Download - Molecular Haematology
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Outline of topics Molecular biology refresher Molecular biology in haematology Quality control issues
Laboratory layout and equipment Case studies Our wish list
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Molecular biology DNA or RNA: when and why Genes and gene structure Methodology
Nucleic acid extraction DNA electrophoresis Polymerase chain reaction (PCR) Quantitative PCR High resolution melting (HRM) DNA sequencing
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Chromosomes are DNA
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Sugar + Phosphate + Base
Sugar + Phosphate form the backbone
DNA: R = HRNA: R = OH
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Base-pairing
PyrimidinesPurines
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DNA or RNA: when and why
One chromosome = 1 dsDNA molecule
Autosome pair = 2 dsDNA molecules Mitochondrial DNA (many
copies/cell) DNA is more stable for analysis
DNases are easily heat denatured DNA autolysis is minimal under normal
pH & temp RNA is less stable
RNases are ubiquitous & difficult to remove
RNA autolysis in mildly acidic pH
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38,310yo 44,450yo
The three bones from Vindija from which Neandertal DNA was sequenced.
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DNA or RNA: when and why
Gene level: DNA there whether expressed or not RNA copies depend on level of
expression in cell Different cell types: Different
expression levels Messenger RNA is an “edited”
version of DNA Shorter Without introns
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… because of large introns and variable genomic breakpoints
mRNA lacks the introns present in gDNA
Fusion genes are more easily amplified from a shorter sequence
Breakpoint clustering and multiple breakpoint clusters
Sizes of PCR products indicate the types of breakpoints present
However, RNA is less stable than DNA
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Gene structure Promoter Transcription start site Exons Poly adenylation site Open reading frame
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
ORF
Poly A tail7-Methylguanosine cap
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Alternative splicing One gene : many mRNA transcripts: many
protein isoformsDNA
Primary RNA transcript
RNA splicing
Skipped exon Skipped exon
Altered reading frame
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Genomic Breakpoints in BCR-ABL
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Sample preparation Purity of sample
Hypotonic lysis of blood or bone marrow
Ficoll density fractionation Speed & sample turnover RNA stabilization DNA extraction
Quick for robust assays (e.g. blood boiling)
High purity DNA for troublesome assays
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RNA Stabilization• Guanidine isothiocyanate • Trizol reagent
– mono-phasic solution of phenol and guanidine isothiocyanate
• RNA-Later– Ammonium sulphate protein
precipitation– Stabilises RNA at ambient temperature– Suitable for transport with minimal
packaging (post)
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RNA-based assays RNA purification from Trizol lysed
cells Synthesis of copy DNA (cDNA) from
RNA Gene-specific PCR of:
Target gene (leukaemia specific fusion gene)
Control gene (“housekeeping” gene) to assess the quality of the RNA
For quantitative assays: Result = fusion gene copies / control gene
copies
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RNA preparation from cells using
Trizol® reagent
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Inherited disorders Thalassaemias Hereditary Haemochromatosis Factor V Leiden, MTHFR
(Ala677Val), Prothrombin gene 20210 mutation
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Clonal disease Clonal markers
X chromosome inactivation (HUMARA assay) Gene rearrangements
Antigen receptor genes T-cell receptor Immunoglobulin
Abnormal fusion genes Mutations Loss of heterozygosity & uniparental
disomy (acquired)
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Qualitative nested PCRFIP1L1-PDGFRA for CEL
M - N 1 2 3 4 5 6 + + W M - N 1 2 3 4 5 6 + + W10-3 10-4 10-3 10-4
Patient samples Patient samples
Assays run in duplicateSlight differences due to stochastic factors e.g. Pt # 4Controls to give an estimate of assay sensitivity
a b
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Transferrin receptor TFRC
RNA quality controlM - N 1 2 3 4 5 6
Patient samples
Positive TFRC indicates RNA quality is acceptable
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Cycle5 10 15 20 25 30 35 40 45 50
Fluo
resc
ence
110
100
90
80
70
60
50
40
30
20
10
0
Cycle5 10 15 20 25 30 35 40 45 50
Fluo
resc
ence
110
100
90
80
70
60
50
40
30
20
10
0
ABL qPCR RNA quality control
Cycle5 10 15 20 25 30 35 40 45 50
Nor
m. F
luor
o.
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Threshold
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ABL qPCR RNA quality control
Concentration
5104.5104103.510310
CT
30
29.5
29
28.5
28
27.5
27
26.5
26
25.5
25
24.5
24
23.5
23
Cycling A.Green (ABL): R=0.99993 R^2=0.99986 M=-3.529 B=40.693 Efficiency=0.92
Concentration
5104.5104103.510310
CT
30
29.5
29
28.5
28
27.5
27
26.5
26
25.5
25
24.5
24
23.5
23
Cycling A.Green (ABL): R=0.99993 R^2=0.99986 M=-3.529 B=40.693 Eff iciency=0.92
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Reduction of T315I mutation
16/2/2011
25/1/2011
~100% T315I
10%-30% T315I
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Residual disease monitoring
Detection of fusion gene or clonal TCR or IgH Highly sensitive Very early detection of molecular
relapse Quantitation of fusion gene
Allows monitoring of treatment effect Detection of relapse
Chimerism in post-transplant patients
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Sensitivity of TCRg PCR analysis
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Cycle5 10 15 20 25 30 35 40
Nor
m. F
luor
o.
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Threshold
Cycle5 10 15 20 25 30 35 40
Nor
m. F
luor
o.
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Threshold
BCR-ABL qPCR
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BCR-ABL IS calculation
BCR-ABL copies (average of 2 cDNA)
BCR copies (average of 2 cDNA)X laboratory correction factorIS=
Correction factor checked annually against reference laboratory(IMVS Royal Adelaide Hospital)
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Laboratory layout and equipment
Measures to ensure quality
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Laboratory Automation
Reducing manual handling reduces chances of sample
mix-up
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Promega Maxwell 16
Automated DNA extraction systemUses magnetic bead cartridges containing lysis and wash reagents
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CAS-1200 Robot
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Rotor Gene™ 6000 (Corbett Research)36 / 72/ 100 well rotor formatThermal uniformity ±0.01°C, Resolution ±0.02°C, HRM data acquisition (read) rate: 20 reads for each 0.02°C increment
5-20 µl Capacity
15 minutes per run (after amplification)HRM, real time PCR and allelic discrimination (5 colours)
Rotor Gene™ 3000 (Corbett Research)36 / 72 well rotor format
5-20 µl Capacity Real time PCR and allelic discrimination (4 colours)
Real time PCR instrumentation
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LABORATORY FACILITIES
• Laboratories configured to minimise the risk of contamination of samples and reagents by amplified material or other samples in the laboratory
• Minimum Standards• Additional Standards for Nested
PCR
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Minimum Standards• area for the extraction of nucleic
acids from samples and for the addition of sample DNA
• dedicated clean area for the preparation of reagents (including dispensing of the master mix)
• a dedicated, contained area for amplification and product detection
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WorkflowReagent
preparation
Detection
Sample preparation
Template addition
Amplification
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Laboratory Layout
1
2
3
Sample preparation
Reagent preparation
Template addition
Amplification
Detection
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Laboratory Layout
1
2
Sample preparation
Reagent preparation
Template addition
Amplification
Detection
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Quality assurance Undertaking a volume of testing that
is sufficient to maintain the knowledge, experience and expertise of staff
Benefits of centralisation versus those of developing local expertise and autonomy
Associations or collaborations between diagnostic laboratories and research laboratories are encouraged for small volume testing
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RCPA QAP programme JAK2 V617F BCR-ABL PML-RARA DNA Chimerism Factor V Leiden, Prothrombin 20210,
MTHFR (A677V) BCL1, BCL2, TCR, IGH Thal a, Thal b Haemochromatosis Cys282Tyr, His63Asp
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Inter-laboratory sample exchanges
Informal regular exchanges of samples with other laboratories
Blinded Comparison of sensitivities
Not generally surveyed by RCPA QAP For establishment of new methods
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Contamination specimen collection or transport handling or testing in the testing or
referring laboratory before nucleic acid detection
during: extraction of nucleic acids from the
sample amplification product detection
by contamination from the reagents used for the test
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Contamination Sources positive samples (cross
contamination); amplified nucleic acid (e.g.
contamination of stock reagents or equipment, or in aerosol droplets);
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Measures to Control Contamination
• the competency of staff at performing laboratory tasks
• the routine use of controls to detect contamination
• Splitting samples• Uracil-N-glycosylase (UNG)• the design of the laboratory
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Uracil-N-glycosylase• If dUTP is used instead of TTP in
PCR• Uracil-N-glycosylase (UNG) cleaves
contaminating PCR products prior to PCR
• Real template lacks dU and therefore is not degraded
• Prevents amplification of minor amounts of contaminating DNA
• Cannot prevent gross contamination
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UNG
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dUTP incorporation
UNG treatment
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Nested PCR• Products from the 1st round of PCR
are used as templates for 2nd round of PCR
• Requires 4th isolated area– Laboratory– Class 2 biosafety cabinet within area 2
• Uracil-N-glycosylase (UNG) :– 1st round: + UNG , - dUTP– 2nd round: - UNG , + dUTP
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Sample processing Hypotonic lysis:
BCR-ABL, AML-ETO, CBFB-MYH11, FIP1L1-PDGFRA, JAK2
Ficoll purification of mononuclear cells: PML-RARA
Ficoll purification of granulocytes: For low level JAK2 V617F
Granulocyte and T-cell isolation: DNA chimerism
DNA-based assays: JAK2, chimerism RNA-based assays: all others
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19/3/2009 51Molecular Haematology
DNA Sequencing Four different fluorophores incorporated
into primer or dideoxy-NTP A dideoxy-NTP terminates strand
extension Cycle sequencing with thermostable DNA
polymerase Four bases electrophoresed in same gel
capillary Multiple capillaries
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19/3/2009 52Molecular Haematology
DNA SequencingATTAGCGCACGCGATATTCCGGGACAT ATGTCCCGGAATATCGCGTG
ATGTCCATGTCCCGGAATATCGCGTGCGCATGTCATGTCCCGGAATATCGCGTGCGCTATGTCCCGGAATATGTCCCGGAATATCGCGTGCGCTAAATGTCCCGGAATATCGCGTGCATGTCCCGATGTCCCGGAATATCATGTATGTCCCGGAATATATGTCCCGGAATATCGCGTGCGCTAATATGTCCCGGAATATCGCATGTCCCGGAATATCGCGTGCGCTAATGTCCCATGTCCCGGAATGTCCCGGAATATCGCGTGCGATGTCCCGGATGTCCCGGAAATGTCCCGGAATATCGATGTCCCGGAATAATGTCCCGGAATATCGCGATGTCCCGGAATATCGCGT
•Random incorporation of ddNTP•Fragments separated by electrophoresis•Fluorescent signals one base apart•Colour indicates sequence of complementary strand
DN
A te
mpl
ate
ATG
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19/3/2009 53Molecular Haematology
Sequencing results
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Somatic cell genetics is complicated by the background of normal cellsand clonal evolution
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Polymerase chain reaction
(PCR)
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DENATURATION PRIMER ANNEALING PRIMER EXTENSION BY POLYMERASE20 to 21 (i.e. 1 copy to 2 copies)
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21 to 22 (i.e. 2 copies to 4 copies)
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22 to 23
(i.e. 4 copies to 8 copies)…and so on,
the number of copies doubling with each cycle
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PCR amplifies a gene of interest
Each cycle of PCR doubles the number of copies of the gene
10 cycles… approx 1000 fold (210) 20 cycles… approx 1,000,000 fold
(220) 30 cycles… approx 1,000,000,000
fold (230) “Invisible” amounts of DNA become
“visible”
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Post-PCR analysisElectrophoresis DNA Sequencing HybridizationRestriction digestionDenaturing HPLCHigh resolution melting
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Loading the samples in the wells of an agarose gel
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Apply voltage to electrophoresis apparatus
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Ethidium bromide stained DNA
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Ethidium bromide stained DNAunder ultraviolet light
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19/3/2009 65Molecular Haematology
Real-time and quantitative PCR
Detection of labelled PCR products while cycling
By using internal standard (gene dilutions) can be used for quantitation
Large variety of detection technologies and instrument platforms
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Common rtPCR Chemistries
SYBR Green ITaqMan (5’-nuclease) probes
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19/3/2009 67Molecular Haematology
dsDNA + Dye + light = Fluorescence
more DNA more incorporated dye more fluorescence
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19/3/2009 68Molecular Haematology
Taqman probe example
GTCGGGTCTTGGGGTCTGGAGCGTTTGGGA...exon 13
TET
AAGACCCGAC
BHQ-1
CAAGCACTAGTCCATCT
Probe is complementary to PDGFRA exon 12/13 junction
TET fluorescence quenched by BHQ-1
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19/3/2009 69Molecular Haematology
5’ nuclease assay
GTCGGGTCTTGGGGTCTGGAGCGTTTGGGA...exon 13
TETBHQ-1 3’-primer
......
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Quantitation by PCR
Determining when to determine how much
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19/3/2009 71Molecular Haematology
Analysing quantitative data
Absolute Quantitation Unknown samples are compared to a
standard curve Standard is a known DNA sample whose
absolute concentration is known Relative Quantitation
Two or more genes are compared to each other; result is a ratio
Endogenous control or a housekeeping gene is compared to a gene of interest
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19/3/2009 72Molecular Haematology
the CT
CT= threshold cycle:the calculated fractional cyclenumber at which the PCR productcrosses a threshold of detection
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Real time quantitative PCR
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. ...
qPCR standard curve
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High Resolution Melt Analysis
Use Saturating dye in PCR
Analysis of fluorescence as amplicon is melted Heteroduplexes and homoduplexes will melt with different profiles
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Molecular diagnostics in the era of targeted
therapies Diagnosis and suitability of targeted
therapy Monitoring of MRD and detection of early
relapse CML, Ph+ ALL, CEL and tyrosine kinase
inhibitors: imatinib, dasatinib, nilotinib, ponatinib
ATRA, arsenic and APL Detection of resistance by mutation
screening (e.g. BCR-ABL T315I, KIT D816V)
Prognostic indicators (e.g. FLT3 ITD & NPM1 mutation)
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Case studies
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Molecular monitoring in APL
a case study
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19/3/2009 79Molecular Haematology
Molecular monitoring in APL
14-D
ec-05
24-M
ar-06
PETHEMAinduction
0.03%
a case study
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19/3/2009 80Molecular Haematology
Molecular monitoring in APL
14-D
ec-05
24-M
ar-06
2-Jul-0
6
10-O
ct-06
18-Ja
n-07
28-A
pr-07
PETHEMAinduction
PETHEMAconsolidation
0.03%neg
startedmaintenance
returned toAustralia
a case study
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19/3/2009 81Molecular Haematology
Molecular monitoring in APL
14-D
ec-05
24-M
ar-06
2-Jul-0
6
10-O
ct-06
18-Ja
n-07
28-A
pr-07
PETHEMAinduction
PETHEMAconsolidation
0.03%0.02%
0.04%neg
startedmaintenance
returned toAustralia
a case study
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19/3/2009 82Molecular Haematology
Molecular monitoring in APL
14-D
ec-05
24-M
ar-06
2-Jul-0
6
10-O
ct-06
18-Ja
n-07
28-A
pr-07
6-Aug-07
PETHEMAinduction
PETHEMAconsolidation
0.03% 1.45%0.02%
0.04%neg
CNSrelapse
startedmaintenance
returned toAustralia
a case study
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19/3/2009 83Molecular Haematology
Molecular monitoring in APL
neg
14-D
ec-05
24-M
ar-06
2-Jul-0
6
10-O
ct-06
18-Ja
n-07
28-A
pr-07
6-Aug-07
14-N
ov-07
22-Feb
-08
PETHEMAinduction
PETHEMAconsolidation
modified CARE+ PBSC harvest
0.03% 1.45%0.02%
0.04%neg
CNSrelapse
startedmaintenance
negnegneg
returned toAustralia
a case study
cyclo-TBIautograft
IT chemo + ATRA+ As2O3
21-Ja
n-09
neg
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Molecular monitoring in
CML2 case studies
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19/3/2009 85Molecular Haematology
0.001
0.01
0.1
1
10
100
1/06/2005
30/11/2005
31/05/2006
29/11/2006
30/05/2007
28/11/2007
28/05/2008
26/11/2008
IS ratio % raw ratio %
Molecular monitoring in CML
2-log
3-log
4-log
IM 400mg/d
IM resistanceBC
R-A
BL/
BC
R (%
)BCR-ABL Quantitative PCR
log reductionfrom std baselineIM 800mg/d
a case study: 1
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19/3/2009 86Molecular Haematology
Mechanisms of Resistance to Imatinib
BCR-ABL dependent
• gene amplification
• kinase domain point mutations
• drug sequestration by1-acid glycoprotein
• OCT-1 mediated druguptake
• MDR1-mediated drugefflux
BCR-ABL independent
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19/3/2009 87Molecular Haematology
base 760: T C
Tyrosine Histidine ( Y253H )codon 253: TAC CAC
Suboptimal response to maximal dose imatinibBCR-ABL : BCR > 0.1 %
750
700 710 720 730
760 780770740
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19/3/2009 88Molecular Haematology
Hughes et al. , 2006Blood 108:28-37.
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Clinical Hematology (eds: Young, Gerson & High; pub: Elsevier). From Tauchi & Ohyashiki: Leuk Res 28[Suppl 1]:S39–S45, 2004, with permission; based on data compiled from Shah, Nicoll, Nagar et al: Cancer Cell 2:117, 2002; and Druker: Semin Hematol 40:50, 2003.
ABL kinase domain
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19/3/2009 90Molecular Haematology
In vitro activity of nilotinib and dasatinib against imatinib-resistant mutations O’Hare et al, Cancer Research 65:4500, 2005
Y253H >5,000 >18 400 27 1.8 3 >5,000 >17 190 27 1.4 2
ABL WT 280 1 15 1 0.6 1 300 1 7 1 0.8 1
T315 I >5,000 >18 >5,000 >333 >10,000 >16,667 >5,000 >17 >5,000 >714 >1,000 >1,250
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19/3/2009 91Molecular Haematology
0.001
0.01
0.1
1
10
100
1/06/2005
30/11/2005
31/05/2006
29/11/2006
30/05/2007
28/11/2007
28/05/2008
26/11/2008
IS ratio % raw ratio %
Molecular monitoring in CML
2-log
3-log
4-log
IM 400mg/d
Das 70mg bd
BC
R-A
BL/
BC
R (%
)BCR-ABL Quantitative PCR
log reductionfrom std baselineIM 800mg/d
lymphadenopathy
a case study: 1
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An answer…Reactive lymphoid hyperplasia
with giant follicles associated with post-therapeutic state of
hematological malignancies: A report of six cases
Masaru Kojima et al. (2006) Leukemia and Lymphoma 47(7):1404
-1406
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19/3/2009 93Molecular Haematology
0.001
0.01
0.1
1
10
100
1/06/2005
30/11/2005
31/05/2006
29/11/2006
30/05/2007
28/11/2007
28/05/2008
26/11/2008
IS ratio % raw ratio %
Molecular monitoring in CMLa case study: 1
2-log
3-log
4-log
IM 400mg/d
Das 70mg bd
IM resistanceBC
R-A
BL/
BC
R (%
)BCR-ABL Quantitative PCR
log reductionfrom std baselineIM 800mg/d
lymphadenopathy
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19/3/2009 94Molecular Haematology
0.01
0.1
1
10
100
BC
R-A
BL/
BC
R (%
)
Molecular monitoring in CMLa case study 2
IM serum trough levels
mutation?No
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19/3/2009 95Molecular Haematology
300n=7
813 ngMedian value
400n=57
1135 ng
600n=21
1709 ng
Daily dose(mg)
Imatinib plasma concentration(ng/mL)
500
4000
3000
2000
1000
0
Figure courtesy of Dr Francois Xavier Mahon (ref. Picard, S., et al., Blood, 2007. 109(8): p. 3496-9).Demonstrating the achievable plasma trough level for imatinib at 3 doses, 300, 400 and 600 mg per day in 75 patients.
270
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19/3/2009 96Molecular Haematology
0.001
0.01
0.1
1
10
100
BC
R-A
BL/
BC
R (%
)
nd
Molecular monitoring in CMLa case study 2
A case of non-compliance
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The molecular lab’s wish list
Blood or bone marrow samples in EDTA Plenty of material (1mL BMA; 9mL PB)
Consider WCC and send more PB if low WCC Unclotted! Mix well and immediately
Pre-treatment specimens Cytogenetics at diagnosis doesn’t tell us
molecular breakpoint of gene rearrangement Assists with future MRD
Tubes labelled with specimen type, collection times, dates, collector, etc.
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The End