Molecular Haematology

Molecular Haematology

10 Mar 2011

Alberto Catalanoemail: [email protected]

Outline of topics Molecular biology refresher Molecular biology in haematology Quality control issues

Laboratory layout and equipment Case studies Our wish list

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

Chromosomes are DNA

Sugar + Phosphate + Base

Sugar + Phosphate form the backbone

DNA: R = HRNA: R = OH

Base-pairing

PyrimidinesPurines

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

38,310yo 44,450yo

The three bones from Vindija from which Neandertal DNA was sequenced.

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

… 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

Gene structure Promoter Transcription start site Exons Poly adenylation site Open reading frame

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

ORF

Poly A tail7-Methylguanosine cap

Alternative splicing One gene : many mRNA transcripts: many

protein isoformsDNA

Primary RNA transcript

RNA splicing

Skipped exon Skipped exon

Altered reading frame

Genomic Breakpoints in BCR-ABL

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

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)

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

RNA preparation from cells using

Trizol® reagent

Inherited disorders Thalassaemias Hereditary Haemochromatosis Factor V Leiden, MTHFR

(Ala677Val), Prothrombin gene 20210 mutation

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)

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

Transferrin receptor TFRC

RNA quality controlM - N 1 2 3 4 5 6

Patient samples

Positive TFRC indicates RNA quality is acceptable

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

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

Reduction of T315I mutation

16/2/2011

25/1/2011

~100% T315I

10%-30% T315I

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

Sensitivity of TCRg PCR analysis

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

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)

Laboratory layout and equipment

Measures to ensure quality

Laboratory Automation

Reducing manual handling reduces chances of sample

mix-up

Promega Maxwell 16

Automated DNA extraction systemUses magnetic bead cartridges containing lysis and wash reagents

CAS-1200 Robot

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

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

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

WorkflowReagent

preparation

Detection

Sample preparation

Template addition

Amplification

Laboratory Layout

1

2

3

Sample preparation

Reagent preparation

Template addition

Amplification

Detection

Laboratory Layout

1

2

Sample preparation

Reagent preparation

Template addition

Amplification

Detection

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

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

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

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

Contamination Sources positive samples (cross

contamination); amplified nucleic acid (e.g.

contamination of stock reagents or equipment, or in aerosol droplets);

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

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

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

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

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


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


Sequencing results

Somatic cell genetics is complicated by the background of normal cellsand clonal evolution

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

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”

Post-PCR analysisElectrophoresis DNA Sequencing HybridizationRestriction digestionDenaturing HPLCHigh resolution melting

Loading the samples in the wells of an agarose gel

Apply voltage to electrophoresis apparatus

Ethidium bromide stained DNA

Ethidium bromide stained DNAunder ultraviolet light


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

Common rtPCR Chemistries

SYBR Green ITaqMan (5’-nuclease) probes


dsDNA + Dye + light = Fluorescence

more DNA more incorporated dye more fluorescence


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


5’ nuclease assay

GTCGGGTCTTGGGGTCTGGAGCGTTTGGGA...exon 13

TETBHQ-1 3’-primer

......

Quantitation by PCR

Determining when to determine how much


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


the CT

CT= threshold cycle:the calculated fractional cyclenumber at which the PCR productcrosses a threshold of detection

Real time quantitative PCR

. ...

qPCR standard curve

High Resolution Melt Analysis

Use Saturating dye in PCR

Analysis of fluorescence as amplicon is melted Heteroduplexes and homoduplexes will melt with different profiles

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)

Case studies

Molecular monitoring in APL

a case study



14-D

ec-05

24-M

ar-06

PETHEMAinduction

0.03%

a case study



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



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


0.03%0.02%

0.04%neg

startedmaintenance


a case study



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


0.03% 1.45%0.02%

0.04%neg

CNSrelapse

startedmaintenance


a case study



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


modified CARE+ PBSC harvest

0.03% 1.45%0.02%

0.04%neg

CNSrelapse

startedmaintenance

negnegneg


a case study

cyclo-TBIautograft

IT chemo + ATRA+ As2O3

21-Ja

n-09

neg

Molecular monitoring in

CML2 case studies


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


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


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


Hughes et al. , 2006Blood 108:28-37.

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


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


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


Molecular monitoring in CML

2-log

3-log

4-log

IM 400mg/d

Das 70mg bd

BC

R-A

BL/

BC

R (%



lymphadenopathy

a case study: 1

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


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


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 (%



lymphadenopathy


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


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


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

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.

The End

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Molecular Haematology