of plasma by method 1 or a raw OD value of 0.082 by method 2.
Volunteer plasma
0
0.5
1
1.5
2
2.5
3
0 100 200 300 400 500 600
L-Asparaginase U/L
0.2
0.4
0.6
0.8
1.2
1.4
1.6
Proprietary calibrator
R2
= 0.9952
0
1.0
0 100 200 300 400 500 600 700
L-Asparaginase U/L
OD
Valu
e
OD
Valu
e
Development of assays
62
Figure3.2: Linear Range of the Indoxine assay. X axis represents
concentrations of ASNase in plasma and Y axis represents the optical density
values obtained by measuring absorbance at 710nm. The error bars
represent ± 1 Standard Deviation. STDEV=standard deviation. CV=
Coefficient of variation.
y = 0.0005x + 0.0653
R2
= 0.9983
0.1
0.2
0.3
0.4
0.5
0 100 200 300 400 500 600 700 800
Plasma L-Asparaginase (U/L)
OD
valu
e
L-Asparaginase Standards: Linearity and Intra Assay Variation
1 2 3 4 Mean
800 0.467 0.438 0.439 0.423 0.442 0.016 3.598
400 0.262 0.277 0.282 0.245 0.267 0.014 5.415
200 0.156 0.158 0.159 0.172 0.161 0.006 3.907
100 0.121 0.117 0.114 0.115 0.117 0.003 2.296
50 0.082 0.093 0.095 0.084 0.089 0.006 6.317
25 0.069 0.069 0.071 0.074 0.071 0.002 2.8920 0.060 0.057 0.068 0.069 0.064 0.005 8.068
Limit of Detection
Method 1=3.3xSD(blank)/slope [U/L] 33.815
Method 2 =LOB+1.645*SD of low conc [OD value] 0.082
Plasma L-Asparaginase (U/L)
Optical Density Values
STDEV CV (%)
A
B
Development of assays
63
Figure3.3: Performance of the Indoxine assay: A Linear Range of the Indoxine
Asparaginase assay. X axis represents concentrations of ASNase in plasma
and Y axis represents the optical density obtained by measuring absorbance
at 710nm. The error bars represent ± 1 Standard Error of the mean (SEM). B
Inter Assay and inter person variation using a uniform patient sample between
the runs as internal control. CV= Coefficient of variation.
y = 0.0005x + 0.0844
R2
= 0.9953
0.1
0.2
0.3
0.4
0.5
0 100 200 300 400 500 600 700 800
OD
valu
e
Plasma L-Asparaginase (U/L)
A
B
Run 1 2 3 4 5 6
R2 Value of the run 0.989 0.994 0.995 0.988 0.991 0.993
Plasma L-Asparaginase (U/L) SEM
800 0.403 0.382 0.462 0.439 0.461 0.449 0.023
400 0.261 0.231 0.288 0.285 0.290 0.293 0.017
200 0.172 0.163 0.193 0.202 0.203 0.196 0.011
100 0.115 0.106 0.139 0.138 0.140 0.150 0.012
50 0.086 0.079 0.108 0.110 0.103 0.115 0.010
25 0.074 0.071 0.089 0.088 0.087 0.096 0.006
0 0.057 0.060 0.074 0.074 0.074 0.087 0.007
Run 1 2 3 4 5 6 CV (%)
Plasma L-Asparaginase (U/L) 235 214 229 205 227 214 3.6
Mean Optical Density Values
L-Asparaginase Standards: Inter Assay Variation
Internal control: Inter Assay Variation
Internal control: Inter Person Variation
Run 1 2 3 4 5 6 CV (%)
Plasma L-Asparaginase (U/L) 227 263 247 210 260 212 11.9
7
243
8
243
Development of assays
64
3.1.4 Pre-analytical effect of sample transport on Asparaginase activity:
All samples have been processed within 8 days of sample collection, Figure
3.4. The median time from sample collection to processing in the initial 18
months of the study was 2 days (n=593). To look at the effect of sample
transport on plasma ASNAse activity, a pilot study was done where 15
randomly selected blood samples were split into three parts on arrival and
processed on the day of arrival or were left on the bench and processed on
day 4 and 5 respectively. All samples that had adequate ASNase activity
(>100U/L, n=13) remained above this threshold even after leaving them on
the bench for upto 4 days. Given that these samples took between 1-4 days to
arrive to us from the participating centres, the processing interval in this pilot
study was well beyond the median of 2 days observed in the parent study. It is
reassuring to conclude that the current standard operating procedures for
sample collection, processing and storage, are suitable for analyses of
ASNase activity for all samples.
3.1.5 Summary: The linear range of the previously published indoxine
method (107) was either 2.5-7.5 U/L or 75-1250 U/L. This was dependent on
the volume of reagents used and the incubation time. Both of these were
changed on a trial and error basis so that the asparaginase standards
achieved a linear range between 0-800 Units of asparaginase/litre of plasma.
An additional strength of this method is that all reactions of the assay until the
step of putting the samples on a spectrophotometer are performed in one
plate without having to transfer volumes of intermediate product into separate
tubes. This makes the assay less cumbersome, less prone to errors and
higher throughput thus making it feasible to test clinical samples in batches of
40 samples over 3 hours.
Development of assays
65
Figure 3.4: Effect of sample transport on ASNase activity A) Box and whisker
plot showing the interval in days between sample collection and processing in
593 clinical samples. Box encompasses values between the 25th and 75th
centiles Lower whisker represents minimum value, upper whisker is the
maximum value that is 1.5 x inter-quartile range (IQR) beyond the 75th
percentile. The bar represents the median (2 days) and + represents the
calculated mean (2.48 days). Outliers are shown as circle (n=4) that are ≤ 3x
IQR and asterix (n=5) that are > 3x IQR. B) Pilot study to determine the effect
of pre analytical phase of sample transport on plasma ASNase activity to
show that samples up to 4 days in transit are suitable for measuring ASNase
activity. The dotted line represents the threshold of adequate activity (≥ 100
U/L).
+
8
4
6
2
Blood samples
Pro
cessin
g tim
e (
da
ys)
n= 593
0
100
200
300
400
500
600
processed on
day of arrival
processed
4 days later
processed
5 days later
L-A
spa
ragin
ase a
ctivity (
U/L
)
*
Development of assays
66
3.2 Expression of AEP by RQ-PCR in cell lines and patient samples:
3.2.1 Quality of the RNA obtained from patient samples: RNA is very
sensitive to degradation. Assessment of the quality of RNA obtained from
clinical samples is a pre-requisite to obtaining meaningful gene expression
data. One of the methods to assess the quality of RNA is to determine its
integrity on Bioanalyser 2100 (Agilent Technologies, USA) (110). RNA
intercalated with a dye undergoes electrophoretic separation on a micro-
fabricated chip. An electropherogram is then generated, figure 3.5 by the
emission of fluorescence from the intercalated dye in presence of laser. The
software of the analyser measures the amount of 18S and 28S ribosomal
RNA and comes up with a ‘RIN’ (RNA integration number) value based on the
fluorescence signal from the peaks and the shape of the curve respectively. A
RIN > 5 is considered good for downstream applications such as RQ-PCR
(110).
To determine if the incubation period between sample collection and
processing affects the RNA, we analysed its chemical purity (A260/280), quantity
(ng/μl) and integrity (RIN) and correlated these with the incubation period as
shown in the table. The majority of samples yielded >100ng/μl of RNA when
RNA precipitates were suspended in standard volume of 50μl of DEPC water.
For patient samples LA 0066, LA0080, LA 0114, LA 0115 and LA0117 the
software of the bio analyser did not give a RIN value.
Development of assays
67
Figure 3.5: Quality of patient RNA. Majority of samples (8/10) yielded >100
ng/μl of RNA and an A260/280 > 1.8 when RNA precipitates were re suspended
in 50μl of DEPC water. N.A= Not available. Electopherogram of RNA samples
from 10 patients showing fluorescent peaks from left to right corresponding to:
background marker, 5S RNA, 18S and 28S rRNA. A high quality RNA has a
clearly visible 28/18S rRNA peak ratio and a small 5S RNA and a RIN value ≥
7.5 (110)
NA2.0567.72LA 0117
9.61.9215.31LA 0087
9.81.9254.12LA 0107
NA1.9160.71LA 0115
NA1.9144.51LA 0114
9.11.8139.81LA 0100
9.31.774.02LA 0079
NA1.721.04LA 0080
NA1.9113.11LA 0066
91.9419.43LA 0069
Processing time (days) RNA (ng/μl) RINA260/280Patient Samples
A
B
Development of assays
68
3.2.2 Dynamic range of the assay: Figure 3.6 shows the dynamic range of
the assay for AEP (gene of interest) & β2 microglobulin (housekeeping gene)
in SD1 cell lines (positive control).
3.2.3 Consistency of the step of reverse transcription: In order to
determine the consistency in the step of reverse transcription, 3 cDNA
samples out of a total of 43 samples were randomly selected and serially
diluted to generate CT values for AEP and β2 microglobulin, figure 3.7. The
error bars represent standard error of the mean CT value obtained for each
sample that was run in triplicates. This shows that the step of reverse
transcription was consistent. In order to determine any variation in the
performance of real time quantitative PCR assay between any two runs of
clinical samples, the cDNA that was generated in above experiment was
pooled, aliquoted in 20μl volumes and stored at -20°C. This cDNA was used
to generate standard curves during each run of the assay for the entire length
of the study.
3.2.4 Amplification efficiency of the assay: The amplification efficiency for
AEP and β2 microglobulin was calculated from the slope of the standard
curves using the equation of Efficiency (E) expressed in percentage = (10-
1/slope-1) x100. The basis of this equation is that if the assay is 100% efficient,
the PCR product will increase by 10 fold every 3.32 cycles. Using this
equation, the efficiency amplification of AEP and β2 microglobulin was 96 and
91% respectively and the co-efficient variation for the efficiencies of AEP and
β2M in the three randomly selected samples was 2.5% and 1.4% respectively.
3.2.5 Selection of control gene: β2 microglobulin was chosen as a control
gene based on its consistent expression in lymphoblasts during micro-array
analysis of 120 patient samples done in the laboratory previously (Prof. V.
Saha, Manchester; personal communication). Moreover, in this study, using a
CT cut off between 18.37 and 21.43, the expression of β2 microglobulin
followed a normal distribution pattern, figure 3.8 A. In 28 samples the
Development of assays
69
amplification efficienciencies of the 2 genes were calculated using LinReg
software and figure 3.8B shows that the amplification efficiencies correlated
with each other, indicating that β2 microglobulin was a good indicator of the
stability of the AEP transcript.
Figure 3.6 Dynamic range of the assay. A: Y axis represents the cycle
threshold values plotted against log nanogram (ng) of the starting amount of
RNA per well in case of AEP and β2 microglobulin on X axis. B: Shows the
data by plotting difference in CT values of AEP and β2 microglobulin on y axis.
The curves are parallel until log ng value of -1.1 which corresponds to 78
picograms of starting concentration of RNA per well. Error bars represent ± 1
SEM.
Log ng of starting RNA
∆C
T
R2
= 0.9958
R2
= 0.9993
5
10
15
20
25
30
35
40
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
CT
Valu
es
R2
= 0.9958
R = 0.9993
5
10
15
20
25
30
35
40
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
AEP
β2
CT
Valu
es
R2= 0.2652R
2= 0.2652
1
2
3
4
5
6
7
8
9
10
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
Log ng of starting RNA
A
B
Development of assays
70
Figure 3.7: Consistency of the RT step. A: Y axis represents the cycle
threshold values plotted against log nanogram (ng) of the starting amount of
RNA per well in case of AEP and β2 microglobulin on X axis. B: Shows the
data by plotting difference in CT values of AEP and β2 microglobulin on y axis.
The curves are parallel. Error bars represent ± 1 SEM.
R2= 0.99
= 0.99
5
10
15
20
25
30
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
SD1 AEP
SD1 β2M
R2
CT
Valu
es
y = 0.0883x + 5.845
R2= 0.3177
1
2
3
4
5
6
7
8
9
10
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
∆C
T
Log ng of starting RNA
Log ng of starting RNA
y=-3.1807x + 25.56
y=-3.269x + 19.715
A
B
Development of assays
71
Figure 3.8 Performance of control gene. A: Histogram showing expression of
β2 microglobulin in clinical samples. CT values of 18.37 and 21.43 represent
±1 standard deviations from the mean expression and these values were
used as cut offs for accepting all samples for data analyses. B & C:
Comparison of the amplification efficiency of β2 microglobulin with that of AEP
in 28 patient samples.
Fre
quency Mean = 19.9
Std. Dev = 1.6C.V. = 7.7%n = 74± 1SD = 18.4 - 21.4
262422201816
20
15
10
5
0
CTValues
R2 = 0.0593
0
0.5
1
1.5
2
2.5
1.65 1.7 1.75 1.8 1.85 1.9
AE
P
β2 microglobulin
β2M AEP
Efficiency (%) 91.05 95.35
mean 1.82 1.90
stdev 0.04 0.15
CV (%) 2.42 8.10
n=28
A
B
C
Development of assays
72
3.2.6 Expression of AEP by RQ-PCR in cell lines: Figure 3.9 shows
expression of AEP in SD1, REH and SUP B15 cells lines. Expression of AEP
in SD1 cells is more than 550 fold of that in REH cells and more than 50 fold
of that in SUP B15 cells.
R
ela
tiv
e fo
ld e
xp
ress
ionX2 fold less
X4 fold less
X8 fold less
X16 fold less
X32 fold less
X64 fold less
X128 fold less
X256 fold less
X512 fold less
0
1
2
3
4
5
6
7
8
9
10
11
SD1 REH SUP B15
X same
Log scale
500 fold less
80 fold less
Figure 3.9: AEP expression by RQ-RTPCR in cell lines. Y axis represents the
relative fold expression of AEP. REH and SUP B15 cell lines expressed less
AEP compared SD1 cells. The negative controls for this assay were no
template sample and genomic DNA from SD1 cells.
3.2.7 Expression of AEP by RQ-PCR in patient samples: Using the criteria
of accepting only samples that have RNA A260/280 of >1.8 and where the CT
values of β2 microglobulin fall within the defined cut offs (19.90 ± 1SD),
normalised expression of AEP is available for 76 patient samples, figure 3.10.
The difference in expression of AEP between the highest and lowest values is
greater than 1000 fold. As we expected to find, there is a small population of
patient samples that highly express AEP as reflected by the outliers (green
circles) and the skew of the mean (+) from the median (bar).
Development of assays
73
Figure 3.10: Expression of AEP by RQ-RTPCR in clinical samples. The data
is shown in box and whisker plots. The box represents the interquartile range.
The bar is the median, + is the calculated mean, circles are the outliers and
the asterisk is an extreme outlier. The whiskers represent the upper adjacent
value (upper quartile + 1.5x interquartile range) and the lower adjacent value
(lower quartile -1.5x interquartile range).
3.3 AEP ELISA:
We decided to additionally quantify the expression of AEP at the protein level
because ultimately it is the AEP protein that degrades ASNase. To quantify
the expression of AEP at the level of protein in clinical samples, a sandwich
ELISA assay was developed. I inherited the ‘first generation’ assay from Dr.
Jizhong Liu that used a chromogenic substrate in the detection system. Over
a period of 3 years, this assay underwent modifications and further
optimization at each step to evolve into a chemiluminescent ELISA that was fit
for clinical use.
Development of assays
74
3.3.1 Developmental history of the assay
3.3.1.1 The first generation assay: This assay was performed on Immuno
Plate F96 MaxiSorp microplates (Scientific Laboratory Supplies Ldt.
Nottingham, UK) and used a monoclonal mouse anti human AEP primary
antibody which only recognised the immature, 56kd form of AEP (R&D
Systems, Minneapolis, US, Catalogue no. MAB2199). The standards,
samples and controls were all diluted in ELISA General Buffer (AbD Serotech,
Oxford, UK, Catalogue no BUF037A) but the samples and control additionally
contained a mixture of a proprietary cell lysis buffer (CellLyticM, Sigma-Aldrich,
Dorset, UK. Catalogue no C2978) and protease inhibitor cocktail. The
detection system consisted of biotinylated polyclonal goat anti-human AEP
antibody (BAF2199), Streptavidin-Horse radish peroxide enzyme and a 1-
StepTM Turbo TMB (3,3’,5,5’-tetramethylbenzidine) chromogenic ELISA
substrate (Thermo Scientific, Rockford, USA). The reaction was stopped after
30 minutes of adding substrate by adding 100ul of 2N H2SO4 stopping buffer
and the absorbance from each well measured at 450 nm using a FLUOstar
OMEGA plate reader. Figure 3.11 summarises the performance of this assay.
The assay was linear when recombinant AEP protein was use to generate the
standard curve. However in order to get a readout from and AEP expressing
SD1 cell line, at least 50μg cell lysate protein had to be loaded per well,
requiring around 108 cells. This was not practically feasible for clinical
samples. Additionally there was >25% variation in the expression of AEP
protein in SD1 cell lystates at this concentration, figure 3.12 A and B. To rule
out other problems besides poor sensitivity of the assay for measuring AEP in
cell lines, a single experiment was performed where I loaded upto 200μg of
SD1 protein lysates. This showed that the assay gave readout only at a higher
protein concentration (Figure 3.12 C).
Development of assays
75
Figure 3.11 Performance of the 1st generation AEP ELISA. A= the linearity of
the assay B= precision and the limit of detection of the assay.
Intra-assay variation: error bars=1SD Inter-assay variation: error bars=1SEDM
R2 = 0.9957
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 20
Recombinant AEP ( ng/ml)
OD
va
lue
OD
va
lue
R2 = 0.9916
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 200
0 5 10 15 20
Recombinant AEP ( ng/ml)
Concentration of the analyte(ng/ml) SEM SEDM
Recombinant AEP 1 2 3
20 0.584 0.535 0.615 0.040 0.023
10 0.356 0.315 0.388 0.037 0.021
5 0.211 0.153 0.232 0.041 0.024
2.5 0.117 0.103 0.126 0.012 0.007
1.25 0.074 0.059 0.079 0.011 0.006
0.625 0.057 0.045 0.062 0.009 0.005
0.3125 0.044 0.036 0.042 0.004 0.002
0 0.042 0.036 0.045 0.004 0.002
Inter assay variation
Optical density values
Limit of detction: Intra assay variation
Concentration of the analyte(ng/ml) Mean SD
Recombinant AEP 1 2 3
20 0.553 0.516 0.535 0.535 0.018
10 0.309 0.321 0.315 0.315 0.006
5 0.145 0.160 0.153 0.153 0.008
2.5 0.103 0.103 0.103 0.103 0.000
1.25 0.058 0.059 0.059 0.059 0.001
0.625 0.047 0.043 0.045 0.045 0.002
0.3125 0.037 0.036 0.036 0.036 0.001
0 0.040 0.033 0.037 0.037 0.004
Optical density values
Linearity and intra assay variation
0.044 OD value0.503 ng/ml
Method 1= 3.3xSD(blank)/slope Method 2= LOB+1.645SD(low conc)
A
B
Development of assays
76
Figure 3.12 Quantification of AEP in SD1 cells by 1st generation AEP ELISA.
A, B and C essentially showing that the first generation assay required at least
50μg of SD1 cell lysates protein to get a readout and there was a wide
variation (CV of >10%) in AEP measurement in SD1 cells if lower (50μg)
amount protein lysates was loaded per well of the ELISA plate.
10
0
1
2
3
4
5
6
7
8
9
SD1 50μgmSD1 100μgmSD1 200μgm
0
0.5
1
1.5
0
0.5
1
1.5
AE
P (
ng
/ml)
SD1 50μgm
CV = 24.8%
AE
P (
ng
/ml)
A
B
C
SUPB15 50μgmREH 50μgm
SD1 50ug lysate Mean SE SEM CV (%)1 2 3 4
ng/ml 0.78 1.03 0.96 1.40 1.05 0.26 0.13 24.75
Days
Development of assays
77
3.3.1.2 2nd generation assay To address the issue of poor sensitivity of the
assay, in the first instance, the capture antibody, MAB2199 was replaced with
a mouse monoclonal anti human AEP antibody, MAB21992 (R&D systems).
This new antibody captured both the immature (56kd) and the mature (36kd)
forms of AEP. Additionally, the commercial cell lysis buffer (Cell LyticM,
Sigma Dorset, UK) was replaced with an in-house NP40 buffer as the
commercial buffer interfered with the assay, figure 3.13 A and B. These
interventions however, failed to improve the detection limit of the assay.
Development of assays
78
Figure 3.13: Optimisation of capture antibody of the 2nd Generation AEP
ELISA. A: Immunoprecipation (IP) experiment to show that MAB21992 (R&D
Systems) used as a capture antibody in ELISA detects both mature and the
immature forms of AEP. 1st three lanes from left to right represent SDS PAGE
of immunoprecipitate of REH cell lines transduced with AEP (AEP+REH).
Lane 1, 2 and 3 represent antibody combinations used in ELISA, standard in-
house antibodies for AEP IP optimized in the laboratory and negative control
respectively. AF21992 (R&D Systems) is a polyclonal goat anti human AEP
antibody. B: Inhibition of the assay when recombinant AEP standards
contained CelLytic M (CLM) but not the same with NP40 based lysis buffer.
MAB21992 AF2199 Mouse IgG -----Pull down Antibody
Recognising antibody
IP (REH transduced with AEP) Immunoblot (SD1)
AF2199 AF2199 AF2199 AF2199
Detecting antibody - goat antibody Donkey anti-Goat
56 kDa
36 kDa
-----
True blot anti- -
0
0.1
0.2
0.3
0.4
5 10
AEP
AEP+PI
AEP+CLM LB
AEP+PI+LB CLM LB
Recombinant AEP ( ng/ml)
OD
valu
e
0
0.1
0.2
0.3
2.5 5 10
AEP+PI+ NP40 LB
AEP
OD
valu
e
Recombinant AEP ( ng/ml)
0
0.1
0.2
0.3
2.5 5 10
AEP+PI+ NP40 LB
AEP
OD
valu
e
Recombinant AEP ( ng/ml)
A
B
(Precursor form)
(Mature form)
Development of assays
79
Each individual steps of the 2nd generation assay were optimised by
performing grid experiments in a 96 well format that interrogated variables at
each step of the assay appendix 5. Performance of the optimised 2nd
generation absorbance assay is summarised in figure 3.14 & 3.15.
Figure 3.14 A Linear range (A) and intra-assay variation (B) of the 2nd
generation AEP ELISA.
Concentration of analyte (ng/ml)
Mean SDRecombinant AEP
1 2 3 4
2.5 0.852 0.825 0.819 0.763 0.815 0.037
2 0.710 0.666 0.727 0.723 0.707 0.028
1.25 0.530 0.510 0.542 0.551 0.533 0.018
1 0.454 0.461 0.443 0.412 0.443 0.022
0.625 0.338 0.344 0.348 0.341 0.343 0.004
0.5 0.301 0.307 0.321 0.304 0.308 0.009
0.3125 0.242 0.257 0.214 0.250 0.241 0.019
0.25 0.265 0.223 0.249 0.237 0.244 0.018
0.15625 0.213 0.213 0.227 0.229 0.221 0.009
0.125 0.205 0.204 0.208 0.214 0.208 0.005
0 0.176 0.161 0.164 0.178 0.170 0.008
Optical density Values
Linearity and intra assay Variation of 2nd
generation assay
Limit of detection
Method 1= 3.3xSD(blank)/slope Method 2= LOB+1.645SD(low conc)
0.191 OD value0.106 ng/ml
y = 0.2625x + 0.1766
R2
= 0.9966
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.5 1 1.5 2 2.5
OD
valu
e
Recombinant AEP (
y = 0.2625x + 0.1766
R2
= 0.9966
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.5 1 1.5 2 2.5
Recombinant AEP (ng/ml)
A
B
Development of assays
80
Figure 3.15: Quantification of AEP in SD1 cells using a 2nd generation AEP
ELISA. The assay could be detected using as little as 12.5μg of SD1 protein
lysates. The figure also demonstrates the appropriateness of the antibody
combinations using in the assay. * represent p=<0.05 and ** represents
p=<0.005. Error bars represent ± 1 SE.
This assay still had one flaw in that it had a high background, figure 3.14.
Attempts to reduce the background were invariably associated with a
reduction in detection limit of the assay. Hence the assay was redesigned into
a 3rd generation chemiluminescent assay.
3.3.1.3 3rd generation assay: The detection system of the assay was changed
to measure end point luminescence instead of absorbance. Performance of
this assay is summarized in figure 3.16
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Row A= Capture Antibody
Row B= Detection Antibody
MAB21992 MAB21992 MAB21992 Mouse -
BAF2199 BAF 2199 BAF2199 BAF2199
IgG
SD1
50 μg
SD1
50 μg
SD1
50 μg
SD1
50 μg
SD1
50 μg
SD1
-
- -
-MAB21992
BAF2199
SD1
25 μg
MAB21992
BAF2199
SD1
12.5 μg
*
* *
*A
EP
(ng/m
l)
A
B
Development of assays
81
Figure 3.16 3rd Generation AEP ELISA. A: Linear range of the assay using the
recombinant protein on the left and in REH cell line transduced with AEP
(REH+AEP), courtesy Dr Alexander. B: Inter-assay variation and the limit of
0
1
2
3
4
5
6
7
8
SD
1 1
0µ
g
RE
H 1
0µ
g
SU
P B
15
10µ
g
SD
1 A
EP
KD
10µ
g
AE
P+
RE
H1µ
g
AE
P+
RE
H 0
.1µ
g
AE
P (
ng/μ
g o
f p
rote
in)
* * *
*
* *
* * *
**
* * *
56 kDa
36 kDa
GAPDH
SD
1 1
0 μ
g
RE
H 1
0 μ
g
SU
P B
15
10
μg
SD
1 K
O 1
0 μ
g
AE
P+
RE
H 1
μg
D
Concentration of analyte (ng/ml) Mean SD
Recombinant AEP 1 2 3
20 21768 22711 21925 22135 505
10 10559 10441 9988 10329 301
5 5141 5137 5028 5102 64
2.5 2618 2537 2683 2613 73
1.25 1605 1654 1552 1604 51
0.625 1049 1014 1028 1030 17
0.3125 858 932 865 885 40
0.15625 789 635 745 723 79
0 644 549 655 616 58
Optical density Values
Linearity and intra assay Variation of 3rd
generation assay
Method 1= 3.3xSD(blank)/slope Method 2= LOB+1.645SD(low conc )
842 RLU0.179 ng/ml
Limit of detection of the 3rd
generation assay
R2= 0.9968
0
5000
10000
15000
20000
25000
5 10 15 20
Recombinant AEP ng/ml
Re
lative
lig
ht u
nits (
RL
U)
R2= 0.9995
0
10000
20000
30000
40000
50000
60000
0.1 0.2 0.3 0.4 0.5REH +AEP cell lysate μg/well
Re
lative
lig
ht u
nits (
RL
U)
B
C
A
Development of assays
82
detection of the assay. C: AEP expression in ng/ml in cell lines using the 3rd
generation ELISA. SD1 AEP KD represents SD1 with a stable transcript
depletion of AEP using a lentiviral shRNA. * represents p= <0.05, **
represents p=<0.005 and *** represents p=<0.0005. Error bars represent ± 1
SE. D: expression of AEP in cell lines by western blotting.
3.4 QUANTIFYING ACTIVE AEP: The inactive AEP zymogen undergoes an
N and C terminal proteolytic cleavage to yield a mature active form, figure
3.17 A. None of the previously described techniques measure specifically the
active (46 and 36 kd) forms of AEP and the ELISA assay does not estimate
the expression of AEP across sub-populations of cells within the same sample.
Flow-cytometry based technique using cell permeable fluorescent labelled
AEP probe (LP-1) (106) had a potential to measure both these parameters
that ELISA could not. Such a technique if developed successfully would be
easy to use, and could be rapidly integrated in clinical practice. The LP-1
probe binds to active AEP Figure 3.17B.
ImageStream (multispectral image flow cytometer) results showed that active
AEP (red) co-localised to lysosomes in SD1 cells, figure 3.18. This was
consistent to previously published finding seen on formalin-fixed cytospins of
SD1 cells(100). Majority (71.5%) of SD1 cells expressed active AEP
compared to REH (4%, data not shown) and there was a skew towards right
of AEP expression in sub-population of SD1 cells, figure 3.19
Development of assays
83
Figure 3.17 Basis of action of LP-1 probe A: The 56 kd zymogen undergoes a
sequential N and C terminal cleavage that is pH dependent to lead to the
46kd intermediate form. The exact mechanism by which this form is further
processed to the mature and 36kd form is unknown. B: Cartoon to describe
the chemistry and specificity of the LP1 probe (Courtesy Dr. Shekhar
Krishnan).
Pre-pro AEP 56kDa
Intermediate
Forms 46 kDa
Mature AEP 36kDa
lysosome
↓pH
Other
proteases
N - ter
AA24D N
297 110
C - ter
Endo lysosomes
Prepro AEP
Mature active AEP
Fluorophore
Aliphatic spacer sequence – influences
cell permeability
Proline residue – stabilises
substrate/enzyme interaction
Azaepoxy-ASN residue –
determines reactivity & specificityCell
membrane
A
B
Development of assays
84
Figure 3.18 Localisation of active AEP in SD1 cells. SD1 cells were dually labelled with LysoTracker® green DND-26 and LP1
probe followed by image acquisition in ImageSteam in 1) bright field, 2) 502nm filter that detects lysosomes stained with
LysoTracker® green DND-26, 3) 633nm filter that detects LP1 probe and finally 4) merged image that shows co-localization of AEP
within the SD1 lysosomes. Courtesy Jeff Barry & Morgan Blaylock.
Selected images
Bright field Overlay Lysotracker green AEP probe Merged
Ch01 br/ch2/ch5 Ch02 Ch05 Ch02/Ch05
37396
Ch01 br/ch2/ch5 Ch02 Ch05 Ch02/Ch05
36145
Ch01 br/ch2/ch5 Ch02 Ch05 Ch02/Ch05
28565
Ch01 br/ch2/ch5 Ch02 Ch05 Ch02/Ch05
29753
Development of assays
85
Figure 3.19 Expression of active AEP by Flow cytometry/Image stream. Tandem flow cytometry acquisition of mean fluorescent
intensity in SD1 cells incubated with LP-1 probe along with single cell imaging showing variable expression of AEP within
subpopulation of SD1 cells.
Intensity_MC_Ch05
Expression of Active AEP Flow Cytometry/Image stream
0-1e3 1e3-1e4 1e5-1e5 1e61e4
0
6
8
4
10
2
Norm
alise
d F
req
ue
ncy
71.54% are positive (4.00% Positive REH)
Majority are weakly fluorescent but with a skew to the right
SD1 AEP
Dim
boosted intensity
Centre
boosted intensity
Bright
Ch01 Ch05 Ch01/Ch05
375
Ch01 Ch05 Ch01/Ch05
66
Ch01 Ch05 Ch01/Ch05
8589
Development of assays
86
Protein electrophoresis of cell lysate of in-vivo labelled cells confirmed that the
probe binds only to the mature (36kd) and intermediate (41kd) forms of AEP,
figure 3.20 A. However, there was no difference in intensity of the AEP band
between SD1 and SD1AEPkd. Live cell labelling with LP1 followed by
measurement of mean fluorescence intensity on flow cytometry also showed
no difference between SD1 and SD1AEPkd cell lines, figure 3.20 B. SD1AEPkd
had less AEP protein compared to SD1 cell by immunoblotting, figure 3.20 C
and by ELISA, figure 3.20 D. However there was still enough AEP protein by
ELISA in 1 million SD1AEPkd cells for the probe to bind unlike in 1 million REH
cells. Additionally there is no difference in the mean fluorescence between
SD1 and REH+AEP despite the latter having greater than 10 times AEP protein
than the former. Thus either the kinetics of the binding of LP1 to active AEP is
not linear or there is saturation of fluorescence. Either way, the LP1 probe is
currently not suitable to quantify AEP in clinical samples.
Though the expression of AEP correlate at the level of transcript and protein
in case of cell lines, there was no correlation in the expression of AEP by
these two techniques in clinical samples (n=16), figure 3.21.
Development of assays
87
Figure 3.20 Suitability of LP1 probe to measure active AEP. A: Protein lysates
were first labelled with LP1 probe following which AEP was
immunopreciptated as described in chapter 2 and subjected to
electrophoresis and visualisation of the bands on LICOR system. It confirms
that the LP1 probe bind to the intermediate and mature (active) forms of the
B Live cell labelling by AEP probe followed by
Flow cytometry
C AEP Western blot cell lysates: 10μg protein per lane except for REH+AEP(0.1 μg )
A Immunoprecipitation of 1 million cell lysates labelled
with AEP probe
SD1REHSD1AEPkdREH+AEP
36 kd
46 kd
0
5000
10000
15000
20000
25000
30000
35000
Rela
tive
Lig
ht
Units
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Re
lative
Lig
ht
Un
its
Media
n F
luore
scent In
tensity
100
150
200
250
300
350
400
450
SD1 REH SD1AEPkd
D AEP ELISA cell lysates: 10μg protein per laneexcept for REH+AEP(0.1 μg )
SD1 REH SD1AEPkd REH+AEP
E One million cells labelled with AEP probe in-vivofollowed by AEP ELISA of cell lysate
SD1 REH SD1AEPkd REH+AEP
Marker
56kd
GAPDH
36 kd
SD1 REH SUPB15 SD1AEPkdREH+AEP
Development of assays
88
AEP protein. B: Flow cytometry of live cells labelled with LP-1 probe showed a
difference in the mean fluorescent intensity between SD1 (AEP expressing
cell line) and REH (AEP non expressing cell line). However there was no
difference in the mean fluorescent intensity between SD1 and SD1AEPkd cells.
C: AEP expression by immunoblotting in the 4 cell lines. Compared to SD1,
SUPB15 shows intermediate and REH shows no expression of AEP whilst
REH+AEP has much greater amount of AEP. Note total protein loaded in the
lane for the REH+AEP lysates was 1/10th of the quantity loaded in lanes of other
cell lines. D: AEP expression in the four cell lines by ELISA. Results mirror
those obtained by immunoblotting. E: One million cells that were labelled live
with LP-1 probe followed by cell lysis and quantification of AEP from 1 million
cell lysates protein by ELISA.
Figure 3.21: Co-relation of expression of AEP at transcript and protein level.
Sixteen samples were selected based on the differences in the expression of
AEP at the level of transcript by TaqMan gene expression assay, X axis. Cell
lysates of these samples were used to measure the expression of AEP at the
level of protein by chemiluminescent ELISA, y axis. There was poor co-
relation of expression of AEP between the two assays (R2=0.2236).
R2= 0.2236
500
0 2 4 6 8 10 12 148
Pg o
f A
EP
/100
μg o
f p
rote
in 400
300
200
100
∆ CT
Value
n=16
R2= 0.2236
500
0 2 4 6 8 10 12 148
Pg o
f A
EP
/100
μg o
f p
rote
in 400
300
200
100
∆ CT
Value
n=16
Development of assays
89
3.5 Summary of assays tested for AEP expression:
1) In cell lines, there was a correlation between AEP expression at the
level of transcript by RQ-RTPCR and at the level of protein by western
blotting and by ELISA.
2) Of these techniques, RQ-RTPCR and ELISA could be used to quantify
AEP.
3) In clinical samples there was poor correlation of expression of AEP
between RQ-RTPCR and ELISA.
4) The only technique that quantifies active AEP is the one using the LP-1
probe but then this technique was not suitable for use in clinical
samples.
In the end, AEP ELISA was taken forward as it measured AEP at a protein
level, accepting the limitation that it measured total AEP and could not
specifically measure the active AEP.
Asparaginase Study: L-Asparaginase activity
90
Chapter 4 Asparaginase Study: L-Asparaginase activity
______________________________________________________________
4.1 BACKGROUND
Clinical trials over three decades have established a clear role for the anti-
leukaemic drug L-Asparaginase (ASNase) in childhood ALL (32, 63-70).
ASNase is an enzyme and its optimal action requires precise dosing to
achieve a consistent therapeutic level of enzymatic activity. Despite this, the
clinical practice of employing ASNase in childhood ALL varies in terms of the
type of preparation of the drug used its dose, the route of administration and
the schedule (Table 4.1) (32, 76, 89, 97, 111-120). As described in the
introduction, the PEG-ASNase offers a more stable pharmacokinetic profile
than the native product. The UK was one of the first countries in the world to
routinely use E.coli ASNase conjugated to polyethylene glycol (PEG-ASNase)
during both induction and post induction phases of the UKALL2003 trial and
ALLR3 protocols (Table 4.1).
Though the drug has been in routine clinical use for over 4 decades, little is
known about the mechanisms by which it is degraded and inactivated. The
enzyme is a bacterial protein. First-pass kinetics suggests that the drug is
possibly eliminated by the reticulo-endothelial system (78). Another method of
inactivation is due to the development of antibodies. Within this mechanism
lies a spectrum from anaphylaxis (IgE or IG4 mediated) to silent inactivation
which is presumably IgG mediated. Work done previously by our group
showed that a dyad of lysosomal cysteine proteases, Asparaginyl
Endopeptidase (AEP) and Cathepsin B (CTSB) expressed in leukaemic blasts,
degraded ASNase in-vitro (100). AEP was additionally over expressed in a
subset of children with precursor B ALL predominantly with poor risk
cytogenetic features (101). We showed that while CTSB cleavage was non-
specific, AEP cleavage inactivated the drug while retaining intact known
antigenic sites. As reticulo-endothelial cells are enriched in lysosomal
proteases, this observation supports the previous conjecture of how ASNase
Asparaginase Study: L-Asparaginase activity
91
Pro
toco
lR
isk g
roup
Schedule
Ris
k g
roup
Phase
Schedule
AIE
OP
-ALL-9
5A
llL-A
spara
gin
ase: 5,0
00 IU
/m2
IM e
very
72 h
ours
; 8 d
oses
All
a)
Rein
duction
pro
tocol II
L-A
spara
gin
ase: 10,0
00 I
U/m
2IM
every
72 h
ours
; 4 d
oses
CC
G1962
SR
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM s
ingle
dose
SR
a)
Dela
yed Inte
nsific
ation
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM s
ingle
dose
vs
vs
L-A
spara
gin
ase: 6,0
00 IU
/m2
IM 3
tim
e a
week;
9 d
oses
L-A
spara
gin
ase: 6,0
00 IU
/m2
IM 3
tim
e a
week;
6 d
oses
CC
G1961
HR
L-A
spara
gin
ase: 6,0
00 IU
/m2
IM 3
tim
e a
week;
9 d
oses
HR
-RE
RS
tandard
thera
py: D
I L-A
spara
gin
ase: 6,0
00 IU
/m2
IM 3
tim
e a
week;
6 d
oses
vs
Incre
ased inte
nsity
Descri
bed in H
R-S
ER
HR
-SE
RIn
cre
ased inte
nsity:
a)
Consolidation
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM tw
o d
oses 4
weeks a
part
b)
Inte
rim
Main
tenance
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM tw
o d
oses 3
weeks a
part
c)
DI
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM s
ingle
dose
d)
Reconsolid
ation
PE
G-A
spara
gin
ase 2
500 IU
/m2
IM s
ingle
dose
ALL-B
FM
95
All
L-A
spara
gin
ase: 5,0
00 IU
/m2
IV 3
tim
e a
week;
8 d
oses
SR
/MR
a)
Rein
duction
L-A
spara
gin
ase: 10,0
00 I
U/m
2IM
every
72 h
ours
; 4 d
oses
or
PE
G-A
spara
gin
ase 1
000 o
r 2
500 IU
/m2
IV s
ingle
dose
HR
a)
Inte
nsiv
e c
onsolid
ation
L-A
spara
gin
ase: 25,0
00 I
U/m
2IV
once p
er
blo
ck;
6 b
locks
b)
Rein
duction
L-A
spara
gin
ase: 10,0
00 I
U/m
2IM
every
72 h
ours
; 4 d
oses
or
PE
G-A
spara
gin
ase 1
000 o
r 2
500 IU
/m2
IV s
ingle
dose
Po
st
Ind
ucti
on
Ind
ucti
on
Ta
ble
4.1
Asparaginase Study: L-Asparaginase activity
92
Pro
toco
lR
isk g
roup
Schedule
Ris
k g
roup
Phase
Schedule
Po
st
Ind
ucti
on
Ind
ucti
on
ALL-B
FM
2000
All
L-A
spara
gin
ase: 5,0
00 IU
/m2
IV 3
tim
e a
week;
8 d
oses
SR
/MR
a)
Rein
duction
L-A
spara
gin
ase: 10,0
00 I
U/m
2IM
every
72 h
ours
; 4 d
oses
or
PE
G-A
spara
gin
ase 1
000 IU
/m2
IV s
ingle
dose
HR
a)
Inte
nsiv
e c
onsolid
ation
L-A
spara
gin
ase: 25,0
00 I
U/m
2IV
tw
ice p
er
blo
ck; 6 b
locks
b)
Rein
duction
L-A
spara
gin
ase: 10,0
00 I
U/m
2IM
every
72 h
ours
; 4 d
oses
or
PE
G-A
spara
gin
ase 1
000 IU
/m2
IV s
ingle
dose
ALL9
All
Win
dow
:N
HR
No f
urt
her
Aspara
gin
ase
PE
G-A
spara
gin
ase 1
000 IU
/m2
IV s
ingle
dose
Induction:
L-A
spara
gin
ase: 6,0
00 IU
/m2
IV t
wic
e a
week;
4 d
oses
HR
a)
Inte
nsific
ation
L-A
spara
gin
ase: 10,0
00 I
U/m
2IV
once a
week,
9 d
oses
CO
ALL-0
6-9
7A
ll N
o A
spara
gin
ase
LR
days 3
1 a
nd 5
9L-A
spara
gin
ase: 45,0
00 I
U/m
2IV
day 8
0P
EG
-Aspara
gin
ase:
2500 I
U/m
2IV
HR
days 3
2,4
6 a
nd 8
7L-A
spara
gin
ase: 45,0
00 I
U/m
2IV
day 1
08
PE
G-A
spara
gin
ase:
2500 I
U/m
2IV
Ta
ble
4.1
co
nti
nu
ed
Asparaginase Study: L-Asparaginase activity
93
Pro
toco
lR
isk g
roup
Schedule
Ris
k g
roup
Phase
Schedule
2
Po
st
Ind
ucti
on
Ind
ucti
on
DF
CI
91-0
1A
ll N
o A
spara
gin
ase
All
a)
Inte
nsific
ation
L-A
spara
gin
ase: 25000 IU
/mIM
every
fort
nig
ht, 1
5 d
oses
random
isation
or
PE
G-A
spara
gin
ase:
2500 I
U/m
2IM
every
week,
30 d
oses
NO
PH
O
ALL-2
000
All
L-A
spara
gin
ase: 6,5
00 IU
every
3-4
days; 4 d
oses
All
a)
DI
L-A
spara
gin
ase:6
,500 I
U e
very
3-4
days;
4 d
oses
Tota
l T
hera
py
XIV
All
L-A
spara
gin
ase:1
0,0
00 U
/m2
IM 3
X w
eek;
9 d
oses
All
a)
Rein
duction
PE
G-A
spara
gin
ase:
2500 U
/m2
IM e
very
week f
or
2 w
eeks
UK
ALL2003
All
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
, fo
rtnig
htly; 2 d
oses
SR
/IR
a)
1 v
s2 D
I ra
ndom
isation
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
once
HR
a)
Aug B
FM
consolid
ation
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
; tw
o d
oses
b)
ICM
IP
EG
-Aspara
gin
ase:
1000 I
U/m
2IM
; tw
o d
oses
c)
DI I
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
; tw
o d
oses
d)
ICM
II
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
; tw
o d
oses
e)
DI II
PE
G-A
spara
gin
ase:
1000 I
U/m
2IM
; tw
o d
oses
Ta
ble
4.1
co
nti
nu
ed
SR
= S
tan
da
rd R
isk
IR
=
In
term
ed
iate
Ris
k
MR
=
Me
diu
m R
isk
HR
=
Hig
h R
isk
RE
R
=
Ra
pid
Earl
y R
espo
nse
SE
R
=
Slo
w E
arl
y R
esp
on
se
DI
= D
ela
yed
In
ten
sific
atio
n
Au
g B
FM
=
Au
gm
en
ted
BF
M
ICM
=
In
teri
m C
ap
izziM
ain
ten
an
ce
Asparaginase Study: L-Asparaginase activity
94
is eliminated by the body. However the selective expression by leukaemic
blasts suggested that this could also be a novel mechanism for drug
resistance. Thus the proteases could lead to early direct inactivation of the
drug by proteolytic activity or late inactivation related to antigen processing
and antibody formation.
This chapter reports on the results of the Asparaginase Study in ALL2003 and
ALLR3. This was a prospective, observational study measuring trough
ASNase activity levels in children receiving PEG-ASNase and correlation with
lymphoblast expression of AEP and CTSB. Patients were recruited, after
consent, from two national trials in childhood ALL: the frontline UKALL2003
and the relapsed ALLR3 trial.
4.1.1 Aims of the Asparaginase Study
1) Evaluate the ASNase activity in newly diagnosed and relapsed patients
receiving 1000 u/m2 of PEG-Asnase
2) To provide recommendations on whether routine pharmacokinetic assay
after PEG-ASNase is required or not in clinical practice and if so, which
subgroup of patients are most likely to benefit from such strategy.
3) To determine whether the expression of AEP and CTSB by lymphoblasts
are predict response to PEG-ASNase.
4) To study the clinical impact of sub-therapeutic response to PEG-ASNase
during the induction and post induction phases.
4.1.2 Objectives of the Asparaginase Study
1) To measure serially ASNase activity during the induction and post induction
phases in order to determine the pattern of drug activity.
Asparaginase Study: L-Asparaginase activity
95
2) To correlate plasma ASNase activity during induction with
a) Baseline biological characteristics such as age, presenting white cell count,
cytogenetics, immunophenotype and the expression of AEP and CTSB
b) Outcome variables such as early response to therapy and the burden of
disease at a molecular level at the end of induction.
3) To correlate plasma ASNase activity during post induction phase with the
development of clinical hypersensitivity or silent neutralising antibodies to
ASNase.
4.1.3 Design of the Asparaginase Study
Figure 4.1 shows the schedule of PEG-ASNase, the sampling time points for
each of the assay done in this study and an overview of the sample
processing. Figure 4.2 gives an overview of the statistical methods used for
correlating the analytical variables such as expression of AEP and CTSB in
leuakemic cells, development of antibodies to PEG-ASNase and native E.coli
Asparginase and ASNase activity levels with outcome variables such as early
response and MRD at the end of induction and with baseline characteristics
such as National Cancer Institute risk category, gender, cytogenetics and
immunophenotype. Data on the baseline variables, outcome variables, patient
characteristics and toxicity to PEG-ASNase was kindly provided by the
Professor Anthony Moorman (Cytogenetics), Dr Jeremy Hancock (MRD) and
Dr Sue Richards and Rachel Wade from the Clinical Trials Office (all the rest
of the data) which also provided the support for the statistical analysis of the
data.
Asparaginase Study: L-Asparaginase activity
96
Figure 4.1 Schedule of L-Asparginase the sample time points for measuring
ASNase activity in UKALL2003 (A), ALLR3 (B) and an overview of the sample
processing and the assays done at each time point in the Asparaginase Study
Regimen A
Weeks:1 6 9 17 24 32 25 -164 ( 1DI)/ 39 -164 (2 DI)
Weeks:1 6 11 19 26 34 26 -164 ( 1DI)/ 41-166 (2 DI)
Regimen B
Weeks: 1 6 15 23 31 39 47 170
= Subject to randomisation
PEG ASNase
Regimen C
DI 1IM 1 MaintenanceConsolidation
d4 d4 d4
Induction DI 1IM 1 Maintenance
d4 d4 d4
Induction IM 2DI 1 DI 2IM 1 Maintenance
d4 d4 d4
Induction IM 2DI 1 DI 2IM 1 MaintenanceStd. BFM Consolidation
d4 d4 d4d18
Induction ICM 2DI 1 DI 2ICM 1 Maintenance
d16 d3d4 d18 d44 d4 d43 d3 d4 d43
Induction ICM 2DI 1 DI 2ICM 1 MaintenanceAug BFM Consolidation
d16 d3d4 d18 d44 d23 d4 d43 d3 d23 d4 d43
L-Asparaginase activity
A: UKALL2003
C: Overview of assay time points
L-Asparaginase Activity &Anti L-Asparaginase Antibody
L-Asparaginase Activity
Anti L-Asparaginase AntibodyFinal sample:
blood
mRNA
Proteins
RQ -RTPCR: AEP
ELISA: AEP, CTSBDiagnosis
Sample:
Blood & bone
marrow
Serial follow up
Samples: blood
Leukaemic cells
Blood plasma
d4d18
(at baseline in selected patients)
B:ALLR3
Phase I
Allo-SCT
SR
HR
Phase IV
<10-3
≥10-3
IR
Phase III Phase VI
1 5
6 9 13
RT* Phase V
14 30 104
Weeks
MRD <10-4
MRD >10-4
Phase I Phase II
Phase IIIPhase IIPhase I
Weeks 1
Weeks 1
4 5 1011 15
d18d15
d3;
w6
d3;
w7d3;
w9
MRD
IM 2 DI 2IM 2 DI 2
d2,4
w11
d2,4
w12d2
w14
d18d3 d18d17
d18d3 d18d17
d2
w6
d2,4
w9
d2,4
w10d2
w12
Erwinase
L-Asparaginase activity on any one occasion 24 hr after Erwinase
Asparaginase Study: L-Asparaginase activity
97
(C). Aug= Augmented, Std= Standard, IM= interim maintenance, DI= delayed
intensification, ICM= interim Capizzi maintenance,
.
Figure 4.2 Overview of the statistical methods used to analyse the results.
4.1.4 Recruitment in the Asparaginase Study: A pilot for sample collection
and for the development of standard operating procedure (SOPs) for sample
processing and storage was opened in December 2007. The study was then
opened to limited centres in May 2008 and then to all paediatric haematology
centres in the UK in September 2008. After doing an initial trial run in the last
quarter of 2008, the study opened to 19 of the 22 paediatric oncology and 8
adult oncology centres in the UK from January 2009. At the point of censoring
February 2011, the study had recruited a total of 593 patients in the
UKALL2003 and 25 patients in ALLR3 studies. The study had to be stopped
because the ALL2003 trial closed and the opening of ALL2011 trial was
delayed Applying the criteria of analysing only those samples that were taken
between 7 and 14 days post PEG-ASNase results are available for 427
patients in the UKALL2003 trial and 22 patients in the ALLR3 trial.
Statistical Analysis
AEP & CTSB
Analytical variables
L-Asparaginase activity
Outcome variables
•Early response (1-2 weeks
Slow/Rapid
•MRD (5 weeks):
Low/Intermediate/High
Baseline Characteristics
•Gender
•National Cancer Institute Risk Category
•Cytogenetics
•Immunophenotype (precursor B or non precursor B)
Anti L-Asparaginase
antibodies
Mann-Whitney Test
Mann-Whitney Test
Mann-Whitney Test for Early Response
Kruskal-Wallis Test for MRD
Chi squared test
Chi squared test
Mann-Whitney Test
Asparaginase Study: L-Asparaginase activity
98
4.2 RESULTS:
The results of the Asparaginase study have been split into two chapters for
ease of reading. The bulk of the work presented in this chapter pertains to the
results and analysis of ASNase activity in 427 patients recruited from the
UKALL2003 trial. The next chapter investigates the biological determinants of
therapeutic response to ASNase.
4.2.1 RECRUITMENT IN UKALL2003 ARM OF THE ASPARAGINASE
STUDY
The study recruited 77% of the target population. Figure 4.3 is a graphical
representation of the recruitment of patients between January 2009 and
February 2011 when the study was fully opened to all the centres
Figure 4.3: Recruitment in the Asparaginase Study- UKALL2003 arm.
Magenta and green colours respectively represent the number of new cases
of ALL diagnosed each month at the participating centres and the number of
cases recruited in the Asparaginase Study. Courtesy Dr Catriona Parker.
UKALL2003: Asparaginase Study Recruitment
0
5
10
15
20
25
30
35
Jan
Feb
Ma
r
Ap
r
Ma
y
Jun
Ju
l
Au
g
Sep
Oct
Nov
Dec
Jan
Feb
Ma
r
Ap
r
Ma
y
Jun
Ju
l
Au
g
Sep
Oct
Nov
Dec
Jan
Feb
2009 2010 2011
Num
be
r o
f P
atien
ts
UKALL2003: Asparaginase Study Recruitment
0
5
10
15
20
25
30
35
Jan
Feb
Ma
r
Ap
r
Ma
y
Jun
Ju
l
Au
g
Sep
Oct
Nov
Dec
Jan
Feb
Ma
r
Ap
r
Ma
y
Jun
Ju
l
Au
g
Sep
Oct
Nov
Dec
Jan
Feb
2009 2010 2011
Num
be
r o
f P
atien
ts
Asparaginase Study: L-Asparaginase activity
99
4.2.1.1 Patient characteristics
As shown in table 4.2 the baseline biological and demographic characteristics
of the patients and the response to induction treatment of patients in the
Asparaginase Study.
Table 4.2 Patient characteristics Characteristics
In Asp Study
n=427
Gender
256 (59.9%)Male
Female 171 (40.1%)
Age (years)
296 (69.3%)
161 (30.7%)
3.2
5.4
11.3
<10
>10
25th
percentile
Median
75th
percentile
Range 1.2 to 23.5
White cell count (x10 9/L)
323 (75.6%)
104 (24.4%)
5.1
14.7
47.1
<50
>50
25th
percentile
Median
75th
percentile
Range 0.5 to 800
Immunophenotype
368 (86.4%)B cell
Non B cell ( T=58; null=1) 59 (15.4%)
Cytogenetic classification of B; n=368
(Moorman et al Lancet Oncology, 2010)
196 (53.4%)
120 (32.5%)
24 (6.5%)
good
intermediate
poor
no result 28 (7.6%)
Regimen at diagnosis
234 (54.8%)A
B 193 (45.5%)
Early Response to therapy
46 (10.8%)slow
rapid 381 (91.2%)
MRD response
95 (22.2%)
172 (40.3%)
low
intermediate
high 160 (37.5%)
Table 4.2Not In Asp
Study n=2705
1521 (56.2%)
1184 (43.8%)
1992 (73.6%)
713 (26.4%)
p value
(heterogeneity)
0.1
0.3
0.4
0.7
2119 (78.3%)
589 (21.7%)
2319 (85.7%)
381 (14.3%)
N.A
N.A
N.A
N.A
Asparaginase Study: L-Asparaginase activity
100
4.2.1.2 ASNase activity at individual time points ASNase activity was
measured in 1074 samples from 427 patients at 6 time points during the
course of therapy. The median number of sample time points per patient was
2.5 (Range: 1-6). Figure 4.4 shows the distribution of activity results in box
and whisker plots at each of the six time points.
Figure 4.4: ASNase activity at individual time points. The box encompasses
ranges between the 1st and 3rd quartiles (Inter-quartile range-IQR; 25th-75th
percentiles). The horizontal bar represents the median value, the lower
whisker represents minimum value, the upper whisker represents maximum
value that lies within 1.5 x IQR of the upper quartile. Outliers (beyond the
2000
1500
1000
500
0
Pla
sm
a L
-Aspara
gin
ase A
ctivity U
/L
TP 6
n=96
TP 5
n=39TP 4
n=206TP 3
n=54
TP 2
n=354
TP 1
n=325
ALL TP
n=1074
Time point (TP)
TP1: 7-14 days post induction day 4 PEG-ASNase 91
TP2: 7-14 days post induction day 18 PEG-ASNase 85
TP3: 7-14 days post Capizzi I day 23 PEG-ASNase 83
TP4: 7-14 days post DI I day 4 PEG-ASNase 96
TP5: 7-14 days post Capizzi II day 23 PEG-ASNase 87
TP6: 7-14 days post DI II day 4 PEG-ASNase 97
% with adequate activity
Overall 90
Asparaginase Study: L-Asparaginase activity
101
upper whisker) are represented by circles (≤ 3 x IQR of the upper quartile and
the asterisk (> 3 x IQR of the upper quartile). The dotted horizontal line
represents cut off for adequate plasma ASNase activity of 100 U/L.
Conclusion 1 – From a trial perspective 85-97% of patients have adequate
trough activity at any time point.
Table 4.3 shows the median activity in those who have adequate ASNase
activity at TP1 and or TP2 and those who don’t. Most patients with inadequate
levels have levels below the level of detection of the assay and much below
the therapeutic threshold. Conversely, most patients with adequate activity
had levels that were well above the therapeutic threshold. Thus increasing the
dose of PEG-ASNase is unlikely to benefit those with inadequate levels and is
likely to increase toxicity for the majority who already have high levels.
Table 4.3 Median ASNase activity levels.
Conclusion 2 - Increasing the dose to 2,500 – 3,500 U/m2 is unlikely to
improve the percentage of patients who will have adequate levels and is likely
to lead to increased toxicity.
Inadequate activityTime point (TP)
TP1: 7-14 days post induction day 4 PEG-ASNase
TP2: 7-14 days post induction day 18 PEG-ASNase
TP3: 7-14 days post Capizzi I day 23 PEG-ASNase
TP4: 7-14 days post DI I day 4 PEG-ASNase
TP5: 7-14 days post Capizzi II day 23 PEG-ASNase
TP6: 7-14 days post DI II day 4 PEG-ASNase
Median level U/L
Adequate activity
Median level U/L
45
Below LOD*
278
349
1027
387
547
687
Table 4.3 L-Asparaginase activity levels in patients split into two group: Those with adequate
and those with inadequate L-Asparaginase activity levels.
LOD*= limit of detection of the assay
Below LOD*
Below LOD*
Below LOD*
Below LOD*
Asparaginase Study: L-Asparaginase activity
102
4.2.1.3 Serial ASNase Activity results: Results of serial ASNase activity
(induction and post induction) are shown for all patients in Table 4.4. A: Five
percent of patients had inadequate activity levels at one or more time points
and 4% had inadequate levels during post induction. Eighty-three percent of
patients had adequate levels during induction and 90% during post induction.
B: shows serial activity results of patients who had inadequate activity
(ASNase activity level of <100 U/litre of plasma) at TP1 &/or TP2 (n=44),
grouped according to the regimen they were assigned to at the end of
induction. All patients were initiated on either regimen A or B based on the
NCI risk stratification. During the course of induction they were assigned to
regimen C if they had poor cytogenetics &/or slow early response. Patients in
regimen A and B who had high MRD at the end of induction underwent a
randomisation of continuing on their assigned regimens or changing to
regimen C. Patients transferred to regimen C showed a higher incidence of
persistent post induction inactivation (p=<0.001). C: shows serial activity
results in patients with adequate activity (ASNase activity level of ≥ 100 U/litre
of plasma) at TP1 & or TP2, again grouped according to the regimen they
were on at the end of induction (n=216). Those who were in regimen C at the
end of induction showed a higher incidence of post induction inactivation
compared to those in regimen A and or B (p=<0.002, Fisher exact test)
Asparaginase Study: L-Asparaginase activity
103
Table 4.4 A: Serial ASNase activity, n=258/427
Induction Post induction n (%)
Adequate Adequate 204 (79)
Adequate Inadequate 11 (04)
Inadequate Adequate 29 (11)
Inadequate Inadequate 14 (05)
Table 4.4 B: Serial ASNase activity in patients who had inadequate levels during induction, n=44/427
Inadequate ASNase in Post induction
induction - Regimen (n) Recovered Not recovered
Regimen C (14) 5 9
Regimen A/B (30) 25 5
Table 4.4 C Serial ASNase activity in patients who had adequate activity during induction, n=216/427
Adequate ASNase in Post induction
induction - Regimen (n) Remained adequate Became inadequate
Regimen C (58) 50 8
Regimen A/B (158) 155 3
Asparaginase Study: L-Asparaginase activity
104
Conclusion 3 - Patients transferred to regimen C at the end of induction have
higher incidence of inactivation of ASNase.
4.2.1.4 Correlation of induction ASNase activity with biological factors
and therapeutic outcome: Table 4.5 shows the correlation of ASNase
activity during induction with baseline characteristics and table 4.6 with
outcome variables. Univariate analysis showed a significant correlation of
inadequate activity with NCI high-risk patients. This was determined by the
age at diagnosis (p=0.0016) and not the presenting white cell count (p=0.6).
Using a Wilcoxon match-pairs signed-rank test, there was a difference in the
age distribution of patients with adequate ASNase activity (Median age 5.08
years, range 1.17-23.2) as compared with those with inadequate ASNase
activity (Median age 7.71 years, range 1.67-22.92), p=0.021.
Table 4.5 Correlation between induction ASNase and baseline characteristics
p value χ2
Adequate Inadequate
Gender 0.2396
Male 209 47
Female 147 24
Immunophenotype 0.5328
B cell 306 63
T cell and null (1 pt) 50 8
Age 0.0016
< 10 years 258 38
> 10 years 98 33
NCI RISK 0.0097
Standard 205 29
high risk 151 42
WCC on presentation 0.6052
<50 x10 9/L 271 52
>50 x10 9/L 85 19
B cell Cytogenetic Subgroups 0.3914
No results 23 5
159 38
104 16
20 4
Table 4.5 Correlation Induction Asparaginase levels with baseline characteristics
No results
Good Risk
Intermediate Risk
Poor Risk
Good Risk
Intermediate Risk
Poor Risk
heterogeneity
Asparaginase Activity (TP1 &or TP2)
Asparaginase Study: L-Asparaginase activity
105
Conclusion 4 - Older patients had increased incidence of inadequate
ASNase activity during induction.
Overall, there was no significant association between response to ASNase
during induction and outcome variables such as early response or the burden
of disease at the molecular level (MRD). However, in the NCI low risk patients,
who receive a three drug induction, inadequate response to ASNase activity
was associated with high MRD at the end of induction, p=0.0289 and
especially so in the NCI low risk patients who belonged to good risk
cytogenetic group, p=0.006 (Table 4.6). Figure 4.5 shows the distribution of
ASNase activity levels in these patients at TP1 and TP2.
Table 4.6 Correlation between induction ASNase and outcome
Patients who had high MRD at the end of induction had significantly lower
ASNase activity at TP2 compared to those with low/intermediate MRD
(p=<0.01). Similar analysis done at TP1 showed no difference in the ASNase
activity levels between the two subgroups (p=<0.871), figure 4.5.
p value χ2
Adequate Inadequate heterogeneity
ALL patients 0.3
SER 36 10
RER 320 61
ALL patients 0.07
MRD low/intermediate 228 39
MRD high 128 32
NCI high risk 0.8
MRD low/intermediate 87 25
MRD high 64 17
NCI Std. risk 0.0289
MRD low/intermediate 141 14
MRD high 64 15
NCI Std. risk, good risk cytogenetics 0.0059
MRD low/intermediate 94 11
MRD high 33 13
Asparaginase Activity (TP1 &or TP2)
Table 4.6 Correlation Induction Asparaginase levels with outcome characteristics
Regimen at end of induction
Assigned - A or B
Changed to C
259 46
96 26
0.12
Asparaginase Study: L-Asparaginase activity
106
Figure 4.5 ASNase activity levels at TP1 and TP2 in NCI SR, precursor B ALL
patients who had good risk cytogenetics. These patients were split into two
groups depending on the MRD result obtained at the end of induction phase
of treatment (TP2). ASNase levels at TP2 co-related with MRD status,
p=0.009.
Conclusion 5 - ASNase activity results at TP2 correlate with MRD at the end
of induction in patients with precursor B ALL who belong to NCI standard risk
and have good risk cytogenetics
4.2.1.5 Time to event analysis: The follow up of patients in the ASNase
study is currently short (median 2 years). Figure 4.6 compares event free,
relapse free and overall survival by univariate analysis of 423/427 patients in
the study divided into two sub groups of adequate and inadequate ASNase
2000
1500
1000
500
0
n= 86 n= 39
MRD
low/intermediateMRD high
Aspara
gin
as
e a
ctivi
ty U
/litre
of pla
sm
a
Asparaginase acti vity at TP1 & T P2 in pati ents who belonged to NCI standard risk & who had good risk precursor B ALL cytogenetics: MRD high vs MRD low/intermediate
p= <0.01
MRD low/intermediate
MRD high
p= <0.871
n= 83 n= 30
TP1 TP2
Asparaginase Study: L-Asparaginase activity
107
activity during induction. Patients with adequate activity had ASNase activity
levels of ≥ 100 U/litre of plasma at one or both available time points during
induction (TP1 &/or TP2). If at either or both time points patients had ASNase
activity level of <100 U/litre of plasma then they were considered to have
inadequate activity. For both, the event free survival and relapse free survival,
there was a trend towards inferior outcome in patients who had inadequate
ASNase activity at TP1 and or TP2. As regards to overall survival, there was
an association between inadequate ASNase activity during induction and an
inferior outcome, figure 4.7C.
Conclusion 6 - Early data seems to indicate the response to PEG-ASNase
during induction is influencing the outcome in children with ALL.
Asparaginase Study: L-Asparaginase activity
108
Figure 4.6: Time to event analysis of patients in UKALL2003 with respect to
response to PEG-ASNase. Kaplan-Meier (K-M) curves were compared with
the log-rank test.
OVERALL SURVIVAL BY ASPARAGINASE ACTIVITY AT TP1/TP2
0 1 2 3 4 50
25
50
75
100
% O
S
Time (years)
95%
68%
At risk:Adequate 398 357 195 60 0 0
Inadequate 25 22 9 1 0 0
No.Patients
No.Events
Obs./Exp.
Adequate 398 13 0·9Inadequate 25 3 3·7
2P = 0·05
Adequate
Inadequate
C
B
EVENT FREE SURVIVAL BY ASPARAGINASE ACTIVITY AT TP1/TP2
0 1 2 3 4 50
25
50
75
100
% E
FS
Time (years)
94%
82%
At risk:
Adequate 398 356 192 60 0 0Inadequate 25 22 8 1 0 0
No.Patients
No.Events
Obs./Exp.
Adequate 398 17 0·9Inadequate 25 3 2·9
2P = 0·09
Adequate
Inadequate
A
RELAPSE FREE SURVIVAL BY ASPARAGINASE ACTIVITY AT TP1/TP2
0 1 2 3 4 50
25
50
75
100
% R
FS
Time (years)
97%
86%
At risk:Adequate398 356 193 60 0 0
Inadequate24 22 8 1 0 0
No.Patients
No.Events
Obs./Exp.
Adequate 398 8 0·8Inadequate 24 2 3·9
2P = 0·09
Adequate
Inadequate
Asparaginase Study: L-Asparaginase activity
109
4.2.2 RESULTS OF PATIENTS RECRUITED IN ALLR3 ARM OF THE
ASPARAGINASE STUDY
4.2.2.1 ALLR3 ASNase activity results: Patients who relapse have already
been treated intensively with ASNase. In the UK, the majority of patients on
the relapsed ALLR3 trial will have received PEG-ASNase. Patients in the
ALLR3 received same dose of PEG-ASNase as patients in the UKALL2003
trial. We hence tested trough ASNase activity levels post PEG-ASNase during
phase 1 and phase 2 of the treatment protocol in 24 patients. In total there
were 35 sampling time points. Twenty of the twenty four patients had
adequate activity at 31/35 of the sampling time points, figure 4.7. Three
patients had inadequate activity during phase 1 and there were no
subsequent samples in these patients. The fourth patient had adequate
activity in phase 1 but inadequate activity in phase 2.
Figure 4.7: ASNase activity in ALLR3. Box and whisker plot showing trough
ASNase activity levels in 24 patients at 35 time sampling time points in phase
1 and phase 2 of the treatment protocol. Asterisk represents an extreme
outlier.
Aspara
gin
ase a
ctivity U
/L
0
1200
1000
800
600
400
200
Patients in the ALLR3 trial, n=35
sampling time points in 24 patients
Asparaginase Study: L-Asparaginase activity
110
4.2.2.2 Outcome analysis of patients in ALLR3 who did not get PEG-
ASNase: We reported a correlation between higher MRD and inadequate
ASNase activity in patients in regimen A. Preliminary survival data for all
patients also suggests a correlation between inadequate ASNase actively and
overall survival (OS). As there were only 4 patients that had inadequate
ASNase activity, to assess the impact of PEG-ASNase at relapse, we
compared the overall and progression free survival (PFS) of all patients who
did not received PEG-ASNase either because of prior hypersensitivity to
PEG-ASNase or the development of ASNase induced pancreatitis (n=16) with
that observed in the matched controls who did receive PEG-ASNase (n= 214),
figure 4.8. The OS and the (PFS) were not different in the two groups.
Figure 4.8: Contribution of PEG-ASNase at relapse. The PFS and OS of
1.0
0.8
0.6
0.2
0.4
0
0 12 24 36 48 60 72 84 96
Pro
po
rtio
n s
urv
ivin
g
Months from registration
Log-rank p = 0.377
46.4 (35.0 – 57.1)
28.6 (18.1 – 40.0)
Progression-free survival: PEG-aspraginase vs. none
All patients on ALL R3
Log-rank p = 0.365
1.0
0.8
0.6
0.2
0.4
0
0 12 24 36 48 60 72 84 96
Pro
po
rtio
n s
urv
ivin
g
Months from registration
53.5 (41.8 – 63.9)
38.1 (27.1 – 49.1)
Overall survival: PEG-asparaginase vs. none
All patients on ALL R3
Asparaginase Study: L-Asparaginase activity
111
patients in the ALLR3 trial who did and did not receive PEG-ASNase
Conclusion 7- The number of patients analysed for ASNase activity in ALLR3
are small but early data indicates that inadequate ASNase activity does not
occur more frequently in patients at relapse. Outcome of patients who did not
received PEG-ASNase at relapse is not statistically inferior to those who did
receive the drug. The dosing strategy in the ALLR3 achieved adequate
ASNase activity in >90% of patients.
Asparaginase Study: L-Asparaginase activity
112
4.3 DISCUSSION:
Age influenced NCI high risk patients have a poorer outcome despite
intensification of treatment. This is related to increase incidence in toxicity and
adverse cytogenetics, lack of compliance to treatment and alterations in the
pharmacokinetics of steroids (2, 121-125). In addition to these possibilities,
our findings show increased incidence of ASNase inactivation in this group.
Routine testing in this group may help identify those who have inadequate
levels. This may be important as the time to event analysis shows a trend to
inferior event free survival and relapse free survival, p=0.09, figure 4.6 and an
inferior overall survival in patients who show sub-therapeutic response to
induction PEG-ASNase, p=0.09. There are limitations in the analysis done
thus far that are important to highlight. The correlation between high MRD and
inadequate induction ASNase activity in patients receiving a 3 drug containing
induction regimen as well as the time to event analysis have been done by
univariate analysis. It remains to be seen if the conclusions derived thus far
hold true on multivariate analysis. The data on time to event analysis will
additionally need to mature over time before it can be safely concluded that
response to ASNase during induction is crucial in determining the outcome of
children with ALL
In this study, PEG-ASNase made the greatest impact in patients belonging to
the NCI standard risk group with good risk cytogenetics. Though this is a
heterogeneous group (126), current prognostic classification used in clinical
practice is unable to further separate the ones in this group who will relapse
from the ones who don’t. Consequently all patients receive a 3 drug induction.
Steroid clearance is possibly enhanced in those who have an inadequate
response to ASNase (72). Thus there is suboptimal exposure to two of the
three drugs used in induction, a possible explanation for the significantly
higher MRD levels. This relationship is now being explored in the current
ALL2011 trial. Monitoring response to PEG-ASNase in this group has the
possibility of improving upon the resolution of the current prognostic
Asparaginase Study: L-Asparaginase activity
113
classification especially in the ones where MRD is indeterminate. These
patients could potentially benefit from post induction intensification of therapy.
The impact of PEG-ASNase in patients who receive a 4 drug induction is less
clear. This might be because of the additional anthracycline used as part of
the 4 drug induction regimen that salvages them from inadequate response to
PEG-ASNase. Alternatively, patients in this group are more likely to be
resistant to one or more drugs used in induction. Patients in this group are
more likely to have high MRD compared to those receiving 3 drug induction
despite treatment intensification.
Patients in Regimen C, not only have a lower chance of recovering PEG-
ASNase activity in the post induction phase if they have inadequate PEG-
ASNase during the induction phase; but they also have higher incidence of
inactivation to PEG-ASNase during post-induction. Patients in Regimen C are
also far more likely to develop clinical hypersensitivity to PEG-ASNase in the
post induction phase (chapter 5). Thus patients in Regimen C with inadequate
activity during induction could be at a double disadvantage of having sub
therapeutic drug levels and increased incidence of drug toxicity. Currently all
patients in regimen C receive post induction intensification with PEG-ASNase.
PEG-ASNase during post induction phase in this group possibly only exposes
them to more sensitising episodes in the context of inadequate levels of the
drug. Thus the importance of intensification of PEG-ASNase in high risk
patients is called into question and needs further evaluation.
Extending the argument further is the effect of ASNase in patients who
relapsed and who were treated in the ALLR3 protocol. Though the majority of
relapsed patients (>80%) showed adequate activity, matched pair analysis of
those who received ASNase and those who did not showed no difference in
either progression free survival or overall survival. Though the number of
patients in this analysis are small it may well be that the lymphoblasts at
relapse are intrinsically resistant to a number of drugs including PEG-ASNase
and so response to PEG-ASNase may not be the only factor that would
Asparaginase Study: L-Asparaginase activity
114
determine outcome. Reassuringly the dosing strategy of PEG-ASNase in
ALLR3 achieved adequate ASNase activity levels in vast majority of the
patients and these patients were not more likely to inactivate ASNase
compared to patients in the UKALL2003 trial.
Asparaginase Study: Determinants of ASNase activity
115
Chapter 5 Asparaginase Study: Determinants of ASNase activity
______________________________________________________________
5.1 BACKGROUND
As mentioned in the previous chapter, the results of the Asparaginase Study
are split into two chapters for the purpose of easy of reading. This chapter
describes:
1) The correlation of expression of AEP and CTSB with
a) ASNase activity
b) The development of immune response to PEG-ASNase.
c) Baseline biological characteristics and
d) Outcome variables
2) The impact of anti-L-Asparaginase antibody on the ASNase activity.
3) The incidence of toxicity such as hypersensitivity and thrombosis after
PEG-ASNase
The first and third sets of results are from patients recruited from the
UKALL2003 arm of the Asparaginase Study, whilst for the second set of
results are from patients recruited from both the UKALL2003 and the ALLR3
arms of the study.
5.2 RESULTS
5.2.1 Role of cysteine proteases as predictive biomarkers to ASNase
therapy:
A total of 144 and 208 patients were tested for the expression of AEP and
CTSB respectively. AEP and CTSB were measured by ELISA in leukaemic
blasts at diagnosis and were correlated with baseline characteristics such as
Asparaginase Study: Determinants of ASNase activity
116
gender, NCI risk, immunophenotype and cytogenetics; response to PEG-
ASNase therapy such as ASNase activity, development of anti-L-
Asparaginase antibodies, occurrence of clinical hypersensitivity; and with
outcome variables such as early response and the residual disease at a
molecular level in Figures 5.1 to 5.4. These figures show expression of
proteases by ELISA in the form of box and whisker plots. To analyse the
expression proteases across each variable on the X axis, patients are split
into two or more groups depending on the nature of the variable. In order to
compare the means in each group the p values were generated by either
Mann-Whitney test (where there are two groups) or Kruskal Wallis Test
(where there are more than two groups). Circles represent outliers and
asterisks represent extreme outliers. The patients were classified into
cytogenetic sub groups based on recently published paper (42).
Asparaginase Study: Determinants of ASNase activity
117
Figure 5.1: Correlation of expression of AEP by ELISA in leukaemic blasts at
diagnosis with baseline characteristics.
M:
n=
97
NC
I ri
sk
P=
.45
9
Pre
B &
T c
yto
ge
ne
tics
P=
.76
0
Pre
B c
yto
ge
ne
tics
P=
<0
.01
F: n=
47
Ge
nde
r
P=
.15
0
Std
: n=
69
Hig
h: n=
75
Poor:
n=
69
Non-p
oor:
n=
75
Int: n
=46
Poor:
n=
10
ngof AEP/μg of cell lysateprotein
126 0
Good: n=
54
Imm
un
op
he
no
typ
e
P=
.79
7
Pre
B: n=
121
T: n=
23
Asparaginase Study: Determinants of ASNase activity
118
Figure 5.2: Correlation of expression of AEP by ELISA in leukaemic blasts at
diagnosis with ASNase levels, antibody, hypersensitivity and therapeutic
outcome
Asp
ara
gin
ase
activity
p=
.73
9E
arl
y r
esp
on
se
p=
.64
9
Rapid
: n=
125
Adq: n=
118
In-a
dq: n=
26
Slo
w:
n=
19
An
ti-A
spa
ragin
ase
an
tibo
die
s p
=.7
10
Pre
: n=
11
Abs:
n=
59
Hyp
ers
en
sitiv
ity
p=
.67
1
Pre
: n=
08
Abs:
n=
136
MR
D p
=.6
05
Low
: n=
37
Int: n
=48
Hig
h: n=
59
ngof AEP/μg of cell lysateprotein
126 0
Asparaginase Study: Determinants of ASNase activity
119
Figure 5.3: Correlation of expression of CTSB by ELISA in leukaemic blasts at
diagnosis with baseline characteristics
ngof CTSB/ 100μg of cell lysateprotein12 8 6 4 2 0
Ge
nde
r
P=
.18
3
M:
n=
132
F: n=
76
NC
I ri
sk
P=
.68
7
Std
: n=
106
Hig
h: n=
102
Pre
B: n=
176
T: n=
32
Imm
un
op
he
no
typ
e
P=
.61
9
Poor:
n=
14
Non-p
oor:
n=
194
Pre
B &
T c
yto
ge
ne
tics
P=
.34
6P
re B
cyto
ge
ne
tics
P=
.71
4
Poor:
n=
12
Int: n
=69
Good: n=
79
Asparaginase Study: Determinants of ASNase activity
120
Figure 5.4: Correlation of expression of CTSB by ELISA in leukaemic blasts at
diagnosis with ASNase levels, antibody, hypersensitivity and therapeutic
outcome
ngof CTSB/ 100μg of cell lysateprotein
Asp
ara
gin
ase
activity
P=
.29
6
Adq: n=
176
In-a
dq: n=
32
Pre
: n=
13
Abs:
n=
74
An
ti-A
spa
ragin
ase
an
tibo
die
s p
=.7
80
Pre
: n=
09
Abs:
n=
199
Hyp
ers
en
sitiv
ity
p=
.87
8
Ea
rly r
esp
on
se
p=
.24
2
Rapid
: n=
184
Slo
w:
n=
124
MR
D p
=.3
50
Low
: n=
47
Int: n
=80
Hig
h: n=
81
8 6 4 2 012
Asparaginase Study: Determinants of ASNase activity
121
Neither the expression of AEP, nor CTSB by ELISA show any correlation with
any of baseline characteristics, response to PEG-ASNase or the outcome
variables.
Conclusion 8 - Cellular expression of AEP or CTSB in leukaemic blasts was
not significantly influenced by any of the know risk factors in childhood ALL,,
ASNase activity levels, development of immune reactions against PEG-
ASNase or other outcome variables such as early response to therapy and
the level of MRD.
5.2.2 Impact of Anti L-Asparaginase Antibodies: Antibodies were
measured against both PEG-ASNase and the native E.Coli ASNase by
indirect ELISA by Medac GmbH, Wedel, Germany. Its impact was studied in
patients recruited from UKALL2003 as well as from ALLR3
5.2.2.1 Anti-Asparaginase antibodies: Patients enrolled in the
UKALL2003 trial (n=129)
5.2.2.1.1 The correlation between serial ASNase activity and antibodies
to L-Asparaginase: This is shown in the table below.
Table 5.1 Correlation between ASNase activity and anti L-Asparaginase
antibodies
Groups Serial ASNase activity Anti-L-Asparaginase antibodies
Induction Post induction n (%) Number tested Number positive
I Adequate Adequate 204 (79) 75 1
II Adequate Inadequate 11 (04) 11 5
III Inadequate Adequate 29 (11) 29 3
IV Inadequate Inadequate 14 (05) 14 7
In the first group of patients, 197/204 had no clinical hypersensitivity. Seven of
the 204 patients had adequate activity despite having reported clinical
hypersensitivity. These will be discussed later. Of the 197 patients; 101/197,
49/197 and 41/197 were in regimen A, B and C respectively. From this group
Asparaginase Study: Determinants of ASNase activity
122
75/197 patients across regimen A, B and C (A=29, B=26 and C=20) were
tested for anti- PEG-ASNase and anti-ASNase antibodies at 92 time points
during induction and post induction. 5/75 patients were tested during both
induction and post induction phases, 7/75 were tested only during induction
phase whilst the majority (63/75) were tested during the post induction phase.
Only one sample in this group of patients tested positive for anti PEG-ASNase
which was transiently detected only during the DI I1 phase and not during the
subsequent Capizzi 2 phase. This suggests that the assay for measuring
antibodies against ASNase is specific.
In the second group of 11 patients who had adequate activity during induction
but inadequate activity during post induction phase, 5 patients developed
antibodies- 4 against both PEG-ASNase and ASNase and 1 against PEG-
ASNase. Four out of these five patients were tested for presence of
antibodies in the diagnostic sample and were all found to be negative.
In the third group of patients who had inadequate activity during induction but
adequate activity during post induction phase (n=29), only 3 patients tested
positive for antibodies during induction. In each the antibody was only against
PEG-ASNase and was not detected subsequently in the post induction phase.
In the last group of patients who never achieved adequate activity (n=14), 7
were positive for anti-L-Asparaginase antibodies. Six of these patients had
both anti- PEG-ASNase and anti-ASNase, whilst one had anti ASNase. None
of these patients tested positive for either of the antibodies in the sample at
diagnosis indicating that the antibodies were acquired during the course of
treatment. In each of these 7 patients samples at multiple subsequent time
points were positive for antibodies indicating that the antibodies were
persistently positive in this group.
In 26 patients at 40 time points, there was simultaneous data for anti-
Asparaginase antibodies and ASNase activity. At 33/40 time points, the
development of anti-ASNase antibodies was associated with inadequate
Asparaginase Study: Determinants of ASNase activity
123
ASNase activity from the sample at the corresponding time points. Four
patients constituted the 7/40 time points where there was a discrepancy
between ASNase activity and anti-ASNase antibodies. All these patients had
clinical hypersensitivity and it was not clear if they were on Erwinase at the
point when sample was sent for ASNase activity. The indoxine assay gives
readout if patients were on Erwinase as it detects ASNase activity. This is
also likely to account for the adequate activity noted in 7 patients who had
clinical hypersensitivity. In this group of patients 6 were tested for antibodies.
Three patients had both anti-PEG-ASNase and anti ASNase whilst 3 had no
detectable antibodies against either PEG-ASNase or ASNase.
Correlation between clinical hypersensitivity and serial ASNase activity was
available for two patients. Both these patients has adequate activity at the first
measured time point followed by a drop in activity level that preceded the
development of hypersensitivity. Both patients recovered ASNase activity
levels on Erwinase, figure 5.5
Figure 5.5 Correlation between hypersensitivity and ASNase activity. Serial
ASNase activity results in two patients who developed clinical hypersensitivity
to PEG-ASNase at time points indicated by arrow. In both these patients,
there was a drop in ASNase activity that preceded the development of
0
100
200
300
400
500
600
700
800
900
1000
Pla
sm
a L
-Aspa
ragin
ase
activity le
ve
ls (
U/L
)
= PEG ASNase
= Erwinase
Induction Post Induction
= Clinical Hypersensitivity
Asparaginase Study: Determinants of ASNase activity
124
hypersensitivity and subsequent replacement of PEG-ASNase by Erwinase
resulted in the recovery of plasma ASNase activity.
5.2.2.1.2 Correlation between anti L-Asparaginase antibodies and
clinical hypersensitivity There were 16 patients who developed clinical
hypersensitivity to PEG-ASNase as shown in the next section. 14/16 patients
were tested for the presence of antibodies against ASNase at one or more
time points. 10/14 patients tested were positive for both anti- PEG-ASNase
and anti ASNase antibodies. In 9/10 patients we looked for the presence of
antibodies in the sample obtained at diagnosis and all of them were negative
Conclusion 9 – Whilst neutralising antibodies against ASNase explains
inadequate ASNase activity during post induction phase in half of the patients,
mechanism of late inactivation in the other half and that of early inactivation of
the drug remains unknown.
5.2.2.2 Anti-Asparaginase antibodies: Patients enrolled in the ALLR3
study (n=16)
Sixteen of the 24 patients were tested for anti-Asparaginase antibodies at one
or more time points. This includes two of the four patients with inadequate
activity. None of the samples tested were positive for anti-Asparaginase
antibodies.
Conclusion 10 - Whilst the numbers are small, there does not appear to be
an increased incidence of anti-Asparaginase antibodies at relapse.
5.2.3 Toxicity after PEG-ASNase: Toxicity to PEG-ASNase was reported in
31 of the 427 patients (7.3%). Of these, 16 (3.7%) developed clinical
hypersensitivity, 10 (3.1%) thrombosis and 5 (1.1%) pancreatitis. Table 5.1
shows the incidence of toxicity to PEG-ASNase across regimen A, B and C.
Patients in regimen C had significantly higher incidence of reported toxicity to
PEG-ASNase (22/122) compared to those in A and B (10/305), p=<0.001.
Hypersensitivity almost exclusively occurred in patients on regimen C. Data
Asparaginase Study: Determinants of ASNase activity
125
on the onset of hypersensitivity is available in 7 patients and the median onset
was 15 weeks into therapy.
Table 5.2 Incidence of toxicity to PEG-ASNase
The overall incidence of hypersensitivity, thrombosis and pancreatitis in this
study are lower to those that reported by other groups (76, 127-129), Table5.3.
Table 4.8 Reported side effects to PEG-ASNase
Hypersensitivity
Thrombosis
Pancreatitis
Regimen A Regimen B Regimen C
1 - 16
4 3 3
2 - 3
Side effect
Table 4.8 Reported side effects to PEG-ASNase
Hypersensitivity
Thrombosis
Pancreatitis
Regimen A Regimen B Regimen C
1 - 16
4 3 3
2 - 3
Side effect
Asparaginase Study: Determinants of ASNase activity
126
Table 5.3 Reported Adverse reactions to ASNase
Patient number
Patient Group Advers even/incidence Type of Asparaginase Comment Reference
ALL-BFM 76 new diagnosis Hypersensitivity (24%) Native E.coli ASNase (128)
Silent antibodies (0%)
Dana-Farber 386 new diagnosis allergic reactions=15% Native E.coli ASNase Older patients higher incidence of thrombosis and pancreatitis
(32)
pancreatitis 7% vs (Randomisation) PEG-ASNase: lower incidence of allergic reactions. Reactions more likely to be mild
thrombosis=4.5% PEG-ASNase
Dana-Farber 463 new diagnosis Allergic reactions=11% Pancreatitis=11% Thrombosis=9%
PEG-ASNase (iv vs im) Doses 2500 U/m
2
(129)
St. Jude 410 new diagnosis Clinical allergy (41%) Silent antibodies (36.9%)
Native E.coli ASNase (130)
St Jude 35 new diagnosis Clinical allergy (62.8%) Native E.coli ASNase (131)
CCG 1001 new diagnosis high risk patients
Silent antibodies (61%) Native E.coli ASNase Vs PEG-ASNase in a
Silent antibodies lead to inadequate ASNase levels in 94% of patients
(79)
randomised setting during post induction
Asparaginase Study: Determinants of ASNase activity
127
CCG 118 new diagnosis L-Asparaginase antibodies: depended on
PEG-ASNase Vs
high titre antibodies: (76)
the product used (Randomisation) PEG-ASNase=2%
Native E.coli ASNase Native E.coli ASNase=26%
Thrombosis= 5.17%
NOPHO 42 new diagnosis silent antibodies: (132)
3/27: Erwiniase Erwiniase
1/15: Native E.coli ASNase
Native E.coli ASNase
NOPHO 39 Relapse silent antibodies: 8% Erwiniase (133)
Meta analysis 1752 new diagnosis Thrombosis: 5.2% 17 prespective trials (134)
Current study 427 New diagnosis Hypersensitivity 3.9% Thrombosis 2.3% Pancreatitis 1.6%
Asparaginase Study: Determinants of ASNase activity
128
Conclusion 11 - The incidence of toxicities to PEG-ASNase in the study was
lower compared to those reported by other international groups. Almost all
hypersensitivity reactions occurred in patients on regimen C
5.3 DISCUSSION
The dose and schedule of PEG-ASNase achieved adequate activity in
majority of patients that were enrolled from both the UKALL2003 and the
ALLR3 arms of the Asparaginase study.
For patients enrolled from the UKALL2003 study, toxicity to PEG-ASNase was
predominantly observed in regimen C, p=<0.001. Overall, the incidence of
toxicity to PEG-ASNase was lower than that reported by other international
groups. This could be due to a the use of PEG-ASNase instead of native
E.coli ASNase and the use of PEG-ASNase at a lower dose of 1000 U/m2 in
comparison to the other groups such as the BFM, CCG and the Dana-Farber
who gave PEG-ASNase at 2500 U/m2.
In those patients who had inadequate ASNase activity, serial activity
measurements revealed 3 major patterns of drug inactivation. These
consisted of early (during induction phase), late (during post induction phase)
and persistent (both early and late) inactivation. Whilst the mechanism for late
inactivation seems to be related to development of antibodies to ASNase in
approximately half of the patients and will be discussed first, the mechanism
of late inactivation in the other half and that of early inactivation remains
unknown.
The incidence of silent neutralising antibodies causing late inactivation in this
study was 4.7%. Both anti-ASNase and anti PEG-ASNase antibodies had the
potential to inactivate PEG-ASNase. These antibodies were not detected in
the diagnostic samples indicating that these were acquired during the course
of therapy following exposure to PEG-ASNase. As they could explain only 50%
of patients who had late inactivation, one possibility is that the assay is not
Asparaginase Study: Determinants of ASNase activity
129
sufficiently sensitive to detect antibodies in all patients. The other possibility is
that there are additional, yet unidentified, mechanisms that account for late
inactivation. Further studies that correlate serial ASNase activity with anti-L-
Asparaginase antibodies are needed to assess the exact incidence of silent
neutralising antibodies and their contribution to the late inactivation of PEG-
ASNase and to study whether this has an impact in the overall outcome. The
latter objective will need long term follow that measures overall survival as
unlike in the early phase of treatment there are no surrogate markers to
assess the impact of inadequate therapy. Such studies may also pick up a
pattern where the drop in ASNase levels precedes clinical hypersensitivity in
which case an earlier intervention could potentially prevent toxicity.
A higher incidence of relapse has been reported in those with anti-ASNase
antibodies (130). In our study there was no increase in the incidence of anti-
Asparaginase antibodies at relapse. This could again be due to the small
cohort of patients in our study or the use of PEG-ASNase in front line UK
protocols after 2003 which could have changed the incidence of anti-
Asparaginase antibodies or due to the difference in the sensitivity of the assay
that measured anti-Asparaginase antibodies.
It is likely that early inactivation is due to yet unexplained biological factors
such as AEP and CTSB that are either secreted or expressed by the
leukaemic cell. Additionally we expected AEP, a protease previously
described to be over expressed in high risk ALL, to play a role in the
development of immune response to PEG-ASNase. The expression of AEP
and CTSB in leukaemic cells by ELISA showed nearly a six (AEP) to twelve
(CTSB) fold variation between individual patients. However, when their
expression was assessed in individual patient subgroups based on the known
risk factors in ALL, their expression in most of the patient subgroups was
statistically uniform. On univariate analysis, the only factor that influenced the
expression of either of these proteases was the presence of poor risk
cytogenetics in patients with pBALL. Patients in this sub-group had lower
expression of AEP compared to those in the good and intermediate sub-
Asparaginase Study: Determinants of ASNase activity
130
groups. There are two points to note. Firstly, the definition of poor risk
cytogenetics referred in this thesis (42) is different to the one use in the
previously published analysis of AEP expression in clinical samples(101).
Secondly, the numbers of patients are small and the AEP expression was
measured at the level of protein.
Though AEP and CTSB have been identified to degrade ASNase in-vitro, they
did not emerge as predictive biomarkers for either the response to PEG-
ASNase or to the development of immune response to PEG-ASNase or to
poor response to induction therapy. This was not surprising given that the
level of inactivation of ASNase was low whilst most of the patient’s leukaemic
cells were found to express AEP and CTSB. One explanation for this could be
choice of the ELISA assay used to study the expression of both AEP and
CTSB. While ELISA was the most suitable technique to study the expression
of proteases in leukaemic blasts, as shown in chapter 3, it measured total
protein and not specifically the active form of the protein. Both AEP and CTSB
are lysosomal cysteine proteases that undergo sequential N and C terminal
cleavage before they become active. The processing of these proteases is
dependent on acidic pH which is likely to influence their in-vivo activity and
possibly limit their action locally to the microenvironment around the acidic
bone marrow mesenchymal stem cell niches. Secondly, we know little about
the half life and stability of AEP and CTSB and the effect of pre analytical
variables such as conditions during sample transport. Thirdly, expression of
AEP and CTSB was measured in the leukaemic cells. If the drug mediates its
effect by extracellular depletion of asparagine and if AEP and or CTSB
expressed by the leukaemic cells degrade ASNase in-vivo, it begs a question
on where the interface between the drug and the proteases is. It is not clear if
measuring cellular expression of AEP and CTSB would be reflective of the in-
vivo capacity of tumour to inactivate the drug that is in the extracellular
compartment.
.
Asparaginase Study: Salient points
131
Salient points of chapters 4 and 5 :
Conclusion 1 - From a trial perspective 85-97% of patients have adequate
trough activity at any time point.
Conclusion 2 - Increasing the dose to 2,500 – 3,500 is unlikely to improve the
percentage of patients who will have adequate levels and is likely to lead to
increased toxicity.
Conclusion 3 - Patients transferred to regimen C at the end of induction have
higher incidence of inactivation of ASNase.
Conclusion 4- Older patients had increased incidence of inadequate ASNase
activity during induction.
Conclusion 5 - ASNase activity results at TP2 correlate with MRD at the end
of induction in patients with precursor B ALL who belong to NCI standard risk
and have good risk cytogenetics.
Conclusion 6 - Early data seems to indicate the response to PEG-ASNase
during induction is influencing the outcome in children with ALL
Conclusion 7- The number of patients analysed for ASNase activity in ALLR3
are small but early data indicates that inadequate ASNase activity does not
occur more frequently in patients at relapse. Outcome of patients who did not
received PEG-ASNase at relapse is not statistically inferior to those who did
receive the drug. The dosing strategy in the ALLR3 achieved adequate
ASNase activity in >90% of patients.
Conclusion 8 - Cellular expression of AEP or CTSB in leukaemic blasts was
not significantly influenced by any of the know risk factors in childhood ALL,
ASNase activity levels, development of immune reactions against PEG-
Asparaginase Study: Salient points
132
ASNase or other outcome variables such as early response to therapy and
the level of MRD.
Conclusion 9 - Whilst neutralising antibodies against ASNase explains
inadequate ASNase activity during post induction phase in half of the patients,
mechanism of late inactivation in the other half and that of early inactivation of
the drug remains unknown.
Conclusion 10- Whilst the numbers are small, there does not appear to be an
increased incidence of anti-Asparaginase antibodies at relapse.
Conclusion 11 - The incidence of toxicities to PEG-ASNase in the study was
lower compared to those reported by other international groups. Almost all
hypersensitivity reactions occurred in patients on regime
Chemoprotection by BMSC derived exosomes
133
Chapter 6 Role of bone marrow stromal exosomes in conferring chemo-
protection to leukaemic cells
6.1 BACKGROUND:
This chapter presents work I did under the supervision of Dr JiZhong Liu in
the laboratory. His work is presented to provide a background for my role in
his project.
6.1.1 Drug resistance in ALL is multi-factorial
Though ASNase inactivity does not seem to influence MRD levels in Regimen
B, nevertheless patients in this group are more likely to have higher MRD than
patients in regimen A (unpublished data from UKALL2003, personal
communication, Rachael Wade). Whilst the interaction between
ASNase/steroids contributes to one part of the spectrum of drug resistance,
there are clearly other factors. Though poor response to treatment has been
intensively investigated (table 6.1) and a number of risk factors for poor
response identified (41, 42, 62, 135, 136), there is yet no unifying mechanism
that explains resistance to combination chemotherapy (61, 62). This may be
related to the fact that many factors contribute to drug resistance, including
the use of multiple drugs and the biological heterogeneity of the disease
Focussing on ASNase, at least two alternative mechanisms of drug resistance
have been proposed. In the first mechanism leukaemic cells show intrinsic
resistance to ASNase mediated by the up-regulation of Asparagine
Synthetase (ASNS). However, the expression of ASNS correlates with in-vitro
sensitivity of ASNase in some but not all genetic subtypes of ALL (93, 94).
Moreover the expression of ASNS in patients failed to correlate with the in-
vivo response to ASNase monotherapy (95). Another proposed mechanism is
an extrinsic mechanism wherein the leukaemic cells when exposed to
ASNase were protected from its cytotoxicity by mesenchymal cells that up-
Chemoprotection by BMSC derived exosomes
134
regulate Asparagine Synthetase (ASNS) resulting in secretion of asparagine
(99). This may result in adequate asparagine levels in the cellular niches
despite successful systemic asparagine depletion following adequate
response to ASNase. Given the lack of intrinsic genetic abnormalities that
can explain therapeutic failure the latter is an area for investigation.
In ALL, significant proportions of late relapses are inherently chemo sensitive
and are cured without the need for a bone marrow transplant (11). This
suggests that they survive due to reasons that are beyond intrinsic drug
resistance. This suggest that extrinsic mechanisms such as microenvironment
meditated chemoprotection may play a significant role in therapeutic failure
(137-139).The microenvironment has been shown to mediate drug resistance
by both contact (140) and non contact mediated mechanisms (99, 141). We
in the group are investigating the role of microenvironment in conferring broad
spectrum chemoprotection.
Chemoprotection by BMSC derived exosomes
135
Table 6.1 Pharmacological heterogeneity in childhood ALL
Drug Clinical group/material investigated for differential response Findings/Explanation for differential response Reference
Steroids a) early precursor B ALL: CR vs no CR ~ no CR-high levels of glucocorticoid receptors (142)
b) cell lines: resistant vs sensitive ~ resistant- higher expression of MCL1 (143)
c) cell lines: resistant vs sensitive ~ resistant- preservation of mitochondrial respiratory function
(144)
d) precursor B ALL patients: resistant vs sensitive by MTT assay ~ resistant- 33 genes differentially expressed (62)
Vincristine a) SR ALL patients: incidence of relapse ~ relapse-higher plasma clearance (145)
b) precursor B ALL patients: resistant vs sensitive by MTT assay ~ resistant- 40 genes differentially expressed (62)
Thiopurines a) ALL patients: Correlation of RBC TPMT with 6 TGN levels and incidence of relapse
~ Children with higher RBC TPMT activity had lower 6TGN levels and a higher incidence of relapse
(146)
b) ALL patients treated in ALL-BFM-2000: MRD SR vs HR ~ TPMT genotype had a substantial impact on the MRD
(147)
c) ALL patients treated in Total Therapy protocol XIIIB: high vs low TGN levels after MP alone
~ higher levels of TGN associated with up-regulation of genes encoding MP metabolic enzymes and transporters (SLC29A1)
(148)
high vs low TGN levels after MP+TGN involved in protein and ATP biosynthesis
d) TEL-AML1 fusion ALL vs other subtypes including T-ALL: ~ TEL-AML patients had (149)
de novo purine synthesis (DNPS) lower DNPS
genes involved in purine metabolism lower expression of 16 genes
Anthracyclines a) precursor B ALL patients: resistant vs sensitive by MTT assay ~ resistant- 20 genes differentially expressed (62)
(Daunorubicin)
Methotrexate a) ALL patients:MTX levels, RFC1 (SLC19A1) polymorphisms & event free survival
~ G80A polymorphism was associated with higher plasma MTX levels and inferior event free survival
(150)
b) Hyperdiploid ALL patients vs T lineage ALL ~ Higher accumulation of MTX-PG in hyperdiploid ALL due to higher expression SLC19A1 as a result of extra copies of Ch 21
(151)
Chemoprotection by BMSC derived exosomes
136
c) Hyperdiploid ALL patients vs other subtypes of pre B and T ALL
~ higher accumulation of MTX-PG in hyperdiploid ALL associated with higher expression of SLC19A1
(152)
~ lower accumulation of MTX-PG in E2A-PBX associated with lower expression of SLC19A1
~lower expression of MTX-PG in TEL-AML1 associated with higher expression of ABCG2
~lower expression of MTX-PG in T ALL associated with higher expression of FPGS
d) ALL patients ~additional Ch 8 associated with higher GGH activity and lower MTX-PG
(153)
e) ALL patients ~genetic variant with triple repeat in promotor region of TYMS is associated with higher TYMS expression
(154)
and an inferior EFS
L-Asparaginase a) precursor B ALL patients: resistant vs sensitive by MTT assay ~ resistant- 35 genes differentially expressed (62)
b) cell lines: resistant vs sensitive ~higher expression of ASNS is associated with resistance
(92)
c) Only in TEL-AML1 negative patients ~higher expression of ASNS is associated with resistance
(94)
Vincristine & a) ALL patients: resistant vs sensitive by LC50 values 139 genes differentially expressed (155)
L-Asparaginase
Induction poly ALL patients treated in ALL-BFM-2000: 54 genes associated with cell cycle progression (156)
chemotherapy MRD Standard vs High Risk & apoptosis differentially expressed
Abbreviations:
Ch Chromosome TGN Thioguanine
CR Complete Remission TPMT Thiopurine methyltransferase
EFS Event free survival TYMS Thymidylate synthetase
FPGS Folylpolyglutamate Synthetase MTX Methotrexate
GGH Gamma glutamyl hydrolase MTX-PG Methotrexate Polyglutamate
MP Mercaptopurine RCF Reduced folate carrier
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
Chemoprotection by BMSC derived exosomes
137
6.2 RESULTS
6.2.1 Bone marrow stromal cell derived conditioned medium (BMSC-CM)
confers chemoprotection to SUPB15 cell line: In order to recapitulate the
in-vivo host tumour interaction in the laboratory, we created an organotypic 3-
D model (157). To develop this model, bone marrow stromal cells (BMSC’s)
were cultured and then alkali stripped to kill the cells but to retain the
cytoskeleton. Next BMSC’s were replated on this 3-dimensional extracellular
matrix (ECM). At about a week, the ECM could be demonstrated to contain
collagen and fibronectin and stromal cells had differentiated into
mesenchymal cells and osteoblasts (Figure 6.1). Growing stromal cells on a
lattice allows a more natural spatial development (158, 159). BMSC grown on
this organotypic 3-D model acquired spindle shape figure 6.1. Cell lines grow
unsupported in this system. Primary cells also survive without the addition of
growth factors for at least 4 weeks.
Figure 6.1: Comparison between 2D model and organotypic 3D culture
systems. Bone marrow stromal cells cultured in a plate (2D model) on the left
and on a cell free extracellular matrix (3D organotypic system) on the right..
Compared to the BMSC grown in the 2D system, the BMSC grown in the 3D
model had a more defined organisation and the cells in 3D system were more
spindle in shape. Both images were obtained on 20x inverted microscope.
Medium obtained from this organotypic 3D model is referred to as bone
marrow stromal cell derived conditioned medium (BMSC-CM). BMSC-CM
Chemoprotection by BMSC derived exosomes
138
protected SUPB15, an inherently sensitive cell line, against number of drugs
used in ALL, figure 6.2. As these drugs have diverse mechanisms of action,
the chemoprotection was broad spectrum and not-selective.
Figure 6.2: BMSC-CM confers non selective chemoprotection to leukaemic
cells. SUPB15 cells were cultured in medium or conditioned medium (CM)
from a human bone marrow stromal cell line HS-5. Following exposure to
drugs between 3-4 days, cell viability was measured by MTT assay. X axis
represent mean values and error bars represent standard error obtained after
3 independent experiments. P values are calculated using Student’s t test; **
represents <0.01, *** represents <0.001. Y axis represents the fold change
viability compared to the control where cells were cultured in normal medium.
6.2.2 Generation of SUPB15MR cells: SUPB15 cells cultured in BMSC-CM
were next exposed to sub-lethal dose of Mitoxantrone. Those that survived
after exposure to Mitoxantrone had a multidrug resistant phenotype and were
called SUPB15MR. These SUPB15MR were subsequently cultrured in normal
medium without further exposure to BMSC-CM and retained the multidrug
resistant phenotype until 50 passages.
0
0.2
0.4
0.6
0.8
1
1.2
cont
rol
Dox
orub
icin
Mito
xant
rone
Idar
ubicin
clof
arab
ine
Aspar
aginas
e
Vincr
istin
e
Dex
amet
haso
ne
ce
ll via
biit
y(%
of
co
ntr
ol) M
TT medium
CM
******
****** ***
*** **
BMSC-CM confers non selective chemoprotection to leukaemic cells
Chemoprotection by BMSC derived exosomes
139
6.2.3 Exosomes in the BMCS-CM contributed to the BMSC-CM mediated
chemoprotection: To further characterise and identify the components in the
BMSC-CM that conferred chemoprotection BMSC-CM was subjected to ultra-
filtration, heating at 95°C for 10 minutes, treatment with proteinase K, or
RNAse. The chemo-protective effect of the BMSC-CM was present within a
trypsin, RNAse and heat resistant, <3kDa fraction (figure 6.3).
Figure 6.3 Protective ability of the <3kd fraction of BMSC. Ability of various
fractions of the BMSC-CM in providing chemoprotection of SUPB15 against
Mitoxantrone was compared against control medium. The Y axis represents
fold change in cell viability of SUPB15 cells exposed to Mitoxantrone for 3
days. Error bars represent standard error. P values were calculated using
Student’s t test. **= p<0.01, ***= p<0.001. Ctrl (Control) represents; </>3kd
represents fractions of the CM; CM-heated is HS5 CM heated at 95°C for 10
minutes; CM-Proteinase K= CM treated with 50μg/ml of Proteinase K at 50°C
for 1 hour and then heated at 95° for 10 minutes to inactivate Proteinase K;
CM RNAse A= CM treated with RNAse A 5 IU/ml for 1 hour at 37°C.
Electron microscopy of the pellet obtained by ultracentrifugation of the <3kDa
fraction of BMSC-CM revealed presence of exosomes (Figure6.4).
0
0.5
1
1.5
2
2.5
3
3.5
4
ctrl CM >3kDa <3kDa CM-heated CM-Proteinase K CM-RNase A
cell
via
bili
ty(r
ela
tive
to c
ontr
ol)
***
**
*** ******
***
Chemoprotection by BMSC derived exosomes
140
Figure 6.4: Transmission electron microscopy of the pellet obtained after
ultracentrifugation of <3kDa fraction of BMSC-CM showing presence of cup
shaped exosomes. (Dr. Jizhong Liu, Dr. Johnson, Dr. Mironov)
6.2.4 HS5 derived exosomes are taken up by SUPB15 and primary ALL
cells In order to determine if exosomes were taken up by leukaemic cells
(figure 6.5), they were fluorescently labelled by PKH67 (green colour) were
incubated with SUPB15 and primary ALL cell lines that were co-stained using
Cell Mask (cytoplasm-red) and Dapi (nucleus-blue).
Figure 6.5 Cellular uptake of BMSC exosomes by SUPB15 (Top panel) and
primary ALL cells (bottom panel) on co-culture. Images were captured on a
low-light system utilising Metamorph software based around a Zeiss Axiover
DAPI MergePKH67Cell Mask
200 nm bar size
30-100nm size membrane limited vesicles (exosomes)
Chemoprotection by BMSC derived exosomes
141
200M microscope with a 300W Xenon light sourse using either a
Photometrics Cascads 512B or Andor iXon DU888+ camera. Original
magnification is x160 provided by x100 oil immersion lens in conjunction with
a 1.6x Optivar magnification enhancer (Dr S Johnson)
6.2.5 BMSC derived exosomes conferred broad spectrum
chemoprotection to SUPB15 cells: In order to determine if the exosomes
conferred chemoprotection, the BMSC-CM was filtered through a 0.2μm
membrane filter and then ultra-centrifuged at 100,000xg for 160 minutes to
pellet the exosomes. The supernatant was named as Exo-depleted CM1.
Exo-depleted CM1 was subjected to further ultracentrifugation step as
described above to give rise to Exo-depleted CM2. The ability of BMSC-CM
to confer chemoprotection progressively decreased as the exosomes were
depleted from the medium, figure 6.6
Figure 6.6 BMSC derived exosomes confer chemoprotection.
Chemoprotection of BMCS CM decreased after depletion of exosomes. The
ability of the BMSC-CM and its two fractions to confer protection to SUPB15
was compared against normal medium (Control). SUPB15 cells were exposed
to 8nM of Mitoxantrone for 3 days and cell viability was measured by MTT
assay. The data on Y axis is presented as fold change compared to the
p=0.0575
p=0.0188
chemoprotection of CM decreased after Exosomes depletion
0
0.5
1
1.5
2
2.5
3
3.5
medium BMSC-CM Exo-depleted CM1 Exo-depleted CM2
ce
ll v
iab
ility
Chemoprotection by BMSC derived exosomes
142
control. Error bars represent standard error from 3 independent experiments.
P values were calculated by using paired t test.
6.2.6 Horizontal transfer of Micro-RNA (miRNA) from BMSC to leukaemic
cell- a mechanism by which BMSC derived exosomes could confer
broad spectrum chemoprotection to SUPB15MR cells: Due to the limited
size of the exosomal sample material, its RNA content was analysed.
Exosome were enriched in small RNA as shown in figure 6.7, (160).
Horizontal transfer of exosomal RNA and miRNA, a subset of small RNA, has
been described to occur between the host and the tumour (161-164). The
transfer of RNA has also been shown to change the biology of the recipient
cell (161, 165-167). To analyse whether there was a transfer of miRNA from
BMSC to the SUPB15 cells, we next examined the miRNA pattern of the
BMSC, SUPB15 and SUPB15MR cells as well as the BMSC derived exosomes.
TaqMan human MircoRNA A+B cards Set v3.0 9Applied Biosystems, part
number 4444913) were used to compare the miRNA expression profile of
SUPB15, SUPB15MR and the BMSC derived exosomes. All miRNAs that had
a cycle threshold (CT) that was within 11 cycles from the endogenous control
genes were included in the analysis.
Chemoprotection by BMSC derived exosomes
143
Figure 6.7 Exosomes contain small RNA. Pictures of RNA electrophoresis on
the left and of electrophoregrams obtained on a bio-analyser from Agilent on
the right. The exosomal RNA is much smaller in size compared to the cellular
RNA (Dr Liu, Dr Johnson).
Table 6.2 shows the list of miRNAs expressed in each of the samples.
Compared to HS5, a BMSC cell line used in the experiment, HS5 exosomes
were enriched in miRNA. Compared to SUPB15, SUPB15MR had 52 unique
miRNA. Of these 6 miRNA were removed as they were either orthologs or
paralogs of a miRNA in the list and the remaining 46 miRNA are coloured in
either plum if they could be traced back to the HS5 exosomes, n= 40 or blue if
Cellular RNA
18S 28S
Pattern of RNA in HS5 cells compared to that in HS5 derived exosomes
BMSC derived Exosomal RNA
RNA ladder sample
Bases
Bases
RNA ladder sample
RNA length
RNA length
Chemoprotection by BMSC derived exosomes
144
not, n=6). Figure 6.8 describes the data by proportion Venn diagrams:
SUPB15 (green circles), SUPB15MR (red circles), HS5 (blue circles with grey
background) and HS5 exosomes (blue circles with interrupted border). The 46
miRNAs uniquely present in SUPB15MR and in HS5 derived exosomes can be
grouped into 35 miRNA families. More than 3/4th (n=40/46; 87%) of the unique
miRNAs found in SUBB15 could be matched to miRNA in the HS5 derived
exosomal (Table 6.2 and figure 6.8).
Table 6.3 summarises key evidence that is currently available that links the
“matched miRNAs” identified in this experiment to cell survival and or
adaptation of cell’s metabolism to stress.
Most of the miRNAs in the SUPB15MR that cannot be matched to the HS5
exosomes are involved in haematopoiesis: miR-150=B and T cell
development (168-172); miR-181= B cell development (173, 174), miR-
223=granulopoiesis (175), haematopoietic cell proliferation(176),
erythropoiesis(177), miR-338= PU.1 dependent haematopoiesis (178).
Chemoprotection by BMSC derived exosomes
145
SUPB15 HS5 HS5 exosomes SUPB15MR
Unique to SUPB15MR
hsa-miR-106a hsa-miR-106a hsa-let-7a hsa-let-7b hsa-let-7b
hsa-miR-126 hsa-miR-138 hsa-let-7b hsa-let-7e hsa-let-7e
hsa-miR-142-3p hsa-miR-146a hsa-let-7e hsa-let-7g hsa-let-7g
hsa-miR-146a hsa-miR-155 hsa-let-7g hsa-miR-106a hsa-miR-106b
hsa-miR-155 hsa-miR-16 hsa-miR-100 hsa-miR-106b hsa-miR-1233
hsa-miR-16 hsa-miR-17 hsa-miR-103 hsa-miR-106b# hsa-miR-125a-5p
hsa-miR-17 hsa-miR-191 hsa-miR-106a hsa-miR-1233 hsa-miR-126#
hsa-miR-186 hsa-miR-19b hsa-miR-106b hsa-miR-125a-5p hsa-miR-1260
hsa-miR-191 hsa-miR-222 hsa-miR-10a hsa-miR-126 hsa-miR-140-5p
hsa-miR-19a hsa-miR-24 hsa-miR-125a-5p hsa-miR-126# hsa-miR-146b-5p
hsa-miR-19b hsa-miR-29a hsa-miR-125b hsa-miR-1260 hsa-miR-149#
hsa-miR-20a hsa-miR-31 hsa-miR-126 hsa-miR-140-5p hsa-miR-150
hsa-miR-218 hsa-miR-484 hsa-miR-126# hsa-miR-142-3p hsa-miR-151-3p
hsa-miR-222 hsa-miR-939 hsa-miR-1260 hsa-miR-146a hsa-miR-151-5P
hsa-miR-24 hsa-miR-1271 hsa-miR-146b-5p hsa-miR-15b
hsa-miR-30c hsa-miR-127-3p hsa-miR-149# hsa-miR-181a
hsa-miR-331-3p hsa-miR-1290 hsa-miR-150 hsa-miR-18b
hsa-miR-454 hsa-miR-130a hsa-miR-151-3p hsa-miR-193b
hsa-miR-484 hsa-miR-130b hsa-miR-151-5P hsa-miR-195
hsa-miR-720 hsa-miR-130b# hsa-miR-155 hsa-miR-196b
hsa-miR-132 hsa-miR-15b hsa-miR-197
hsa-miR-134 hsa-miR-15b# hsa-miR-20b
hsa-miR-138 hsa-miR-16 hsa-miR-223
hsa-miR-140-3p hsa-miR-17 hsa-miR-26a
hsa-miR-140-5p hsa-miR-181a hsa-miR-26b
hsa-miR-145 hsa-miR-186 hsa-miR-29a
hsa-miR-146a hsa-miR-18a hsa-miR-30b
hsa-miR-146b-5p hsa-miR-18b hsa-miR-30d
hsa-miR-149 hsa-miR-191 hsa-miR-320
hsa-miR-149# hsa-miR-193b hsa-miR-338-5P
hsa-miR-151-3p hsa-miR-195 hsa-miR-342-3p
hsa-miR-151-5P hsa-miR-196b hsa-miR-345
hsa-miR-152 hsa-miR-197 hsa-miR-34a#
hsa-miR-155 hsa-miR-19a hsa-miR-374a
hsa-miR-15b hsa-miR-19b hsa-miR-374b
hsa-miR-16 hsa-miR-19b-1# hsa-miR-378
hsa-miR-17 hsa-miR-20a hsa-miR-425
hsa-miR-186 hsa-miR-20b hsa-miR-520D-3P
hsa-miR-18b hsa-miR-218 hsa-miR-590-5p
hsa-miR-191 hsa-miR-222 hsa-miR-625#
hsa-miR-192 hsa-miR-223 hsa-miR-766
hsa-miR-193a-5p hsa-miR-24 hsa-miR-92a
hsa-miR-193b hsa-miR-26a hsa-miR-93
hsa-miR-195 hsa-miR-26b hsa-miR-93#
hsa-miR-196b hsa-miR-29a hsa-miR-939
hsa-miR-197 hsa-miR-30a-5p hsa-miR-99b
hsa-miR-199a-3p hsa-miR-30b
hsa-miR-19a hsa-miR-30c
hsa-miR-19b hsa-miR-30d
hsa-miR-20a hsa-miR-320
hsa-miR-20b hsa-miR-331-3p
hsa-miR-21 hsa-miR-338-5P
hsa-miR-210 hsa-miR-342-3p
Table 6.2 Expression of micro-RNAs
Chemoprotection by BMSC derived exosomes
146
SUPB15 HS5 HS5 exosomes SUPB15MR
Unique to SUPB15MR
Table 6.2 continued
hsa-miR-214 hsa-miR-345
hsa-miR-214# hsa-miR-34a#
hsa-miR-218 hsa-miR-374a
hsa-miR-221 hsa-miR-374b
hsa-miR-222 hsa-miR-378
hsa-miR-222# hsa-miR-425
hsa-miR-23a hsa-miR-454
hsa-miR-24 hsa-miR-484
hsa-miR-25 hsa-miR-520D-3P
hsa-miR-26a hsa-miR-590-5p
hsa-miR-26b hsa-miR-625#
hsa-miR-27a hsa-miR-708
hsa-miR-27a# hsa-miR-720
hsa-miR-28-3p hsa-miR-766
hsa-miR-29a hsa-miR-92a
hsa-miR-29c hsa-miR-93
hsa-miR-301a hsa-miR-93#
hsa-miR-30a-3p hsa-miR-939
hsa-miR-30b hsa-miR-99b
hsa-miR-30c
hsa-miR-30d
hsa-miR-30e-3p
hsa-miR-31
hsa-miR-31#
hsa-miR-320
hsa-miR-320B
hsa-miR-323-3p
hsa-miR-331-3p
hsa-miR-339-3p
hsa-miR-339-5p
hsa-miR-342-3p
hsa-miR-345
hsa-miR-34a
hsa-miR-34a#
hsa-miR-34b
hsa-miR-365
hsa-miR-370
hsa-miR-374a
hsa-miR-374b
hsa-miR-376a
hsa-miR-376c
hsa-miR-378
hsa-miR-409-3p
hsa-miR-411
hsa-miR-425
hsa-miR-431
hsa-miR-432
hsa-miR-454
hsa-miR-483-5p
hsa-miR-484
hsa-miR-494
hsa-miR-539
hsa-miR-574-3p
hsa-miR-590-3P
Chemoprotection by BMSC derived exosomes
147
SUPB15 HS5 HS5 exosomes SUPB15MR
Unique to SUPB15MR
Table 6.2 continued
hsa-miR-590-5p
hsa-miR-625#
hsa-miR-629
hsa-miR-650
hsa-miR-664
hsa-miR-720
hsa-miR-744
hsa-miR-766
hsa-miR-886-3p
hsa-miR-886-5p
hsa-miR-9
hsa-miR-92a
hsa-miR-92b#
hsa-miR-93
hsa-miR-93#
hsa-miR-935
hsa-miR-939
hsa-miR-99a
hsa-miR-99b
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Figure 6.8 miRNA expression profiles in host and tumour-drug sensitive &
drug resistant. Proportion Venn diagrams that compares the miRNA
expression profiles of SUPB15 (green circles), SUPB15MR (red circles), BMSC
cell line-HS5 (blue circles with grey background) and HS5 derived exosomes
(blue circles with interrupted border). Top left: HS5 compared to SUPB15. Top
right: HS5 compared to HS5 exosomes. Bottom Left: HS5 exosomes
compared to SUPB15MR. A vast majority of miRNAs present in SUPB15MR,
59/72 (82%), match with the list of miRNAs present in HS5 exosomes. Bottom
right: Of the 52 unique miRNAs in SUPB15MR compared to SUPB15. This
figure trims further to 46 after removing 6 orthologs and paralogs. Again, a
vast majority of unique miRNA in SUPB15MR, 40/46 (87%) matches to the list
of miRNAs present in the HS5 exosomes
10 10 0414112
52 205967 13
SUPB15 HS5
SUPB15MR
SUPB15
HS5 exosomes
HS5
HS5 exosomes
SUPB15MR
miRNA unique to
SUPB15MR n=52
Orthologs/paralogs n=6
n=46
Match to HS5 exosomes
n=40/46 (87%)
Do not match to HS5 exosomes
n=6/46 (13%)
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Table 6.3 Previously described role of miRNA in chemoprotection and or cell survival and or cell cycle and or cell metabolism Unique to SUPB15
MR miRNA family described role Reference
let7 family protects hepatocytes against oxidant injury (179) let7 family histone demethylase KDM2B suppresses let-7b: regulation of ageing, proliferation (180) let7 family essential for panabinostat mediated downregulation of HMGA2 (181) let7 family let-7 family involved in glucose homeostasis and insulin sensitivity (182) let7 family modulates acquired resistance to taxanes (183) hsa-miR-106b miR-17 family functions as oncogene by suppressing p21 and Bim in oesophageal Ca (184) suppresses p21, overrides doxorubicn induced DNA damage checkpoint (185) funtions as oncogene by targeting PTEN (186) targets Smad, activates TGFβ signalling, induces EMT; breast cancer (187) dysfunction of p53 pathway in Hodgkins lymphoma cell lines (188) increases E2F1 expression and is upregulated in hepatocellular Ca (189) modulates E2F in neuronal lineage differentiation (190) targents retinoblastoma in laryngeal carcinoma (191) hsa-miR-125a-5p targets key proteins regulating apoptotis, immunity, inflammation (192) hsa-miR-126# plays a role in angiogenesis (193) hsa-miR-140-5p mediates chemoresistance in osteosacroma and colon cancer cells (194) hsa-miR-150 promotes gastric cancer proliferation by negatively regulating ERG2 (195) hsa-miR-151-3p miR-151 facilitates tumour cell migration & spreading by downregulating RhoGDIA (196) hsa-miR-195 implicated in acquired teomzolomide resistance in glioblastoma multiforme cells (197) hsa-miR-196b targets both HOXA9/MEIS1 and FAS in MLL rearranged leukemia (198) hsa-miR-20b mirR-17 family modulates VEGF expression by targeting HIF-1 α & STAT3 in breast cancer cells (199) hsa-miR-223 promotes gastric cancer invasion and metastasis by targeting EPB41L3 (200) hsa-miR-26a represses PTEN in a murine glioma model enhances de novo tumor formation (201) hsa-miR-34a# miRNA34 links p53 with Wnt (202)
Chemoprotection by BMSC derived exosomes
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Abbreviations used in Table 6.3: KDM2B= lysine (K) specific demethylase 2 B;
HMGA2= high mobility group AT- hook 2; PTEN= phosphatase and tensin
homolog; EMT= epithelial to mesenchymal transformation; E2F= eukaryotic
transcription factor; HOXA9/MEIS1= homeobox gene A9/myeloid ectopic viral
integration site 2; ERG= Ets related gene; HIF= hypoxia inducible factor;
signal transducer and activator of transcription; EPB41L3= erythrocyte
membrane protein band 4.1 like protein 3 and VEGF= vascular endothelial
growth factor.
6.2.7 BMSC derived exosomal mi-RNAs target ROS pathway in
leukaemic cells
Ingenuity pathway analysis was done on the uniquely expressed miRNAs in
SUPB15MR cells. The core analysis was performed using Ingenuity miRBase
18 (hosted at Manchester) and interrogates > 600.00 known miRNA targets. It
used all available data sources (Ingenuity IPA; Application: Build 140500 and
Content: Version12710793). Analysis considered direct and indirect
relationships between miRNA and endogenous chemical and genes and set
the confidence interval of the data sources at “experimentally observed” and
“high(predicted)” for miRNA in humans, mice and rats. Analysis revealed 9
networks, 3 of them were overlapping with each other and contained 86% of
all miRNAs. These 3 networks were merged and are shown in figure 6.10.
H2O2, an indicator of production of reactive oxygen species (ROS), emerged
as a major node of the network. The connections were with a) 13 miRNAs
identified by the analysis (red) and 2 that were unidentified (miRNA28 &
miRNA342) and b) 7 proteins that included Insulin, Vascular endothelial
growth factor (VEGF), RAC1, oestrogen and progesterone receptors, DNA
damage inducible transcript 3 and CD86.
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Figure 6.9 Connection between miRNAs and ROS. Map showing connections
between miRNAs, proteins and endogenous chemicals present in three
overlapping networks generated by Ingenuity Pathway Analysis software
using a list of unique miRNAs that were present in SUPB15MR cells. It shows
H2O2 to be a major node having connections to 15 miRNA and 7 proteins.
6.2.8 Further characterisation of SUPB15MR cells: Gene expression
analysis of SupB15 and SupB15MR (U133 v2.0 array showed an overall down
regulation of the global gene expression (left panel figure 6.10) but an up-
regulation of histones (right panel, figure 6.10) in SUPB15MR cells compared
to SUPB15. This suggests epigenetic down regulation of gene expression in
resistant cell lines. Four sets of predefined genes related to AKT, ROS,
ATPase and apoptosis pathways were chosen and gene set enrichment
analysis (GSEA) was used to measure enrichment scores between SUPB15
and SUPB15MR cells, figure 6.10 B. All four set showed high scores
suggesting differential expression of these genes in the two cells lines.
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Figure 6.10 Gene expression in SUPB15MR cells. A Gene expression
analysis using U133 v2.0 array- Left panel: Overall, the expression of genes is
higher in Sup B15 Darker colours reflect increased fold expression. However
(right panel) expression of histones is up-regulated in SupB15MR. B
Enrichment plots comparing the expression of predetermined set of genes
SupB15 SupB15MR SupB15
Histones
SupB15 MR
A
B GSEA analysis: Genes up- regulated in SUPB15 MR compared to SUPB15
Gene expression analysis (U133 v2.0 array): SUPB15MR compared to SUPB15
Chemoprotection by BMSC derived exosomes
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identified in each of the 4 pathways involving AKT, apoptosis, ROS and ATP
pathways between SUPB15 and SUPB15MR cells.
Finally, SUPB15MR had lower levels of ROS compared to SUPB15 cells, figure
6.11, that was coupled with cell quiescence (personal communication, Dr. Liu).
Figure 6.11 ROS levels in SUPB15 cells compared with SUPB15MR.
Dichlorofluorescein diacetate (DFC-DA) was used to measure cellular ROS
levels. DFC-DA is cleaved by intracellular esterases to yield fluorophore,
intensity of which is measured by flow cytometry.
Lower levels of ROS in SUPB15MR compared to SUPB15
0
0.2
0.4
0.6
0.8
1
1.2
SUPB15 SupB15-MR
Me
an
F
luo
resce
nce
(fo
ld)
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6.3 Discussion:
This chapter highlights a number of important discoveries that will potentially
change our understanding of how leukaemic cells survive and proliferate in a
normal microenvironment and how this milieu can in fact protect the blast cells
from the effect of chemotherapy. Traditional models have examined the
mechanisms of resistance in the cell itself as is detailed in table 6.1. These
models have failed to provide a unifying mechanism of cell survival in the
context of multiagent chemotherapy. Though cell to cell contact is indeed
viewed as a mechanism of cell survival, the loss of which leads to anoikis,
leukaemic cells have been traditionally viewed as non-adherent and thus
unable to use these mechanisms for survival. This chapter shows that stromal
cells are able to use paracrine mechanisms to regulate lymphoblast cell
survival. Indeed our observations suggest that microvesicle/exosomes
mediated cell signalling is a key component of normal and transformed cell
behaviour.
Dr Liu’s work clearly shows that stromal cell derived exosomes are taken up
leukaemic cells and this process confers chemoresistance. Exosomes are
exocytosed 30-100 nm size intraluminal vesicles of endosomal origin (203).
Figure 6.12 summarises key steps involved in production of exosomes
suggesting that the generation of exosomes is an active process. Exosomes
are one of the several ways by which cells can communicate with one
another(204).
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Figure 6.12 Origin of exosomes. Alphabets A to H sequentially depict the key
steps involved in the production of exosomes. The key factors associated with
each step are shown in blue legends. Little is known about the process that
determines the fate of sorting endosomes. Adapted from (204-207).
We now show that the exosomes are enriched in a miRNA population.
Lymphoblasts once exposed to these miRNA, sustain the pattern of miRNA
Origin of Vesicles: Exosomes = A to H; Microvesicles =1
Clathrin-dependent endocytosis Clathrin -independent endocytosis
Ubiquitination of transmembrane proteins
Lipid rafts: cholesterol, SYT I, VAMP2, SYN*
Binding of AP2 to SYN*
Early endosomes
Sorting endosomes
ESCRT dependent MVB
Degradation
Lysosome
Recycling endosomes
TGN
Clathrin- ACAP1
Clathrin- AP1B Clathrin-GGA3
Retromer
SNX27
SNX17
RAB5EEA1ESCRT 0
ESCRT 0/I/II/IIIVps4 ComplexTsg101AlixLBPACD63LAMP
ESCRT independent MVB
Proteolipidprotein cargoCeramidenSMase2LBPACD63CD9CD81Rab27,11 & 35
LBPACD63LAMP
Ptdins3P
overlap
Rab27ASlp4- aSNARESNAP-25Syntaxin (SYN)
C
D
E
G Exosome secretion
Budding microvesicle
*
ACAP1
AP
EEA
ESCRT
LAMP
LBPA
nSMase2
Ptdins3P
SNARE
SYN
SYP
SYT
TGN
Tsg
Vps
A
B1 B2
F2F1
H
=described with clathrin dependent endocytosis
=ADP ribosylation factor GTPase-activating protein with Coiled coil ANK repeat & pleckstrin homology domains
=adaptor protein complexes
=early endosome antigen
=endosomal sorting complex required for transport
=lysosome associated membrane protein
=lysobisphosphatidic acid
=neutral sphingomyelinase 2
=phosphatidly inositol 3 phosphase
=soluble n-ethyl-maleimide-sensitive factor attachment protein receptors
=syntaxin
=synaptophysin
=synaptotagmin
=transgolgi network
=tumour susceptibility gene
=vacuolar protein sorting
1
VAMP
SYP
cholesterol
Chemoprotection by BMSC derived exosomes
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expresson for many months and retain chemoresistance. Additionally these
cells enter a G1/G0 phase and down regulate oxidative phosphorylation. The
gene expression data suggests that the exosomes miRNA lead to an
epigenetic down regulation of global gene expression, possibly via higher
order regulation at the level of histones (131, 208-210). Such epigenetic
programming has been shown to generate prolonged but ultimately reversible
chemoresistance in cancer cells (211).
Thus we can explain both early and late relapses with this mechanism. In
early relapses, gene expression suggests that cell cycle genes are over
expressed, while late relapses do not appear to have any clear differences in
GEP(212). Cancer cells that have mutations in cell cycle checkpoint genes
such as p53 and CDKN1B (p27) however maintain their ability to proliferate
(personal communication, Dr Liu). Both early and late relapses have been
rendered chemoresitance via the exosome/miRNA transfer, but the early
retains the ability to proliferate and thus this disease is relatively incurable
with conventional chemotherapy. The late relapses, slowly lose
chemoresistance and thus are now vulnerable to intensive therapy. Thus one
approach would be to add drugs that target the epigenome – an approach that
has recently been described to have successful results(213)
In our model, the uniquely acquired miRNAs in SUPB15MR can be broadly
separated into 2 groups. The first one consists of 36 miRNAs that match to
the BMSC derived exosomes and the second one which contains 6 miRNA
that are unique to SUPB15MR cells. The miRNAs in the first group have
previously been shown to target a number of pathways associated with
alteration in cell metabolism and/or chemoprotection, table 6.3. The miRNAs
in the second group have a pivotal role in haematopoiesis and in particular the
B cell development suggesting that cells hold on to their original
developmental programme. The next step would be to individually assess the
role of uniquely identified miRNAs in conferring protection via epigenetic
reprogramming of the cell. This can be done by first measuring their
expression by RQ-RTPCR in clinical samples from patients that show poor
response to therapy as well as in cells that survive chemotherapy in an in-vivo
Chemoprotection by BMSC derived exosomes
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mouse model, followed by knockdown and enforced expression experiments
involving individual miRNA to study their effects on conferring broad spectrum
chemoprotection. Such an approach has a potential to find a mechanistic
explanation behind broad spectrum chemoprotection when current strategies
using genome wide studies have failed to provide an explanation (61, 62).
Several of the unique miRNAs indentified in our experiment have been
reported to be differentially expressed by primary lymphoblasts (214-217).
However, as yet the mechanism behind their differential expression remains
unknown. Our findings suggest that the differential expression of miRNAs in
lymphoblasts may well be due to the effect of the stroma.
Finally, results from the gene expression analysis and miRNA array show that
a number of miRNAs are involved in regulating a number of genes related to
diverse pathways. Thus targeting one gene, miRNA or pathway may not
necessarily yield desired therapeutic results. If dynamic regulation of ROS is
indeed an important downstream effect whereby the leukaemic cells acquire
broad spectrum chemoprotection, then it increasing the cellular ROS level
could become a potential therapeutic strategy. This could be achieved by
combining ASNase with β-phenethyl isothiocyanate (PEITC). ASNase
depletes glutamine; one of the principle building block in the production of
antioxidant glutathione which in turn is a key mediator in maintaining cellular
redox balance(218, 219). Glutamine is also a principle source of energy for a
cell that is deprived with glucose and it plays a crucial role in cell survival in
this setting which in the field of cancer metabolism is also referred to as the
beyond Warburg effect (220). PEITC is a compound known to increase ROS
generation and suppress the BCL-2 family molecules at the same time that is
shown to selectively kill cancer cells. The combination of these two agents
thus shows promise in overcoming multidrug resistance in childhood ALL.
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Chapter 7 Work in progress
______________________________________________________________
7.1 BACKGROUND
As discussed in chapter 5, we could not demonstrate an association between
the expression of AEP and/or CTSB in the leukaemic cells with early
inactivation of PEG-ASNase. Additionally, if these proteases were indeed
mediating the in-vivo degradation of PEG-ASNase, we do not know the exact
interface between the proteases and PEG-ASNase where the degradation
may occur. As the drug leads to extracellular asparagine depletion, the
interaction between PEG-ASNase and the proteases may well be taking place
in the extracellular compartment. Proteases in their active forms are packaged
into vesicles inside the leukaemic cells (100). It is possible that these vesicles
are then secreted into the extracellular compartment. Microvesicles (MV), one
of the subtypes of extracellular membrane limiting vesicles, do indeed contain
proteases (221-224). Thus, MV may well represent the interface between the
drug and the proteases. To investigate this was beyond the scope of this
thesis but in this chapter I describe early results of a technique I developed to
enrich B cell derived MV from cell supernatant. This method is also suitable
for harvesting MV from clinical samples.
7.1.1 Nomenclature of Membrane-limited vesicles:
The extracellular compartment contains mobile membrane-limited vesicles,
variably referred to as “extracellular vesicles”(225) or “membrane
vesicles”(203) or “extracellular organelles”(226). They range from 50 nm to 5
μm in size and are sub-classified into smaller (<100 nm) size vesicles that
include exosomes (227, 228), prominin 1- enriched membrane particles (229)
and exosomes-like vesicles(230); and larger (>100 nm) size heterogenous
vesicles that include MV,100-1000 nm(231-236); ectosomes (237, 238), 50-
200 nm; and apoptotic bodies. (203, 239). Perhaps a biologically more
meaningful classification is the one that is based on their origin. Whilst
exosomes and exosomes like vesicles originate from intra-luminal vesicles of
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the multivesicular bodies (MVB); MV, ectosomes and apoptotic bodies are
“shed” directly from the plasma membrane (203). There is a lack of
standardised nomenclature to define membrane-limited vesicles (225). For
example, the term “MV” has also been used as an umbrella term to describe
all extracellular vesicles (240). To confuse the matter further “MV” as defined
by (203) are also referred to as “shedding MV” by (226), shedding vesicles by
(240) and ectosomes by (204). In this thesis exosomes are defined
exocytosed 30-100 nm size intra-luminal vesicles of endosomal origin and MV
as 100-1000nm size vesicles produced by direct plasma membrane blebbing.
Table 7.1 shows defining features of MV, exosomes and apoptotic bodies.
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Table 7.1 Key features of membrane limited vesicles:
Exosomes Microvesicles Apoptotic bodies
Size 30-100 nm 100-1000nm 50-500nma; >1000nm
b
Mechanism of generation Exocytosis of MVB Budding from plasma membrane Budding from plasma membrane,
Mode of extracellular release
constitutive and regulated Regulated Regulated
Main protein markers Tetraspanins (CD63, CD9, CD81) Proteases: MMP2, MMP9, uPA, CTSB Histones
Alix Membrane associated: VAMP3, β1 integrin
TSG101 Cytoskeleton associated: Actin, myosin
Rab11, Rab27a, Rab27b, Rab35
Lipid composition Enriched in cholesterol, ceramide, SM, enriched in cholesterol and DAG Not determined
nSMase2, lipid rafts, LBPA high phosphatidylserine exposure high phosphatidylserine exposure
low phosphatidylserine exposure
Density in Surcose 1.13-1.19g/ml Not determined 1.16-1.28
EM appearance Cup shpae Irregular and electron dense Heterogeneous
Isolation Differential gradient ultracentrifugation Differential centrifugation Established protocols lacking
Sucrose gradient ultracentrifugation 18,000-20,000g
1,00,00-2,00,000 g
Table 7.1 Adapted from (203, 225, 226, 240-242). MVB= multivesicular bodies; nSMase2=neutral sphingomyelinase 2; LBPA=
lysobisphosphatidic acid, EM= electron microscopy; SM=sphingomyelin; MMP= matrix metalloprotinases; CTSB=Cathepsin B;
VAMP= vesicle associated membrane protein; uPA= urokinase plasminogen activator; DAG=Diacylglycerol; a= as described in
(203) ; b= as described in (204).
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7.1.2 Functional role of vesicles
Release of membrane vesicles is a process that is evolutionarily conserved
across the prokaryotic and eukaryotic cells. Table 7.2 highlights some of roles
of vesicles that have been identified in health and disease including cancer
(225, 241). Vesicles have been identified as one of the mechanism by which
cells communicate (204). Table 7.3 highlights the example of cargo contained
in the MV that affects a number of processes in cancer.
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Table 7.2 Vesicles in health and diseases
Categories References
Physiological Processes: (203, 204, 225, 243-245)
Actin cytoskeleton/integrin/
Rho A/synaptic signalling,
Caveolar mediated endocytosis,
Acute phase response, conveying of
immune response, bone mineralisation,
angiogenesis, intercellular communication
Diseases
Autoimmmune diseases (246-256)
(Systemic lupus erythromatosis,
anti phospholipid antibody syndrome,
rheumatoid arthritis, vasculitis,
systemic sclerosis, type 1 diabetes
Cardiovascular diseases (257-266)
Acute coronary syndrome, hypertension,
pulmonary hypertension, Buerger's
disease, atherosclerosis, deep vein
thrombosis, congestive cardiac failure
Cerebrovasuclar diseases (267-269)
Transient ischaemic attack, multi-infarct
dementia, subarachnoid haemorrhage
Haematological diseases (270-273)
Paroxysmal nocturnal haemoglobinuria,
sickle cell crisis, immune thrombo- cytopenia, thrombotic thrombocytopenia
Cancer (161, 274-279)
lung adenocarcinoma, glioblastoma, oral/ovarian/prostatic/colorectal/
gastric cancers, melanoma,
cancer associated thrombosis
Other diseases (280-285)
Alzheimer's disease, metobolic syndrome,end stage renal disease,
sepsis, pre-ecclampsia, obstructive sleep apnoea
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Table 7.2 Adapted from (204, 225, 242, 245)
Table 7.3 Microvesicle cargo in cancer
Microvesicle Cargo Function References
Proteins
Soluble Factors: Angiogenesis, inflammatory (161, 286-288)
(VEGF, FGF, IL8, IL6 cytokines, regulators of
MMPs, TIMPs) Proteolysis
Membrane receptors: Chemokine receptor, receptor (230, 289-295)
(CCR5, CCR6, TNFR1, p55, EGFR, AXL, FasL)
tyrosine kinase, Death ligand
Oncoproteins and tumour suppressors:
Oncogenic EFGR, RTK, GTPase
(161, 290-292, 295-297)
(EGFR, EGFRvIII, HER2, Mutant EGFR
MET, K-ras, Akt, PTEN) Tumour suppressor
Lipids cell signalling (298)
(Sphingomyelin) Angiogenesis
Nucleic acids tumour growth (161, 279, 299-301)
(mRNA, micro RNA, DNA, gene regulation
mt DNA, gDNA) cell communication
Table 7.3 Adapted from (242). VEGF= vascular endothelial growth factor;
FGF= fibroblast growth factor; IL= interleukins; MMP= matrix
metalloproteinase; TIMP= tissue inhibitor or metalloproteinase; TNFR=
tumour necrosis factor receptor; CCR= chemokine receptor; EGFR=
endothelial growth factor receptor; HER2= human epidermal growth factor
receptor 2
7.2 PRELIMINARY RESULTS- Harvesting B cell derived MV:
Technique described in this thesis to isolate MV consists of two stages. In the
first stage cell supernatant and plasma is subjected to a series of
centrifugation steps based on previously published method to pellet MV from
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cell free supernatant and patient plasma. The differential centrifugation steps
consisted of 750 g for 5 minutes and 1500 g for 15 minutes to remove cells
and debris respectively; followed by a final spin of 15000 g for 45 minutes to
pellet the MV (302). As the method did not employ ultra-centrifugation
procedure, exosomes were not expected to be in the pellet (234). The
second stage of the technique added specificity to the process by isolating
just the B cell derived MVs. This is important in the context of isolating tumour
derived MV from plasma of patients with precursor B ALL. In this stage the
pellet was first labelled with a green fluorescent lipid staining dye PKH-67 and
the labelled MV were re-suspended in PBS. These labelled MV “pulled out”
from the PBS solution and immobilised on to the protein A/G Ultralink resin by
using a modified immunoprecipitation technique. Immunoprecipitation was
originally used to precipitate proteins of interest from a solution. Instead of
precipitating the protein, mouse anti human anti-CD19 antibody (clone HIB19,
BD Biosciences, catalogue number 555410) was used as a “pull down
antibody” to harvest the MV.
7.2.1. Pellet obtained after stage one of the method was enriched in
microvesicles markers and contained CD19
The pellet was lysed and its protein content was analysed by immunoblotting.
Immunoblotting showed presence of VAMP3, a marker described to be
associated with MVs (241) and absence of proteins associated with
exosomes, such as LAMP1, TSG101, CD63 and CD81, figure 7.1A.
Immunoblotting revealed the presence of CD19, a pan B cell marker, figure
7.1A. The pellet was re-suspended in PBS and analysed for surface
expression of phosphatidylserine by flow cytometry using Annexin V
conjugated to FITC (Annexin V-FITC). Annexin binds to phosphatidylserine
and when tagged with a fluorophore (FITC), it can be use to study the surface
expression of phosphatidylserine. The shift in mean fluorescent intensity seen
between the two populations, figure 7.1 B, shows that at least 55% of the
pellet vesicles have phosphatidylserine externalisation. Phosphatidylserine
externalisation is a defining feature of microvesicles (303-305).
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Figure 7.1 Characterisation of pellet obtained after stage one of MV isolation
method. A: pellets were lysed and immunoblotted for a number of markers.
The lysates expressed CD19 (pan B cell antigen) and VAMP3 (vesicle
associated membrane protein, characteristically shown to be associated with
MV). There was an absence of detectable CD63, LAMP1, TSG101 and CD81
(markers known to be associated with exosomes). B: Mean fluorescent
Fluorescent intensity (FI) FITC 502nm
SD1 vesicles
SD1 vesicles Annexin V-FITC labelled
Co
un
ts %
B FLOW CYTOMETRY
250 kd
150 kd
100 kd
75 kd
50 kd
37 kd
25 kd
20 kd
15 kd10 kd
1 2
1 VAMP3 13 kd
2 CD19 95 kd
GAPDH 36 kd
3 CD63 50 kd
4 LAMP1 120 kd
5 TSG101 44 kd
6 CD81 26 kd
3 4 5 6
A IMMUNOBLOTTING
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intensity by flow cytometry of pellets re-suspended in phosphate saline buffer
with and without prior staining with Annexin V-FITC, a dye that stains
phosphatidylserine on the membranes. The shift in mean fluorescent intensity
of the labelled vesicles compared to the unlabelled vesicles indicates that 55%
of vesicles had externalisation of phosphatidylserine, a feature that is
characteristic of MV. As the sensitivity of the flow cytometer was pushed to its
limits to count all the MV, the data set would have picked any particles in the
fluid stream. Hence the actual proportion of MV showing phosphatidylserine
externalisation is likely to be far higher.
7.2.2 Immobilisation of CD19 expressing SD1 microvesicles on Ultralink
A/G resin:
As the microvesicle pellet lysate expressed CD19 antigen and as
microvesicles are shed from B cell surface, it was possible that CD19 was
expressed on MV surface. We hence used anti-CD19 antibody to immobilise
MV on to Protein A/G Ultralink resing. Figure 7.2 A shows PKH67 dye labelled
SD pellet (green) immobilised onto the Protein A/G Ultralink resin using anti-
CD19 antibody. The negative controls used to demonstrate the specificity of
interaction between CD19 antigen on the MV and the “pull down” mouse anti
human anti-CD19 antibody consisted of mouse anti human anti CD3 antibody
(Figure 7.2 B) and protein A/G Ultralink resin alone (Figure 7.3 C) Images
were acquired on the time lapse system that consisted of Zeiss 200M inverted
microscope and a Zeiss 20x lens for images. ImageJ software showed that
immobilied round bodies had a vesicular shape and their size varied between
380 nm to 850nm in diameter.
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Figure 7.2 Immobilisation of SD1 pellet microvesicles on to protein A/G
Ultralink resin. Images from left to right represent bright field images using
phase contrast (Left); images using a Sedat 488nm filter from Chroma and a
metamorph acquisition software from Molecular Devices (centre) and merged
image created on ImageJ software (right). A, B & C are generated using Zeiss
20x lens.
C: No antibody
B: anti-CD 3 antibody
A: anti-CD19 antibody
50μ scale bar
Ultralink A/G resin PKH67(green) labelled vesicles
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7.2.3 Quantifying PKH67 labelled cell line MV by flow cytometry:
Replacing larger beads in protein A/G UltraLink resin with smaller 6.0-8.0 μm
protein G polystyrene particles, SPHERTM (Catalog no PGP-60-5, Spherotech,
Illinois, US) permitted us to quantify the amount of PKH labelled vesicles
immobilized on to the beads. Figure 7.3 A, shows the mean fluorescent
intensity of PKH67 labelled MV obtained from 1 ml of SD1 and REH debris
free supernatants. Both the cell lines were cultured under identical conditions
that included a starting cell concentration of 1 million per ml and the MV were
harvested 48 hours later. Compared to REH cell supernatant, SD1 cell
supernatant had greater concentration of PKH labelled MV. Figure 7.3 B,
shows increase in mean fluorescent intensity that was proportional to the
amount of MV.
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Figure 7.3: Quantification of MV in cell supernatants A Shift in mean
fluorescent intensity (MFI) seen PKH67 labelled MV of SD1 and REH cell
lines on flow cytometry when using anti CD19 antibody (blue) compared to the
MFI obtained with anti-CD3 antibody (red, negative control). For identical
culture conditions, there were more MVs in SD1 supernatant that REH
supernatant. B: Increase in mean fluorescent intensity that is proportional to
the amount of labelled MV: bead alone (red), MV from 1 ml (blue) and 6 ml
(orange) of debris free SD1 supernatant.
MFI
anti CD3 1475anti CD19 2061
27%17%
SD1 MVREH MV
Fluorescent intensity (FI) PKH67 502nm
Co
un
t (%
) MFI
anti CD3 1410anti CD19 1528
Co
un
t (%
)
Fluorescent intensity (FI) PKH67 502nm
6ml of SD1 supernatant MV: 54% positive compared to beads alone
1ml of SD1 supernatant MV: 34% positive compared to beads alone
Beads alone
SD1 MV anti CD19
A
B
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7.2.4 Immobilised vesicles expressed microvesicles markers : In order to
further demonstrate that the vesicles immobilised to protein A/G UltraLink
resin were indeed MV, the vesicles were lysed and the protein content was
blotted for presence of markers previously shown to be associated with MV:
matrix metalloproteinase-2 and 9 (MMP2, MMP9) (223), Cathepsin B (CTSB)
(224), Actin and Leukocyte Function Adhesion (LFA) molecule (CD11a),
figure 7.4.
Figure 7.4 Protein content of immobilised MV. Immunoblot showed presence
of markers previously been described to be associated with MV (introduction).
All results were generated from a common microvesicle lysates.
7.2.5 MV from patient plasma: Figure 7.5 A shows the appearance on
electron microscopy of a pellet obtained after subjecting the bone marrow
plasma to the same spin employed to pellet the MV. It shows presence of
membrane bound vesicles that are heterogeneous with respect of size and
shape. Figure 7.5 B shows PKH67 labelled vesicles immobilised on the
protein A/G UltralLink resin using anti CD19 antibody. The size of the vesicles
range from 350 nanometers to 1.4 micrometers
MMP9 92-86 kd
MMP2 77-66 kd
LFA 160 kd
CTSB 43-46 kd
31 kd
24-25 kd
Actin 37kd
Work in progress
171
Figure 7.5 Isolation of MV from patient plasma. A: Transmission electron
microscopy image of MV pellet obtained from a bone marrow plasma of a
patients at presentation of ALL. B: Images from left to right represent bright
field images using phase contrast (Left); images using a Sedat 488nm filter
from Chroma and a metamorph acquisition software from Molecular Devices
(centre) and merged image created on ImageJ software (right). A, B & C are
generated using Zeiss 20x lens.
200 nm scale bar 500 nm scale bar
5000 nm scale bar
Ultralink A/G resin PKH67(green) labelled vesicles
A: Transmission electron microscopy image of a pellet obtained from bone marrow plasma at
diagnosis
B: Images acquired on the time lapse system of bone marrow plasma derived, PKH labelled
vesicles immobilised on Ultralink A/G resin using anti CD-19 antibody
Work in progress
172
7.3 DISCUSSIO|N:
The in-vitro and in-vivo isolation, detection, quantification and characterisation
of MV is complicated by the heterogeneity in their size, the lack of
standardised methods/protocols that selectively harvest them from other
vesicles and the potential influence of pre-analytical variables such as shear
stress caused during venepuncture, anticoagulant in the sample, sample
storage, processing time and the temperature conditions during transit (225).
The most common approach to isolate MV employs differential centrifugation
followed by flow cytometric detection. However detecting smaller (<500nm)
MV using forward scatter and convincingly separating them from the
background instrument noise created by salt crystals, microparticles, plasma
lipoproteins and platelet fragments remains a challenge for most modern flow
cytometers even after using calibrated sub-microscopic beads (306-309).
Optical methods such as transmission electron microscopy, atomic force
microscopy and dynamic light scattering whilst are able to give an accurate
size of particles, are not suitable for routine clinical practice or laboratory
research. None of the techniques can specifically separate tumour derived
MV from non tumour derived MV in clinical samples. The method described in
this chapter attempts to address some of the above mentioned difficulties that
limit accurate isolation of tumour derived MV. The anti CD-19 antibody was
chosen primarily because CD19 is specific for pre-B cells. Thus in diagnostic
plasma, CD19 positive MV are more likely to originate from lymphoblasts.
This approach could in the future be used to relatively quantify the amount of
tumour derived MV in clinical samples using different concentrations SD1
supernatant MV to create a standard curve.
The technique described in this chapter needs further validation. Firstly, the
immunoblotting results shown in this chapter need appropriate controls to
demonstrate that the antibodies used for exosomal markers are suitable. This
can be done by pelleting exosomes by ultracentrifugation of MV free cell
supernatant, followed by lysis of pellet and performing immunoblotting using
Work in progress
173
the same antibodies to demonstrate the presence of exosomes. Secondly, the
anti CD3 antibody used as negative control for immobilising SD1 (precursor B
cell line) MV on the protein A/G UltraLink resin, needs to be validated. This
can be done by demonstrating its ability to immobilise MV from a T ALL cell
line where the anti CD19 antibody would serve as a negative control. Thirdly,
it is possible that not all precursor B ALL derived MV bear CD 19 antigen and
hence the technique may not catch all tumour derived MV. Fourthly, MV
cannot be reliably differentiated from apoptotic bodies at this point. Apoptotic
vesicles bleb off from the cell membrane, show phosphatidylserine
externalisation and contain fragmented DNA(310). A strategy that employs
Annexin V or Hoescst staining is unlikely to discriminate the two as both show
phosphatidylserine externalisation and the presence of mitochondrial DNA in
the MV is likely to give a false positive signal. I thought of immunoblotting MV
lysates for histones. Mitochondrial DNA (mtDNA) unlike the mammalian DNA
is packaged into discrete units know as “nucleoids” that are reported to be
devoid of histones(311, 312). However other publications have isolated
histones from mitochondria(313-315). An explanation for this apparent
discrepancy is due to the binding of histones to the outer mitochondrial
membrane where they have an unidentified function (315).
Nevertheless, the technique described in this chapter provides a starting point
in the attempt to isolate tumour derived MV from clinical samples. Once
validated, the technique offers the option of characterising tumour derived MV
in terms of its RNA, protein or lipid content. Such an approach may advance
our understanding of the potential role of MV in mediating a number of
biological processes such as cell survival and cell to cell communication.
Concluding remarks
174
Chapter 8: Concluding remarks
______________________________________________________________
Childhood acute lymphoblastic leukaemia is one of the great success stories
in the field of cancer treatment as well as in the field of translational research
aimed at understanding the tumour biology to improve outcome.
Therapeutically the regimens devised for childhood ALL have give rise to the
principles that drive current chemotherapeutic protocols for patients with
cancer and provide the rationale for combination therapy. Leukaemia biology
led to the first identification of somatic genetic change associated with cancer,
the fusion of BCR-ABL genes. It also remains the most successful example of
targetable genomic mutation, with the use of tyrosine kinase inhibitor in
Philadelphia chromosome positive leukaemias.
Serendipity has played a major role in the success story. By and large almost
all early chemotherapy agents proved to be effective as single agents in
childhood ALL. Initially the remission was short lived, but we discovered that
the prolonged use of combination chemotherapy could sustain remissions. No
new drugs have entered routine clinical use since the 1970’s. Risk
stratification and progressive intensification with the same agents has lead to
over 80% survival with current chemotherapeutic regimens (3), This success
comes at a price; that of treatment related short and long term toxicity (316-
321) that is balancing the relative risk of relapse (322). Over 25% of NCI
standard risk and 40-50% of high risk patients now report therapy related
severe adverse events whilst on therapy (personal communication, Professor
Ajay Vora). Treatment related deaths now match the numbers who relapse.
(personal communication, Professor Ajay Vora). Additionally, the disease is
heterogeneous with cure rates in cytogenetic high risk sub-groups (42) , still
less than 50% (323). Thus additional intensification with existing drugs is
unlikely to improve cure rates further. The focus has therefore shifted towards
finding new drugs and compounds, both to improve outcome and decrease
toxicity. So far, apart from the afore-mentioned tyrosine kinase inhibitors,
there has been little success in finding more effective anti-leukaemic agents.
Concluding remarks
175
Broad-spectrum new anti-cancer agents such as Clofarabine occupy a niche
in relapsed disease, though there is little data to show that any of the new
chemotherapeutic drugs have improved outcome when compared to the
existing ones. Given that ALL relapses occur after multi-agent therapy, it is
likely that the returning leukaemic cells have acquired broad-spectrum
chemoresistance. Thus non-specific chemotherapeutic agents are more likely
to increase toxicity than effect a cure..
As with other cancers, most pharmaceutical companies are now trying to
develop targeted therapy. This can be broadly classified into two types. The
first group consists of antibodies that target molecules expressed on the cell
surface. For example, we recently described the expression and targeting of
5-T4 in high risk childhood ALL(324). The more interesting result has been
with the use of a bi-specific anti CD19 antibody, Blinatumomab. In a phase I
study in adult patients with relapsed ALL, as a single agent, it achieved a 72%
CR rate. Almost all patients in CR achieved a molecular remission. However,
in a phase II study, the emergence of CD19 negative clones has been
reported. One of the disadvantages of Blinatumomab is that it is dependent on
the presence of active T cells and thus cannot be given with other cytotoxic
chemotherapeutic agents. It remains to be seen whether resurgent CD19
negative cells will respond to subsequent therapy or not.
The second group of targeted therapy consists of compounds that are directed
at somatic mutations that are peculiar to the cancer cell. An example is the
use of Gefitinib to target epidermal growth factor receptor (EGFR) expressing
lung and breast cancers. Whole genome sequencing in childhood ALL reveals
relatively few non-random mutations, they occur at about 6-8 per genome.
Mutations described occur primarily in genes regulating B-cell development
(e.g. IZKF, PAX5), cell cycle (p53, Rb) or are involved in normal cell signalling
(e.g. RAS). Thus clear targets for therapy in lymphoblasts have yet to be
identified.
Concluding remarks
176
Our data suggests that while we wait for more effective new agents, it is
possible to optimise therapy using current existing drugs, both to reduce
toxicity and to improve efficacy. The first one pertains to the drug ASNase.
Routine monitoring of ASNase could play a role in both the low risk and high
risk groups. In the former it might help in identifying patients who might benefit
from post induction intensification of therapy whereas in the latter it identifies
patients not achieving adequate ASNase activity levels after post induction
intensification of ASNase. In those with inadequate activity, one could
consider using alternative PEG formulations, the use of recombinant PEG-
ASNase or for example using additional daunorubicin in those receiving a 3-
drug induction. Additionally, the current UKALL2011 trial presents with the
opportunity to prospectively study the interaction of PEG-ASNase and
Dexamethasone and we may be able to further optimise the potential synergy
by optimising the scheduling of the two drugs.
An alternative approach at improving the outcome is directed to targeting the
microenvironment. In childhood ALL, there are a number of observations that
suggest the role of bone marrow microenvironment in supporting the tumour.
(325-327). ALL is peculiar amongst cancers where the best results have been
obtained by intensive use of the same class of drugs in a repetitive fashion,
followed by a prolonged phase of thiopurine therapy. The best outcomes are
observed in patients who develop repeated episodes of uncomplicated
cytopenias. This is illustrated by our observations with Mitoxantrone in
relapsed ALL (11).
At least a third of patients who experience late relapse can still be cured using
essentially chemotherapeutic agents belonging to the same classes as in
frontline protocols, albeit in more intensified relapse regimens. Thus treatment
failure cannot be attributed to intrinsic chemoresistance alone. Those patients
who relapse early require allogeneic transplant which not only provides fresh
source of haematopoietic stem cells but the conditioning therapy first disrupts
the haematopoietic stem cell niches. Our data shows BMSC mediated
chemoprotection to ALL cells involves chromatin regulation and pathways that
Concluding remarks
177
governs cell’s AKT activity, oxidative metabolism and apoptosis. Figure 8.1
proposes a conceptual model of how the bone marrow stromal cells might
protect leukaemic cells from chemotherapy by alteration of above mentioned
processes and suggests non cytotoxic approaches that can be combined with
conventional therapy to improve outcome and reduce toxicity.
Concluding remarks
178
Figure 8.1: Proposes a central role of BMSC derived soluble factors in conferring chemoprotection to the leukaemic cell. Blue
legends refer to molecules and blue arrows to their connections for which evidence exists for their role in altered cancer cell
1: Asparaginase
induced extracellular
Asparagine depletion
2: BMSCs secrete soluble factors: Primarily
or in response to chemotherapy
2a Asparagine ϯ 2b Cysteine*2c: Other: amino acids (AA), lipids, RNA, cytokines, MV, exosomes,
Exosomal: miRNA, small RNA, lipids, proteins, AA
Repletion of Asparagine
3a:Reprogramming of cell
metabolism & alteration of gene
expression, possibly mediated by
epigenetic changes #
BMSC
Leukaemic cell
Glutamate
α KGOAA
Aspartate
CS
Citrate
Isocitrate
NADPH NADP+
GSHGSSG
IDH2
3b: Increased uptake of
glutamine- number of pathways
(Beyond Warburg effect)
4a:GSH-Antioxidant
Glycine
GLS
ROS H2O
CysteineNAC
↓ROS
IDH2
α KG
3 c:IDH
Mutant
Type
effect
Glutamine
Glutamine
depleting
ASNasePEITC
TET2 DNA
hydroxylase
JmjC histone
demethylase
2HG2HG
3d:DNA CpG
hypermethylation
Hypomethylating agents
TET2 inhibitors
IDH inhibitors
NUCLEUS
CYTOPLASM
MITOCHONDRIA
Concluding remarks
179
metabolism and/or cell survival, either in ALL or in other cancers. In response to ASNase induced extracellular depletion of
asparagine, BMSC up-regulates and secretes asparagine: Ϯ =(99). BMSC confer chemoprotection by secreting cysteine, one of the
three building block of glutathione (GSH): * =(328). Work done by our group show the role of BMSC derived exosomes in
mediating chemoprotection. While this may not be the sole mechanism by which cells acquire chemoprotection, we propose a role
of BMSC derived exosomal miRNA in mediating downstream effects such as low levels of ROS, global decrease in gene
expression, up-regulation of histones (black dotted lines). The exact sequence of events is currently unknown. A number of
pathways such as PI3K and AKT are affected. We propose a central role of glutamine, one of the most important sources of energy
to a cancer cell after glucose and a key mediator of the beyond Warburg phenomenon(220) in mediating the downstream effects of
exosomal miRNA. While mutations in IDH resulting in generation of an oncometabolite, 2-hydroxyglutarate (2-HG) followed by DNA
hypermethylation is described in myelodysplastic syndromes (329-331), exosomal miRNA induced repression of wild type IDH may
induce similar downstream effects in ALL.
Legends in red propose new therapeutic targets. Attempts to block exosomal secretion by targeting Rab27a (figure 6.12) would
lead to unacceptable side effects as all cells secrete exosomes. Besides exosomes may not be the only secreted soluble factor that
mediates chemoprotection as suggested in figures 6.3 and 6.6. Likewise there are a number of miRNAs (figure 6.9) that could
target ROS and hence would not be an easy therapeutic target. Both the PI3K & AKT pathways are linked to a number of cellular
physiological processes. Targeting them is unlikely to be tumour specific and might be associated with unacceptable toxicity.
However, both the epigenetics and the oxidative metabolism of the leukaemic cells could be targeted to disrupt the host tumour
interactions. Use of hypomethylating agents such as Azacytidine have shown promise in acute myeloid leukaemia and
myelodysplastic syndrome (332) Early results show agents such as Piperlongumine and Phenethyl isothiocyanate (PEITC) can
Concluding remarks
180
selectively kill cancer cells by upregulating ROS levels (219, 333). These could be combined with agents such as ASNase that is
engineered to have enhanced glutaminase activity (334). Such an approach might deplete extra cellular glutamine and further
decrease the ability of tumour cells to scavenge ROS. Figure was adapted from:(220, 335, 336)
Concluding remarks
181
The work carried out in this thesis, shows that we still have a lot to learn about
the drug ASNase. In addition to asparagine depletion, our work suggests the
glutaminase activity of ASNase is essential for lymphoblast toxicity. The work
presented in this thesis also suggests that the glutaminase activity of ASNase
is potentiated by drugs that increase ROS. Of the currently used agents, only
the anthracyclines elevate ROS. Thus the conjunctive usage of anthracyclines
with ASNase in induction and intensification may well be a fortuitous one.
We describe that 1000 units/m2 of PEG-ASNase is sufficient for trial purposes
in childhood ALL. This results in adequate levels in most children and a lower
incidence of inactivation than previously reported. In this study, antibodies
were reported both against the PEG as well as against the native E.coli
ASNase. The incidence of silent inactivation was 4.7%. As all patients with
inactivation benefit from therapy with the Erwinia carotovera derivative
(Erwinase), this is an argument for the routine use of ASNase activity assays
in patients on therapy.
How do we decrease the incidence of antibodies against PEG-
ASNase? A new recombinant ASNase that is more tightly linked to PEG
reportedly has a longer half-life and may also be less antigenic. We have
previously shown that ASNase can be engineered to be less degradable and
more potent (98). Another option would be to identify the antigenic epitopes of
PEG-ASNase by exposing overlapping peptide fragments of PEG-ASNase to
T cells and performing T cell activation assays in patients who have
completed therapy. These antigenic epitopes can then be modified so that the
drug becomes less antigenic while retaining its efficacy at the same time.
Why the inactivation of PEG-ASNase and the development of hypersensitivity
are both more commonly observed in patients in regimen C remains
unresolved. One possibility is the frequency of administration, but the
experience from EsPhALL where Ph+ patients continue to experience a high
rate of hypersensitivity reactions but do not receive frequent ASNase suggest
Concluding remarks
182
that the tumour biology, by yet unidentified mechanisms, determines the
development of immune response as well. The UKALL 14 trial
(Clinicalrials.gov no NCT01085617) for adult ALL patients is studying the role
of anti-CD 20 antibody (Rituximab) is reducing the incidence of antibodies to
PEG-ASNase (personal communication, Professor David Marks). This is an
interesting approach as Rituximab could have a dual role of working as a
targeted therapy and being an immunosuppressant at the same time.
What are the mechanism of inactivation of Asparaginase which are unrelated
to antibody formation? Currently this remains unknown. A clue that proteases
may govern the pharmacokinetics of ASNase comes from animal studies.
Mice have a more complex degradome compared to humans (337) and the
half life of ASNase in mice is much shorter compared to that in humans. To
answer the question of whether proteases such as AEP and CTSB do mediate
in-vivo inactivation of ASNase, one can develop techniques that can
specifically quantify catalytically active proteases in leukaemic cells. Future
studies need to additionally address the question of where the in-vivo
inactivation occurs. Proteases containing, secretory, tumour derived
microvesicles may well mediate the degradation of ASNase in the extracellular
compartment.
A number of other clinical features remain unresolved, particularly the issue of
toxicities peculiar to ASNase. Hypersensitivity we have discussed.
Development of pancreatitis remains a problem as the drug in all forms has to
be discontinued, potentially affecting survival. We do not why some patients
develop pancreatitis (338, 339), though there is evidence from animal models
to suggest that the pancreas is one organ in the body that suffers the most
from asparagine depletion.
The other problem is that of thrombosis. I would like to speculate on the
mechanism of thrombosis in childhood ALL and the possible role of MV in
supporting venous thrombosis, not only in ALL but also in other cancers.
Concluding remarks
183
Figure 8.2 depicts a concise summary of normal processes involved in
regulating coagulation during homeostasis and Figure 8.3 show mechanisms
involved in clot formation in response to vessel injury.
Currently there are no clinical predictive biomarkers for thrombosis seen in
childhood ALL. A common pre-requisite for thrombin generation is a
phosphatidylserine platform on which both the tenase and prothrombinase
complex are formed (Figure 8.3). In the context of either a low or normal
platelet count, the question is who substitutes the physiological role of
activated platelets during induction phase of the treatment? MV have been
shown to have phosphatidylserine externalisation (303-305) and to express
tissue factor on their surface (340-342). I decided to investigate if the tumour
derived MV can substitute the role of activated platelets. If this was the case
then quantifying tumour derived MV in diagnostic bone marrow plasma by
flow cytometry may predict the risk of thrombosis in children undergoing ALL
therapy. I chose to test this hypothesis using dilute Russell Viper Venom Time
(dRVVT). To the best of my knowledge, such an approach has not been
reported before. The result shown in table 8.1 is only a pilot experiment done
once to demonstrate the proof of the concept. MV used in this experiment
were pelleted from 10 ml of SD1 supernatant. The experiment will clearly
have to be repeated, ideally with titrating amount of SD1 MV and then after
spiking standard plasma with patient derived MV. However, if this approach
works, its advantage over measuring parameters such as tissue factor or
generation of thrombin on the surface of tumour derived MV is that this would
be a functional assay that not only measures the thrombogenic potential in
patient plasma but offers an explanation to the mechanism of thrombosis in
childhood ALL. The other priority would be to validate a flow cytometry based
assay in order to quantify the MV. Once this is done, spiking standard plasma
with a fixed amount of MV will generate a more meaningful dRVVT times that
would hopefully be able to predict thrombosis that occurs during induction
phase in ALL.
Concluding remarks
184
Table 8.1: MV participate in coagulation. Standard plasma when spiked with
MV result in shortening of dRVVT times and by blocking the
phosphatidylserine sites by prior incubation with Annexin V, their efficiency in
coagulation as measured by dRVVT times, is limited. dRVVT test was done
on Stago STA-R Evolution Analyser. The dRVVT reagent (STA®-Staclot
dRVV Screen 5-ref –00333) was supplied by Stago. The standard plasma (SP)
pool was supplied by Siemens.
dRVVT (sec)
Standard plasma (SP) 48.9
Standard plasma plus SD1 MV (SPMV) 40.9
Standard plasma plus SD1 MV blocked with annexin (SPMV-A) 43.1
% correction of dRVVT time [(SP-SPMV)/SP]*100
SPMV 18%
Effect of Annexin 11%
Concluding remarks
185
Figure 8.2: Concise summary Central role of intact endothelial to preventing
thrombin generation. Tissue factor bearing extra-vascular cell generates
activated IXa, Xa and limited amount of thrombin (basal, priming phase of
coagulation). IXa enters the vascular compartment but is unable to form a
tenase complex with VIII. VIII is bound to globular form of vWF. Xa that
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Concluding remarks
186
generates limited amount of thrombin is inactivated by TFPI in the
extravascular space. Endothelium prevents platelet activation directly by
secretion of NO & PGI2 and indirectly via endonucleotidase that inactivates
ADP. In resting state, platelet membrane has phosphatidlyserine on the inside
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Concluding remarks
187
Figure 8.3: Mechanisms involved in clot formation in response to vessel injury.
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conformational change in vWF which mediates initial indirect binding of
platelets to collagen and “slowing” of platelets. This is followed by change in
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late
let in
teg
rin
rece
pto
r
=P
late
let
vW
Fre
ce
pto
r
(Gp
1b
/V/I
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Concluding remarks
188
platelet shape and a stable, direct binding of platelets to collagen via integrin
receptors; inside out and outside in signalling in platelets that results in
generation of platelet agonists such as ADP, thromboxane A2 and thrombin.
These agonists can then activate other platelets. Thrombin forms a crucial
role in generating Va and VIIIa. Activated platelets having phosphatidlyserine
externalisation, in association with Ca (not shown) provides a crucial platform
on which Gla domains of Factor IXa and Va dock to sequentially generate
tenase (IXa/VIIIa) and prothrombinase (Va/Xa) complexes. Prothrombinase
complex is responsible for the final ‘burst’ of thrombin that cleaves of the
Fibrinopeptide A and B (not shown) from fibrinogen to form fibrin and
eventually a fibrin clot.
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329. Kosmider O, Gelsi-Boyer V, Slama L, Dreyfus F, Beyne-Rauzy O, Quesnel B,
et al. Mutations of IDH1 and IDH2 genes in early and accelerated phases of
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330. Thol F, Weissinger EM, Krauter J, Wagner K, Damm F, Wichmann M, et al.
IDH1 mutations in patients with myelodysplastic syndromes are associated with an
unfavorable prognosis. Haematologica. 2010 Oct;95(10):1668-74.
331. Yoshida K, Sanada M, Kato M, Kawahata R, Matsubara A, Takita J, et al. A
nonsense mutation of IDH1 in myelodysplastic syndromes and related disorders.
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332. Bhatla T, Wang J, Morrison DJ, Raetz EA, Burke MJ, Brown P, et al.
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of cancer cells by a small molecule targeting the stress response to ROS. Nature.
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335. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even
warburg did not anticipate. Cancer Cell. 2012 Mar 20;21(3):297-308.
336. Aon MA, Cortassa S, Maack C, O'Rourke B. Sequential opening of
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338. Minowa K, Suzuki M, Fujimura J, Saito M, Koh K, Kikuchi A, et al. L-
Asparaginase-Induced Pancreatic Injury is Associated with an Imbalance in Plasma
Amino Acid Levels. Drugs in R&D. 2012;12(2):49-55 10.2165/11632990-
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339. Tokimasa S, Yamato K. Does octreotide prevent l-asparaginase-associated
pancreatitis in children with acute lymphoblastic leukaemia? Br J Haematol.
2012;157(3):381-2.
340. Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, et al.
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Achievements
213
PUBLICATIONS:
2010 Lucas G, Culliford S, Green F, Sidra G, Calvert A, Green A, Harrison P,
Harvey J, Allen D, Smilie D, Masurekar A, Marks D, Russell N, Massey E.
Recipient-derived HPA-1a antibodies: a cause of prolonged thrombocytopenia
after unrelated donor stem cell transplantation. Transfusion, Feb;50(2):334-9.
2010 Parker C, Waters R, Leighton C, Hancock J, Sutton R, Moorman A,
Ancliff P, Morgan M, Masurekar A, Goulden N, Green N, Revesz T,
Darbyshire P, Love S, Saha V. Effect of Mitoxantrone on outcome of children
with first relapse of acute lymphoblastic leukaemia (ALL R3): an open-label
randomised trial. The Lancet, Dec; 376 (9757):2009-17.
2011 Holland M, Castro F, Alexander S, Smith D, Liu J, Walker M, Bitton D,
Mulryan K, Ashton G, Blaylock M, Bagley S, Connolly Y, Bridgeman J, Miller
C, Krishnan S, Dempsey C, Masurekar A, Stern P, Whetton A, Saha V.
RAC2, AEP, and ICAM1 expression are associated with Central Nervous
System (CNS) disease in mouse model of pre-B childhood acute
lymphoblastic leukemia. Blood. Jul;113 (3):638-649.
PEER-REVIEWED ABSTRACTS
2010 Masurekar A, Parker C, Choudhuri S, Leighton C, Hancock J, Sutton
R, Moorman A, Ancliff P, Morgan M, Goulden N, Green N, Revesz T,
Hoogerbrugge P, Darbyshire P, Love S, Saha V. Mitoxantrone
improves the outcome of children with central nervous system involvement at
First relapse of acute lymphoblastic leukemia-results of the international
ALLR3 study. Abstract number 3303. American Society of Hematology
Annual Meeting. For presenting this poster, I received a 2010 ASH Travel
award.
2011 Fong C, Parker C, Hussain A, Liu J, Essink M, Kuehnel H, Lancaster D,
Pridham G, Payne D, O’Horo C, Krishnan S, Hancock J, Goulden N,
Achievements
214
Moorman A, Richards S, Vora A, Saha V, Masurekar A. Intramuscular PEG-
Asparaginase (PEG-ASNase) at 1000 U/m2 achieves adequate trough activity
levels in the majority of patients treated on the UKALL 2003 Childhood Acute
Lymphoblastic Leukaemia (ALL) protocol. Abstract. American Society of
Hematology Annual Meeting. The abstract will be presented by Caroline
Fong a 4th year medical student, who I supervised in the laboratory during her
MRes project. She has received a 2011 ASH Travel Award and her MRes
project report was ranked as first..
CHAPTER IN BOOK
2011 Krishnan S, Masurekar A and Saha V. Identifying targets for new
therapies in children with acute lymphoblastic leukemia. New Agents for the
treatment of acute lymphoblastic leukemia; Saha V, Kearns P (eds.); Springer
Science, (ISBN: 978-1-4419-8458-6)
MANUSCRIPT UNDER PREPARATION
Masurekar A, Parker C, Choudhuri S, Leighton C, Hancock J, Sutton R,
Moorman A, Ancliff P, Morgan M, Goulden N, Green N, Revesz T,
Hoogerbrugge P, Darbyshire P, Love S, Saha V. Mitoxantrone improves the
outcome of children with central nervous system involvement at First relapse
of acute lymphoblastic leukaemia-results of the international ALLR3 study.
Liu J, Masurekar A, Holland M, Johnson S, Krishnan S, Alexander S, Parker
C, Dempsey C, Saha V. Bone Marrow microenvironment regulates drug
resistance in acute lymphoblastic leukaemia via exosomal transfer of miRNA
and regulation of reactive oxygen species.
Masurekar A, Fong C, Parker C, Hussain A, Liu J, Essink M, Kuehnel H,
Lancaster D, Pridham G, Payne D, O’Horo C, Krishnan S, Hancock J,
Goulden N, Moorman A, Richards S, Vora A, Saha V. Intramuscular PEG-
Achievements
215
Asparaginase (PEG-ASNase) at 1000 U/m2 achieves adequate trough activity
levels in the majority of patients treated on the UKALL 2003 Childhood Acute
Lymphoblastic Leukaemia (ALL) protocol.
Masurekar A, Fong C, Parker C, Hussain A, Liu J, Essink M, Knehnel H,
Lancaster D, Pridham G, Payne D, O’Horo C, Krishnan S, Saha V. Role of
Asparaginase in Relapsed Acute lymphoblastic leukaemia (ALL).
Appendix
216
Appendix 1: Reagents Reagents Source
0.1 M DTT, D0632 Sigma Aldrich
10mM AHA A6508 Sigma Aldrich
8 hydroxyquinoline H6878-100G Sigma Aldrich
Acetic Acid 3% with methylene blue stored at RT 07060 Stem Cell Technologies
Aldehyde/Sulfate Latex, 4%w/v 4μm A37304 Invitrogen
Ammonium persulphate 17-1311-01 Amershem Biosciences
Annexin V-FITC, catalog number 556547 BD Pharmingen
Beta mercaptoethanol M7522 Sigma Aldrich
Biotinylate polyclonal goat anti-human Legumain Antibody; 50μg; lyophilized Catalog no BAF2199 R&D Systems
bovine serum albumin Sigma Aldrich
bromophenol blue, B8026 Sigma Aldrich
CelLytic M C2978 Sigma Aldrich
Chloroform:Isoamyl alcohol 24:1 C0549-1QT VWR International Ltd
Complete Protease Inhibitor Cocktail , Catalogue number 11697498001 Roche
DEPC treated water, AM9915G Ambion
Diluent C CGLDIL-6X Sigma Aldrich
DMSO D8418 AnalaR
dNTP Mix 10mM , PCR Grade Catalog no 18427-013 Invitrogen
Ethanol absolute E/0650DF/17 Thermo Scientific
First-Strand Buffer (250mM Tris-HCl, pH 8.3 at R.T; 375 mM KCl; 15mM MgCl2) Invitrogen
FITC Annexin V, Catalogue number 51-65874X BD Biosciences
Foetal bovine serum, S1830500 Biosera
Glycerol 100mL Cat No G5516-100mL Sigma Aldrich
Glycine G7126 Sigma Aldrich
Horseradish Peroxidase conjugated Donkey Anti-goat IgG antibody (sc-2020) Santa Cruz Biotechnology
Hydrochloric acid; 43,557 Sigma Aldrich
Igepal CA-630, Catalogue number 11 697 498 001 Roche
Appendix
217
Iodoacetamide, 1149-56 Sigma Aldrigh
Lymphoprep TM
stored at RT wrapped in foil
Axis-Shield PoCAS
LysoTracker® Green DND-26, Catalogue number L7526 Invitrogen
Methanol M/4000/17 Fisher Chemical
Monoclonal mouse anti human Actin antibody, Catalogue number A5441, Clone AC-15 Sigma-Aldrich
Monoclonal mouse anti human anti-CD3 antibody, Catalogue number MA1-7639, Clone RIV9 Thermo Scientific
Monoclonal mouse anti human anti-MMP2 antibody, Catalogue number 303602, Clone F14P4D3 Biolegend
Monoclonal mouse anti human anti-MMP9 antibody, Catalogue number 635002, Clone F11P2C3 Biolegend
Monoclonal mouse anti human CD19 antibody, Catalogue number 555410, Clone HIB19 BD Pharmigen
Monoclonal mouse anti human CD81 antibody, Catalogue number 349501, Clone 5A6 Biolegend
Monoclonal mouse anti human TSG101 antibody, Catalogue number ab8319, Clone 4A10 Abcam
Monoclonal mouse anti-human Legumain Antibody; 100μg; lyophilized Catalog no MAB2199, Clone 312114 R&D Systems
Monoclonal rabbit anti human CD63 antibody, Catalogue number ab8319, Clone MEM-259 Abcam
PBS tablets P44171 Sigma Aldrich
Phosphate-buffered saline, Mg/Ca free, stored at RT Laboratory Services, PICR
Polyclonal goat anti human CD19 antibody, Catalogue number SC-8498 Santa Cruz Biotechnology
Polyclonal rabbit anti human Cathepsin B antibody, Catalogue number BML-SA361 BioMol Alexis
Polyclonal rabbit anti human CD11a antibody, Catalogue number ab52895 Abcam
Polyclonal rabbit anti human LAMP1 antibody, Catalogue number ab24170, Abcam
Polyclonal rabbit anti human VAMP3 antibody, Catalogue number PA1-767A Thermo Scientific
Pro-Cathepsin B Assay Diluent RD1-34 Concentrate (Part 895865) R&D Systems
Pro-Cathepsin B Calibrator Diluent RD5-34 Concentrate (Part 895828) R&D Systems
Pro-Cathepsin B Color Reagent A ( Part 895000; Stabilized hydrogen peroxide) R&D Systems
Pro-Cathepsin B Color Reagent B (Part 895001; Stabilized chromogen- tetramethylbenzidine). R&D Systems
Pro-Cathepsin B Conjugate (Part 892537) R&D Systems
Pro-Cathepsin B Microplat (Part 892536) coated with mouse monoclonal antibody against pro-Cathepsin B R&D Systems
Pro-Cathepsin B Standard (Part 892538) R&D Systems
Pro-Cathepsin B Wash Buffer Concentrate (Part 895003) R&D Systems
Propan-2-ol p/7490/17 Fisher Chemical
Protein A/G UltraLink resin PN53132 Thermo Scientific
Appendix
218
ProtoGel 30%(w/v) Acrylamide 0.8% (W/v) bis-acrylamide solution AA EC 810 National Diagnostics
Purified recombinant Annexin V, Catalogue number 51-65871A BD Biosciences
Random Hexamers 20μg, Catalogue no C1181 Promega
Reagent A Alkaline copper tartrate solution 500-0113 Biorad
Reagent B Dilute folin reagent 500-0114 Biorad
Reagent diluent concentrate DY995 R&D Systems
Reagent S 500-015 Biorad
RestoreTM
western blot stripping buffer, 46430 Thermo Scientific
RNAase Zap, R2020 Sigma Aldrich
RPMI- 1640 ultra glutamine 5965 Lonza
SDS. Cat number L3771-100g Sigma Aldrich
Sodium Bicarbonate 99.7-100.3% Catalogue number S7277 Sigma Aldrich
Sodium Carbonate Catalogue number S7795 Sigma Aldrich
Sodium chloride, Catalogue number S7653 Sigma-Aldrich
Streptavidin-Horse Radish Peroxidase (HRP) Catalogue no DY998 R&D Systems
Sulphuric acid; 25,810 Sigma Aldrich
Super Signal West Pico (Reagent A&B) 37070 Thermo Scientific
SuperScript™ II Reverse Transcriptase, Conc: 200U/μl, Cataloge No. 18064-071 Invitrogen
SuperSignal®West Dura Extended Duration Enhanced Chemiluminescent Substrate Kit 34076 Thermo Scientific
TaqMan Universal PCR Master Mix Part number 4304437 Applied Biosystems
Trichloroacetic acid, T9159 Sigma Aldrich
Trizma base 1KG, catalogue no T1503 Sigma Aldrich
TRIzol® (store at 4°C) 15596-C18 Invitrogen
TRIzol® LS (store at R.T.) 10296028 Invitrogen
Trypan blue 0.4%, T8154 Sigma
Tween® 20 P1379 Sigma Aldrich
Appendix
219
Appendix 2: Buffers: 1) 0.1% NP-40 lysis buffer/Protease inhibitor solution:
(100ml solution)
Tris base 50mM 0.606g
Sodium chloride 150mM 0.88g
Adjust pH to 8.0 with concentrated HCl
Add distilled/deionised (D/D) water to make up the volume to 100ml
Add 100μl Igepal CA-630
Filter the solution through a 0.2 micron filter
Add 2 Complete Protease Inhibitor Cocktail tablet (Roche), mist to dissolve
and store in 1ml aliquots in microcentrifuge tube at -80°C
2) ELISA coating buffer, 0.05 M Carbonate buffer pH 9.6:
Dissolve 0.795 g of Sodium Bicarbonate and 1.465 g of Sodium Carbonate in
500 ml of D/D water.
Filter the solution through 0.2 micron filter.
Store the buffer in aliquots of 10mls at -20°C
3) Phosphate Buffer Saline (PBS)
Dissolve 5 tablets of PBS in 1 litre of D/D water.
Filter the solution through a 0.2 micron filter
Store the solution at 4°C for a maximum of 2 weeks.
4) ELISA wash Buffer (PBST):
Add 500μl of Tween® 20 to 1 litre of PBS.
Filter the solution through a 0.2 micron filter.
Store the solution at 4°C for up to 2 weeks.
5) ELISA blocking buffer (1% Bovine Serum Albumin PBST)
Add 27.7μl of Tween® 20 to 50ml of D/D water.
Pass through a 0.2 micron filter.
Take 45mls of the above solution and add 5ml of reagent diluent concentrate.
Appendix
220
6) 10% SDS
Dissolve 10 g of SDS in D/D water to make up a total volume of 100ml. Store
at R.T.
7) Resolving gel buffer (1M Tris HCl pH 6.8)
Dissolve 12.1g of Tris base in 80 ml of D/D water.
Adjust pH to 6.8 with concentrated HCl.
Bring up the total volume to 100 ml by adding D/D water.
8) Staking gel buffer (1.5M Tris HCl pH 8.8)
Dissolve 27.23 g of Tris base in 80 ml of D/D water.
Adjust pH to 8.8 with concentrated HCl.
Bring up the total volume to 150 ml by adding D/D water.
Pass through a 0.2 micron filter.
9) Resolving gel (10%)
For a 20 ml volume, add
D/D water 8.00 ml
Protogel 30% bis-acrylamide solution 6.60 ml
Resolving gel buffer 5.00 ml
SDA 10% 0.20 ml
Ammonium persulphate (10%)*+ 0.20 ml
TEMED* 0.008 ml
10) Stacking gel
For a 10 ml volume, add
D/D water 6.80 ml
Protogel 30% bis-acrylamide solution 1.70 ml
Stacking gel buffer 1.25 ml
SDS 10% 0.10 ml
Ammonium persulphate 10%*+ 0.10 ml
TEMED* 0.01 ml
* Added just before use
Appendix
221
+ 0.5g in 5 ml of D/D water, stored at -20°C in 500 μl aliquots.
11) SDS Reducing buffer/Loading buffer
For a 10ml of x5 solution
2M Tris HCl pH 6.8 1.25 ml
10% SDS 1.00 ml
Glycerol 3.00 ml
0.5% Bromophenol blue 0.20 ml
D/D water 5.55 ml
Store at R.T. Add 50 μl of β mercaptoethanol to 950 μl of SDS reducing buffer
just before use. One part of above buffer is mixed with 4 parts of sample
lysates diluted in water to ensure loading of at least 10 μg of protein in 20 μl
volume per well.
12) Running buffer x10
Dissolve in 800 ml of D/D water
Tris base 30.3 g
Glycine 144.0 g
SDS 10.0 g
Bring up the total volume to 1 litre by adding D/D water
Before use, dilute with D/D water to achieve running buffer x1
13) x10 Transfer buffer (Before use dilute 100 mls to 700 ml of D/D water and
add 200 mls of methanol and 3.75ml of 10% SDS)
Dissolve 58.2g to Tris Base and 29.3 g of Glycine in 1L of D/D water. Store at
R.T.
14) Blocking buffer - 5% (w/v) solution of Non-Fat Milk
Dissolve 2.5 g of non fat skimmed mild powder (Marvel, Premier Foods, UK)
into 50 ml wash buffer (PBS-Tween® 20 0.1%).
15) Blocking buffer when using biotinylated antibodies
Dissolve 5 gm of BSA in 100 ml of wash buffer
Appendix
222
16) Blocking buffer when using the LICOR detection system
Dissolve 5 g of BSA into 100 ml of PBS (no tween)
Appendix
223
Title: Appendix 3: ALL2003 and ALLR3 Sample Processing
Document
No:
Version
No:
2 CopyNo/Hold
er:
Issue Date: 22/12/20
08
Review
Date:
Issued by: Rebecca
Cole
INTRODUCTION
Serial research samples are collected at diagnosis and various time-
points throughout treatment:
Samples included peripheral blood-ACD, bone marrow aspirate, and
cerebrospinal fluid.
Processed aliquots included:
Peripheral blood plasma + PIC (PBP+)
Peripheral blood plasma + TRIzol® (PBPT)
Bone marrow plasma + PIC (BMP+)
Bone marrow plasma + TRIzol® (BMPT)
Peripheral blood cells + TRIzol® (PBCT)
Peripheral blood cells + CelLytic M + PIC (PBCLB)
Peripheral blood cells + cryoprotectant (PBC)
Bone marrow cells + TRIzol® (BMCT)
Bone marrow cells + CelLytic M + PIC (BMCLB)
Bone marrow cells + cryoprotectant (BMC)
Cerebrospinal fluid supernatant (CSFS)
Cerebrospinal fluid cells + TRIzol® (CSFCT)
Appendix
224
This SOP applies to samples received with the following proformas:
Asparaginase and AEP Study Form ALL2003 – Samples stored for the
ALL2003 LASP study. Additional samples not currently
required will be banked for future studies.
UKALL2003 Storage Request Form – Samples to be banked for future
studies. These should ONLY be received from the Royal
Manchester Children’s Hospital.
Asparaginase and AEP Study Form ALLR3 – Samples stored for the
ALLR3 LASP study. Additional samples not currently
required will be banked for future studies.
ALLR3 Storage Request Form – Samples to be banked for future
studies.
EsPhALL Storage Request Form – Samples should be sent to The
Patterson Institute. Contact Shekhar or Ashish (see 1.4.2).
Duplicate samples taken on the same day but with different
timepoints must NOT be pooled. This includes UKALL2003 Day
28/ALL2003 LASP Day 30 samples.
Contact information for the Patterson Institute:
Catriona Parker – for issues concerning samples
0161 446 3093
catriona.parker@cancer.org.uk
Ashish Masurekar – for issues concerning the protocol
0161 446 3234
amasurekar@picr.man.ac.uk
Scope
This SOP applies to all personnel processing samples from the
ALL2003, ALLR3, and EsPhALL studies.
Responsibilities
Sample storage and documentation:
All samples awaiting collection at reception should be stored at 4C.
Appendix
225
Sample forms must be completed and filed in external reference order,
together with corresponding proformas.
Samples must be logged in before being stored.
Aliquot ID’s must be scanned into the correct location of the
corresponding box plan.
Aliquots must be correctly located in LIMS.
PBC and BMC aliquots to be left in ‘Mr Frosty’ at -80C overnight
must be located to the correct position within freezer 31 freezer
and then moved to freezer 31 the following day. Aliquots must
be correctly located in LIMS and scanned into the corresponding
box plan.
General guidelines:
Always work in a class II biosafety cabinet.
Observe strict aseptic techniques including wearing gloves and lab
coats. Use dedicated pipettes and filter tips.
Ensure appropriate labelling of all samples.
related documents
LAB/005/5 Sample Receipt, Destruction and Sample Requests (NOT sections
6.1, 6.2 or 6.9).
LAB045/2 Allocating Samples to Locations.
health and safety
Observe standard precautions regarding organic solvent, biohazard,
and sharps disposal.
Observe standard safety precautions when storing samples in liquid
nitrogen.
Equipment/Materials/Reagents
Equipment
Equipment Manufacturer CIGMR ID#
Refrigerated bench-top centrifuge Jouan 275/276
Refrigerated microcentrifuge Sigma 438
Appendix
226
Dedicated 1ml, 100l and 20l pipettes Star Labs
(Nichiryo)
Unknown
Light microscope Olympus 40
‘Mr Frosty’ containing 250ml isopropanol
(refreshed after every 5 uses. Store at R.T
when not in use)
Nalgene n/a
Liquid nitrogen freezer (-192C) MVE Cryogenics 78
-20◦C freezer Liebherr 325
-80◦C freezer Sanyo 298
Refrigerator Wolf Labs (Lech) 470
Materials
Material Supplier Cat#
1.5ml microfuge tubes (sterilised by
autoclaving)
Greiner Bio-One 616201
Sterile 15ml screw-cap (Falcon) tubes Greiner Bio-One 188271
Sterile Cryo.sTM
2ml cryovials Greiner Bio-One 122263
1ml, 200l and 20l filter tips Star Labs (Nichiryo) S1122-1830
S111-1806
S1120-1810
Sterile pastette® pipettes (10 pack) Alpha Labs LW4113
C-Chip disposable haemocytometers Labtech DHC-N01
Biohazard incineration carton Scienceware 132050001
Designated phenol-chloroform (TRIzol) waste
container
VWR Unknown
Ice n/a n/a
Reagents
Reagent Supplier Cat#
Protease inhibitor cocktail in molecular biology
Grade H2O (store in 20µl and 25µl aliquots in
cryovials at-20C). Remaining PIC should be
stored in 2ml aliquots at -20C to prevent
multiple freeze-thaw cycles
Sigma P2714
LymphoprepTM
(store at R.T wrapped in foil) Axis-Shield PoC AS 1053980
Mg/Ca-free Phosphate-buffered saline (store at
R.T)
Invitrogen 20012019
Acetic acid 3% with methylene blue (store at
R.T wrapped in parafilm)
Stem Cell
Technologies
07060
0.4% Trypan blue Sigma
T8154
TRIzol® LS (store at R.T) Invitrogen
10296-010
CelLytic M (store at 4C) Sigma
C3228
Appendix
227
Cryoprotectant: 10% DMSO 90% foetal calf
Serum (store at -20◦C in 5ml aliquots). Foetal
calf serum should be stored in 45ml aliquots at
-20C
AnalaR
Sigma
103234-L
F9665
Gigasept®FF Schulke & Mayr 19227-A
PROCEDURE
Samples should be KEPT ON ICE throughout.
Sample documentation
Log samples into the file S:/childhood leukaemia/incoming samples.xls
(password ‘chl’). Include the following information:
Week beginning
Date
Study (LASP Diagnostic, LASP F/U, UKALL2003, ALLR3
LASP, ALLR3 Storage, or ESPHALL)
Centre name
Patient initials
External reference
Timepoint
Date sampled
Time arrived
Advance notification (if any)
Any notes (e.g if a follow-up sample arrived by courier
instead of Royal Mail)
Complete the corresponding patient information list (ALL2003 or
ALLR3) at S:/childhood leukaemia/study name patient list.xls
(password ‘chl’) as follows:
New samples: patient initials, trial, trial number (if known),
date sampled, and centre name. Assign the next
consecutive external reference in the format: LA001 for
ALL2003 patients or 001 for ALLR3.
Follow-up samples: date sampled.
Appendix
228
Any issues with the sample/proforma or for samples which
arrive > 48 hours following collection, contact Catriona (see
1.4.1) and make a note on the patient list.
Complete a sample information form. This can be obtained from
L:/CIGMR/project folders/childhood leukaemia/sample
information form.doc. File in the corresponding folder in
external reference order, together with the accompanying
proforma. Include the following information:
Trial
Trial number (if known)
Patient initials
Timepoint
Date sampled
Date of last asparaginase treatment (not necessary for
diagnostic samples or for tissue banking)
External reference (see 7.1.2)
Date of birth (if known)
Date sample arrived and processed
Your initials
Processing samples where ONLY plasma is required
Process as soon as possible. Keep at 4C prior to processing.
Centrifuge the sample tube at 2,000g for 10 minutes at room
temperature to obtain a plasma supernatant (programme 31).
Remove the upper ¾ of the plasma supernatant with a sterile Pastette
and dispense 1ml aliquots into sterile cryovials containing 20 µl
of PIC. Label tubes as follows:
External reference
Sample type (see 1.1.2 for abbreviations)
Date sampled
Login and store samples as per 7.6.
Processing samples where plasma AND cells are required
Appendix
229
Process as soon as possible, however samples may be left up to 48 hours
following collection. Keep horizontally at room temperature prior
to processing.
Centrifuge the sample at 200g for 10 minutes at room temperature to
obtain a plasma supernatant (programme 39 on centrifuge
#276).
Remove the upper ¾ of the plasma supernatant with a sterile Pastette
and dispense into sterile CONICAL-BOTTOMED 1.5ml
microcentrifuge tubes.
Centrifuge plasma in a chilled microcentrifuge at 15,000g for 10
minutes.
Carefully aspirate plasma supernatant into sterile labelled (see 7.2.3)
cryovials as follows:
1 x 300l. Add 1ml TRIzol and vortex mix.
900l aliquots into cryovials containing 20l of PIC.
Keep plasma tubes on ice and proceed to 7.4.
Isolating mononuclear cells (MNCs)
Process immediately following step 7.3.
Add 3-4 ml PBS to the cellular sediment and transfer to a fresh sterile
15ml Falcon tube. Add PBS to a final volume of 10ml. Gently
invert to mix.
Using a sterile Pastette, gently layer the 10ml cell suspension over 5ml
of LymphoprepTM
in a 25ml sterile universal.
Centrifuge at 400g for 30 minutes at 20C using a NO BRAKE
deceleration setting (programme 35 centrifuge #276).
Using a sterile Pastette, carefully isolate the MNC layer formed at the
interface of Lymphoprep with PBS and place contents in a
fresh sterile 15ml Falcon tube. Add PBS up to 15ml. Note:
isolate as much of the MNC layer as possible - RBC
contamination is acceptable (make a note on the sample
information form if this is particularly apparent).
Remove 50l from each cell suspension and place into sterile
microcentrifuge tubes.
Appendix
230
Centrifuge the Falcon tubes containing cell suspensions at 400g for 10
minutes at 20C (programme 36).
Aliquot samples from 7.4.6 into sterile microcentrifuge tubes for cell
counting as follows:
Normal MNC interface (most PB/follow-up BM samples):
Dilute 10l of cell suspension with 10l of 3% acetic acid-
methylene blue for a 1 in 2 dilution
Thick MNC interface (most diagnostic BM samples): Dilute
10l of the cell suspension with 40l of PBS in a sterile
microcentrifuge tube. Further dilute 10l of this sample in
10l of 3% acetic-acid methylene blue for a 1 in 10 dilution
Greater dilutions may be required if the cell count exceeds 50-
60 cells per large square
To assess cell viability, dilute 10l of the cell suspension with 10l of
0.4% Trypan blue in a sterile microcentrifuge tube.
Dispense 10l of the diluted sample from 7.4.8 into chamber A of a
disposable haemocytometer.
Dispense 10l of the diluted sample from 7.4.9 into chamber B of the
same disposable haemocytometer.
Determine the total cell count as follows:
Count the total number of cells within the 4 large squares
of chamber A (red blood cells are lysed by the methylene
blue and so should not be visible)
Divide by 4 to obtain an average count
Multiply by the dilution factor (e.g 2 or 10)
Multiply by the total volume of the cell suspension (usually
15ml)
Multiply by the volume correction constant of 104
Record cell count on the sample information sheet
Determine cell viability as follows:
Count 100 cells within one of the large square of chamber B
– note the number of blue (unviable) cells
Record the percentage of viable cells on the sample
information sheet
Appendix
231
Low viability (<90%) should be recorded in the ‘comments’
section of LIMS during sample login
Cells are processed dependent upon cell count:
Discard the supernatant
Resuspend the cell pellet in the volume of PBS specified in
table 1
Split cell suspension into sterile 15ml Falcon tubes as
described in table 1
Note: Cells extracted from the final follow-up sample
(intrathecal MTX) for the ALL2003/ALLR3 LASP studies
should ONLY be stored in cryoprotectant
Split cell suspension as follows:
Cell Count Volume of PBS to
resuspend pellet in
Cells +
TRIzol®
Cells +
CelLytic M
Cells +
cryoprotectant
10×106 1ml 1 x 1ml (10x10
6)
10-20×106 2ml 1 x 1ml (10x10
6) 1 x 1ml (10x10
6)
>20×106 10x10
6 cells/ml 1 x 1ml (10x10
6) 1 x 1ml (10x10
6) 1 x Xml
Table 1
Centrifuge the cell suspensions at 400g for 10 minutes at 20C
(programme 36).
Discard the supernatant, leaving behind a small volume (≤150l).
Gently resuspend the pellet with a sterile Pastette.
Process aliquots as follows:
Cells + TRIzol®
Add 1ml of TRIzol® to the aliquot and vortex mix
Dispense into a sterile labelled (see 7.2.3) cryovial
Cells + CelLytic M
Appendix
232
Add 125l of chilled CelLytic M to the cells and mix well
Dispense into a sterile labelled (see 7.2.3) cryovial containing
25µl of PIC and place immediately on ice
Cells + cryoprotectant
Estimate the volume of cryoprotectant required:
For cell counts <100x106 add 1.5ml per 10-20x10
6 cells
For cell counts >100x106 add 1.5ml per 30-50x10
6 cells
Do not exceed 50x106 cells per 1.5ml of cryoprotectant
Using a sterile pastette add 1 drop of cryoprotecant to the cell
suspension every 10 seconds for 1 minute and then slowly add
the remaining volume along the sides of the tube. Regularly
mix the suspension by gently agitation
Dispense 1ml aliquots into sterile labelled (see 7.2.3) cryovials
Appendix
233
Make a note of cell count per vial on the sample information form.
Login and store samples as per 7.6.
Processing cerebrospinal fluid (CSF)
Process as soon as possible. Keep at 4C prior to processing.
Aspirate contents into a sterile 1.5ml microcentrifuge tube.
Centrifuge in a chilled microcentrifuge at 10,000g for 10 minutes.
Record on the sample information form whether a pellet was seen and
if it was tinged with blood.
Dispense the supernatant into 500μl aliquots in sterile labelled (see
7.2.3) cryovials. If the sample volume exceeds 1ml, dispense
into 1ml aliquots.
Resuspend the pellet in 100μl TRIzol® by vortexing. Should the
solution remain excessively viscous add an additional 400μL of
TRIzol® and vortex. Dispense into a sterile labelled (see 7.2.3)
cryovials.
Login and store samples as per 7.6
Sample login and storage
Samples must be kept on ice and logged in as soon as possible.
Log aliquots into LIMS by following the corresponding workflows:
Peripheral blood – ACD
Bone marrow aspirate
Cerebrospinal fluid
Aliquots should be logged into the relevant study in LIMS:
ALL2003 samples into the L-asparaginase and AEP study
ALLR3 samples into the Childhood leukaemia study
Input the date sampled into LIMS
Input the following information in order into the ‘comments’ section
in LIMS:
Timepoint: ‘Diagnosis’, ‘Day 28’ etc
Appendix
234
Proforma type if sample is for tissue banking:
‘UKALL2003’ or ‘ALLR3 storage’
Date of last asparaginase treatment: ‘Date of last asp
01/01/01’ or ‘Date of last asp unknown’
Samples must be correctly stored as soon as possible:
PBC and BMC + cryoprotecatant: Place in ‘Mr Frosty’
overnight in freezer 10 (-80C). Record the location, both
in LIMS and the corresponding freezer-box plan at
S:/childhood leukaemia/freezer 31 locations/box locations.xls,
where you will store the samples within the liquid nitrogen
freezer 31 (-192C)
The following morning move the samples from ‘Mr Frosty’
to the correct positions within freezer 31
Document history
This is the second version of the SOP.
Document changes
Plasma aliquots without PIC are no longer required as PIC was not
found to have an adverse effect on downstream applications.
Cells containing cryoprotectant are now stored as 1ml instead of
1.5ml aliquots.
Appendix
235
Appendix 4 Leukaemic cell lines
SD1 SUPB15 REH
Type B precursor ALL B precursor AL B precursor ALL
Genetic subtype BCR-ABL1, p190 BCR-ABL1, p190 ETV6-RUNX1
Karyotype human near-
tetraploid karyotype
- 92(88-
92)<4n>XXXX,
t(9;22)(q34;q11)x2 -
carries two
balanced Ph
translocations -
tetraploid derivate
of original diploid
karyotype
Pseudodiploid -
46<2n>XY,
der(1)t(1;1)(p11;q3
1), add(3)(q2?7),
der(4)t(1;4)(p11;q3
5),
t(9;22)(q34;q11),
add(10)(q25),
?del(14)(q23q31),
der(16)t(9;16)(q11;
p13)
46(44-47)<2n>X, -
X, +16, del(3)(p22),
t(4;12;21;16)(q32;p
13;q22;q24.3)-
inv(12)(p13q22),
t(5;12)(q31-
q32;p12),
der(16)t(16;21)(q24
.3;q22) - sideline
with
inv(5)der(5)(p15q31
),+18 - carries
t(12;21) and del(12)
Growth medium RPMI + 10% FCS RPMI + 10% FCS RPMI + 10% FCS
AEP expression Positive Negative Negative
Doubling time 28-32 hours 60-72 hours 60-72 hours
Source CRUK Central
services
DSMZ CRUK Central
services
Appendix
236
Appendix 5: Example of a grid experiment to optimise AEP ELISA:
04/0
3/2
010
05/0
3/2
010
07/0
3/2
010
08/0
3/2
010
09/0
3/2
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2.5
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200µ
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200µ
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time
2hrs
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3hrs
3hrs
3hrs
3hrs
3hrs
3hrs
3hrs
3hrs
3hrs
3hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
2.5
hrs
tem
pR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
T
Pro
tein
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
03/0
3/2
010
reag
en
t1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI1
% B
SA
+ P
BS
+ L
B+
PI1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I1
% B
SA
+ P
BS
+ L
B+
PI
1%
BS
A+
PB
S+
LB
+ P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0
.5%
BS
A +
PB
S+
LB
+P
I0.5
% B
SA
+ P
BS
+ L
B+
PI
0.5
% B
SA
+ P
BS
+ L
B+
PI
vol
100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l
time
1.5
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
tem
p37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
Se
co
nd
ary
03/0
3/2
010
reag
en
t1
% B
SA
+ P
BS
1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS
1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0.5
% B
SA
+ P
BS
0.5
% B
SA
+ P
BS
vol
100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l
co
n4
00
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
200
ng
/ml
200
ng
/ml
400
ng
/ml
400
ng
/ml
200
ng
/ml
200
ng
/ml
400
ng
/ml
200
ng
/ml
400
ng
/ml
100
ng
/ml
400
ng
/ml
200
ng
/ml
100
ng
/ml
400
ng
/ml
300
ng
/ml
200
ng
/ml
100
ng
/ml
400
ng
/ml
300
ng
/ml
300
ng
/ml
100
ng
/ml
100
ng
/ml
100
ng
/ml
100
ng
/ml
100
ng
/ml
100
ng
/ml
100
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
400
ng
/ml
time
2.5
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
1.5
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2h
rs2
hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
2hrs
tem
p37C
37C
37C
37C
37C
37C
37C
Str
ep
HR
P
co
mp
an
yR
&D
R&
DR
&D
DA
KO
DA
KO
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DD
AK
OD
AK
OR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DD
ak
oD
ak
oD
ak
oD
ako
Da
ko
Da
ko
Da
ko
Da
ko
Dak
oD
ak
oD
ak
oD
ako
reag
en
t1
% B
SA
+ P
BS
1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1
% B
SA
+ P
BS
1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S1%
BS
A+
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0
.5%
BS
A +
PB
S0.5
% B
SA
+ P
BS
0.5
% B
SA
+ P
BS
vol
100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l
co
n1to
200
1to
200
1to
500
1to
1000
1to
1000
1 to 2
00
1 to 5
00
1to
200
1to
200
1to
200
1to
350
1 t
o 3
50
1to
500
1to
500
1to
200
1to
200
1to
400
1to
200
1to
1000
1to
800
1to
400
1to
200
1to
1000
1to
800
1to
1000
1to
1000
1to
200
1to
200
1to
400
1to
400
1 to 2
00
1 to 4
00
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
1to
750
time
45m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in3
0m
in3
0m
in3
0m
in3
0m
in3
0m
in3
0m
in3
0m
in3
0m
in3
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in1
hr
1h
r1
hr
1h
r1
hr
1h
r1
hr
1h
r1
hr
1h
r1h
r1
hr
tem
p37C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
37 C
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
Su
bs
tra
te
co
mp
an
yR
&D
R&
DR
&D
Pie
rce
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
R&
DR
&D
PO
D r
och
R&
DP
OD
R&
DP
OD
PO
DP
ierc
eP
ierc
eR
&D
R&
DP
ierc
eP
ierc
eR
&D
R&
DP
ierc
eP
ierc
eR
&D
R&
D
vol
100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100
µl
100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100µ
l100ul
100ul
100ul
100ul
100ul
100ul
100ul
50
µl
100µ
l50µ
l100µ
l50
µl
100
µl
50
µl
100
µl
50µ
l100µ
l50µ
l
time
20m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
0m
in1
7m
in1
7m
in1
7m
in1
7m
in2
5m
in2
5m
in2
5m
in2
5m
in2
5m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in2
0m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in1
5 m
in15 m
in1
5 m
in
tem
p37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37C
37 C
37 C
37 C
37 C
37 C
37 C
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
Sto
p
So
lutio
n2
N H
2S
O4
volu
me
time
Co
mm
en
tsH
igh b
ackg
rou
nd
R&
D s
ubstr
ate
bett
er
tha
n P
ierc
eco
mb
inatio
n o
f 400ng/m
l o
f 2°
with
1 to 2
00 o
f H
RP
giv
e a
hig
h b
ackg
rou
nd
last
co
mb
inatio
n is
the
be
st
2.5
ug
/ml p
rim
ary
, 20
0n
g/m
l se
c,
1to
500
hrp
, r&
D s
ubstr
ate
od v
alu
es o
f b
est
co
mb
inatio
ns a
re h
ighlit
ed in
ita
lics a
nd
bo
ld le
tte
r
stil
l w
ould
like
to
re
duce
th
e b
ackg
rou
nd,
ad
d t
we
en
to
blo
ckin
g b
uff
er,
re
du
ce
prim
ary
co
mp
are
co
lum
n 5
with
9:o
ld p
rim
ary
is p
erf
orm
ing le
ss w
ell
co
mp
are
d t
o n
ew
eve
n t
ho
ug
h it
wa
s s
tore
d a
t -8
0
Pla
nre
du
ce
1°
co
mp
are
old
prim
ary
with
new
one
, co
mp
are
low
er
co
nc o
f n
ew
prim
ary
, co
mp
are
2ug/m
l 1°,
200ng/m
l o
f 2°,
1to
350
/400 H
RP
incre
ase
blo
ckin
gco
mp
are
diffe
ren
t co
nc o
f se
co
nd
ary
and
HR
P vs
incre
ase
pro
t2ug/m
l 1°,
100ng/m
l o
f 2°,
1:2
00 H
RP
redu
ce
2°
redu
ce
str
ep/H
RP
tim
eto
eva
luate
diffe
ren
t co
nce
ntr
atio
ns o
f H
RP
co
mp
are
R&
D s
ubsta
te v
s P
ierc
e
co
mp
are
Dako
HR
P t
o R
&D
Appendix
237