Molecular Haemato-Oncology at Bristol Genetics Laboratory Kayleigh Templeman .

Molecular Haemato-Oncology at Bristol Genetics Laboratory

Kayleigh Templeman

http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

Condition


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.


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


Test











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.


Condition


Test











Ig/TCR Clonality Assessment


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


Test











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.


0

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10000

15000

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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


Test











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


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.


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


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.


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


Test











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.


120

140

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E S C G T G C T G T G

G:0.0%T:100.0%

5

110

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140


G:100.0%T:0.0%

5

110

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140

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G:89.1%T:10.9%

5

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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


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)


Thank you for listening. Any questions?

MRD team

Dr Jeremy HancockService lead

Paul ArcherLead Technician

Alison Stevens

Adiela Chudley


Molecular Oncology team

Dr Paula Waits (Mat Leave)Jennifer Corfield / Rebecca Wragg

Kayleigh Templeman

([email protected])

Molecular Haemato-Oncology at Bristol Genetics Laboratory Kayleigh Templeman .

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Transcript of Molecular Haemato-Oncology at Bristol Genetics Laboratory Kayleigh Templeman .