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Molecular Haemato-Oncology at Bristol Genetics Laboratory
Kayleigh Templeman
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
Minimal Residual Disease (MRD) analysis
Used to monitor disease levels in children with ALL (acute lymphoblastic leukaemia).
Remission is defined as leukaemic cells being no longer detectable by light microscopy, but there could still be up to 5 x 1010 cells – this is the minimal residual disease.
MRD status following induction therapy is the single most important prognostic indicator in children with ALL.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
It can be assumed that these junctional regions are unique in each lymphoid precursor cell.
In theory, all of a patient’s leukaemic cells originated from a single clone, and therefore all the malignant cells should have identical Ig/TCR rearrangements.
Patient’s diagnostic sample is screened for Immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements.
This gene rearrangement is a normal process in the development of lymphocytes.
Minimal Residual Disease (MRD)
From the diagnosis sample we are aiming to identify 2 MRD markers that can be used to quantitate disease to a level of 1 leukaemic cell in 10,000 normal cells (10-4).
Rearrangements identified are sequenced, and patient-specific primers created that can be used to detect disease in follow-up samples.
Assays are carried out by real-time PCR, using a dilution series created from the patient’s diagnostic DNA, in order to quantitate any disease detected.
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
IgVH mutation testing in CLL
Disease progression is very varied, so prognostic indicators are very important.
IgVH mutational status in the leukaemic clone affects prognosis.- Mutations = 295 month median survival- No mutations = 95 month median survival
(Somatic hypermutation ≥ 2% divergence from germline sequence)
Clonal gene rearrangements are identifed and sequenced using a very similar method to the first stages of MRD analysis.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
Ig/TCR Clonality Assessment
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
0250005000075000
100000125000150000
50 100 150 200 250 300 350 400 450
C H C .1 1 .0 9 8 9 8 IG H B L Y M P H O M A 4 9 .B 0 4 _ 1 1 1 1 0 4 0 9 L 1
Size (nt)
Dye
Sig
nal
246.84
0
50000
100000
150000
200000
250000
50 100 150 200 250 300 350 400 450
P O S C O N IG H B L Y M P H O M A 4 9 .E 0 4 _ 1 1 1 1 0 4 0 9 L 4
Size (nt)
Dye
Sig
nal
285.82
05000
100001500020000
25000
50 100 150 200 250 300 350 400 450
N E G C O N IG H B L Y M P H O M A 4 9 .F 0 4 _ 1 1 1 1 0 4 0 9 L 5
Size (nt)
Dye
Sig
nal
Patient
Requested when there is uncertainty as to whether a lymphoid mass is malignant.
Test detects Ig/TCR gene rearrangements.
A normal, not malignant (polyclonal) lymphoid cell population will contain the whole repertoire of gene rearrangements, and when analysed PCR products will give a polyclonal spread of peaks.
A malignant cell population will be clonal, and produce a single peak.
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
0
10000
20000
30000
40000
100 200 300 400 500 600
G M C .1 1 .1 0 1 7 6 F L T 3 .N P M 1 P C R 8 .A 0 3 _ 1 1 1 1 1 0 1 0 5 I
Size (nt)
Dye
Sig
nal
231.34330.10
FLT-3 and NPM1 testing in AML
An internal tandem duplication (ITD) in the FLT-3 gene is found in ~25% of adult AML and ~15% of childhood AML.
Poor prognosis
Mutations in the NPM1 gene occur in ~35% of AML patients.
Good prognosis.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
0
5000
10000
15000
20000
25000
100 200 300 400 500 600
P O S C O N F L T 3 .N P M 1 P C R 8 .B 0 3 _ 1 1 1 1 1 0 1 0 5 J
Size (nt)
Dye
Sig
nal
231.29
235.53330.05
390.05
0
2500
5000
7500
100 200 300 400 500 600
P O S C O N F L T 3 .N P M 1 P C R 7 .B 1 0 _ 1 1 1 1 0 3 0 9 C Y
Size (nt)
Dye
Sig
nal
231.28
235.46
330.13378.28
05000
100001500020000250003000035000
100 200 300 400 500 600
S N C .1 1 .0 9 6 9 7 F L T 3 .N P M 1 P C R 7 .A 1 0 _ 1 1 1 1 0 3 0 9 C Y
Size (nt)
Dye
Sig
nal
231.28
408.58
Patient 1
Patient 2
Patient 3
Patient 4
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
BCR-ABL1 Qualitative Analysis (RT-PCR)
The BCR-ABL1 fusion gene is formed by a reciprocal translocation between chromosomes 9 (ABL1) and 22 (BCR)
Philadelphia (Ph) chromosome
90% of CML
20% of adult ALL
5% of childhood ALL
1% of childhood AML
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Patient Patient Patient +ve -ve1 2 3 control control
385bp
(b3a2)
BCR-ABL1 Quantitative Analysis (RQ-PCR)
Molecular monitoring of BCR-ABL1 is vital to the management of Ph +ve CML/ALL.
Residual disease monitoring usually commences once a patient is in cytogenetic remission and allows for the assessment of response to treatment and identification of patients at risk of relapse.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Level of BCR-ABL1 normalised to ABL1
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
10
26/0
5/200
9
01/0
7/200
9
02/0
9/200
9
09/0
9/200
9
17/0
3/201
0
15/0
6/201
0
13/0
7/201
0
14/0
9/201
0
28/0
9/201
0
04/0
1/201
1
08/0
3/201
1
24/0
5/201
1
21/0
6/201
1
13/0
9/201
1
Sample Date
Rat
io o
n lo
g s
cale
BCR-ABL1: ABL1ratioSensitivity of detection
Target sensitivity
ABL1 kinase domain (AKD) mutation screening
Recommended when a patient is not optimally responding to treatment, or when there is a loss of response to treatment so disease levels start to rise.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Level of BCR-ABL1 normalised to ABL1
0.00001
0.0001
0.001
0.01
0.1
1
16/07/10 12/08/10 07/10/10 02/12/10 11/08/11 15/09/11
Sample Date
Rat
io o
n lo
g s
cale
BCR-ABL1: ABL1ratioSensitivity of detection
Target sensitivity
Condition
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Test
ALLChildhood Minimal Residual Disease (MRD) analysis
Ph+veBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain (AKD) mutation screening
CLL IgVH mutation testing
Suspect lymphoproliferations
Ig/TCR clonality assessment
AML FLT-3 and NPM1 mutation testing
CMLBCR-ABL1 analysis (Quantitative and qualitative)
ABL1 kinase domain mutation (AKD) screening
MPN JAK2 and MPL mutation testing
JAK2 Val617Phe (V617F) mutation~98% of patients with Polycythaemia Vera (PV)
~50% with Essential Thrombocythaemia (ET) or Idiopathic Myelofibrosis (IMF).
Testing carried out by pyrosequencing.
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
120
140
160
E S C G T G C T G T G
G:0.0%T:100.0%
5
110
120
130
140
E S C G T G C T G T G
G:100.0%T:0.0%
5
110
120
130
140
150
E S C G T G C T G T G
G:89.1%T:10.9%
5
110
120
130
140
150
E S C G T G C T G T G
G:10.3%T:89.7%
5
JAK2 Exon 12 mutation screen
A small proportion of JAK2 V617F –ve patients have been shown to have mutations in exon 12.
Mutations are detected by HRM and then characterised by direct sequencing.
Difference Plot Normalised Melt Curve
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Patient
MPL mutation testing
3-4% of ET patients and 4-8% of IMF patients have mutations within Exon 10 of the MPL gene.
MPL gene encodes thrombopoietin receptor.
Patient – G T mutation at residue 1544 of codon 515 (most common mutation)
TGG TTG = tryptophan leucine (MPLW515L)
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Thank you for listening. Any questions?
MRD team
Dr Jeremy HancockService lead
Paul ArcherLead Technician
Alison Stevens
Adiela Chudley
http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory
Molecular Oncology team
Dr Paula Waits (Mat Leave)Jennifer Corfield / Rebecca Wragg
Kayleigh Templeman