Identification and RQ-PCR
monitoring of CML patients
with rare variant BCR-ABL
transcripts
Chris Bowles
West Midlands Regional
Genetics Laboratory
Chris Bowles WMRGL
Chronic Myeloid Leukaemia
• Chronic Myeloid Leukaemia is a stem cell cancer
representing about 15-20% of adult leukaemia.
• Chronic phase, which if left untreated will progress into
an accelerated phase followed by blast crisis.
• 750 new cases every year. ELN guidelines: patients
monitored by RQ-PCR every 3 months.
Normal
CML
Chris Bowles WMRGL
Genetics of
CML
• 95% of cases have a common t(9;22)(q34;q11)
chromosome translocation, resulting in an abnormally short
chromosome 22
• Results in the fusion of two genes:
– BCR on chromosome 22
– ABL on chromosome 9
• BCR-ABL fusion found in some ALL – different clinical
course. Poor prognostic indicator
• Fusion protein codes for a constitutively active tyrosine
kinase. High level of successful treatment with drugs such
as imatinib
Chris Bowles WMRGL
Genetics of CML
•
98% of time exon 13 or 14 of BCR fuses with exon 2
of ABL (e13a2/e14a2)
BCR
Exon 13
ABL
Exon 14
Exon 13
Exon 2
Exon 3
Exon 2
Exon 3
•
Each patient has unique genomic breakpoint
•
Use RNA to allow streamlined monitoring
Chris Bowles WMRGL
Monitoring residual disease
• RT-PCR
– Endpoint monitoring to determine whether or not fusion gene
is present
– Can be influenced by quality of sample
• RQ-PCR – Real Time Quantitative
– Measure quantity of gene in exponential phase of PCR
– Calculate ratio of BCR/ABL to housekeeping gene to remove
variation of sample quality
RT-PCR
RQ-PCR
Chris Bowles WMRGL
Rare Variants
• 2% of cases are result of a different BCR-ABL fusion
• e6a2, e8a2, e13a3, e14a3, e19a2
• Can not be monitored by standard RQ-PCR system
– Missing exons where RQ-PCR primers bind
• Non quantitative RT-PCR only
• No comparison between successive samples
• No response data, no early warning of relapse/treatment
failure
Chris Bowles WMRGL
Aims of project
• Characterise rare variants at WMRGL
– 9 CML & 1 ALL BCR-ABL rare variant
patients
– Sequence breakpoints & characterise
gene fusions
• Set up RQ monitoring for rare variants
– Design new assays for monitoring MRD
– Retrospective patient study
Chris Bowles WMRGL
RT-PCR
????
243bp
Marker
Negative
Patient 5
Patient 4
Patient 3
Patient 2
Patient 1
Control
168bp
•
3 patients with 243bp band
•
6 patients with 168bp band + extra band
Chris Bowles WMRGL
Sequencing of rare variants
• Sequence 243bp sized band – e14a3
• Sequence 168bp sized band – e13a3
BCR exon 14
ABL exon 3
e14a3
BCR exon 13
ABL exon 3
e13a3
• Sequence additional bands – fusions of BCR intron 13
to ABL
intron 2 BCR intron 13
ABL intron 2
Variation of fusion
point between
patients
Chris Bowles WMRGL
Origin of additional band
• Genomic contamination of RNA extraction
• Sequence genomic DNA stored on one patient
• Looking at original genomic breakpoint for fusion gene
• Why extra bands in e13a3 patients only?
Chris Bowles WMRGL
RQ-PCR design
e13a2
e14a2
• Currently use primers located in BCR exon 13 and ABL
exon 2
• Deletion of exon 2 prevents use with rare variant patients
• ABL used as housekeeping gene
• Use ABL primers and probes with original BCR/ABL
forward primer
Chris Bowles WMRGL
Other rare variants
•
One additional rare variant – 290bp
•
Sequencing revealed truncated BCR exon 13 with
insertion of 7 bases
BCR exon 13
ABL exon 2
•
Sequence genomic DNA
•
Extra bases from ABL intron at point of fusion in ABL
intron 1
•
Removal of RQ primer site – design new forward
primer specific to this patient
Chris Bowles WMRGL
Validation
• Normally ensure quantitative accuracy using
plasmid DNA
• PCR efficiency, different monitoring methods,
diagnostic ratios
1. Comparison of PCR efficiency
•
Can determine PCR efficiency using RQ-PCR
•
Accurate high and low quantification
•
Similar for comparison between genes
PCR Efficiency
ABL = 93% PCR efficiency
Rare variants = 90% PCR efficiency
Chris Bowles WMRGL
Validation
2. Comparison with other monitoring methods
Patient A
Cytogenetics
RT-PCR
RQ-PCR
Patient B
Cytogenetics
RT-PCR
RQ-PCR
Diagnosis
100% M
Not done
Not done
Diagnosis
100% M
SS +ve
1.11376
3 months
0% M
Not done
Not done
3 months
84% M
SS +ve
1.34918
6 months
11% M
Failed
0.02308
6 months
0%wIF, 0%gIF
SS +ve
0.00555
9 months
0% M
SS +ve
0.00094
9 months
Failed
N+ve
0.00131
12 months
Not done
Negative
0
12 months
Not done
N +ve
0.00124
15 months
Not done
Negative
0
15 months
Not done
N +ve
0
18 months
Not done
Negative
0
18 months
Not done
N +ve
0
3. Comparison of diagnostic ratio values
•
Typical BCR/ABL fusion ratio are similar for all diagnosis
samples
Typical BCR/ABL patient diagnostic mean ratio
1.3
Rare variant BCR/ABL diagnostic mean ratio
1.4
Chris Bowles WMRGL
Retrospective patient
monitoring
• Archive of patient RNA throughout disease
• Test using new assay
Patient A
Patient B
10
Ratio
on
log
scale
1
Ratio
on
log
scale
0.1
1
0.1
0.01
0.01
0.001
0.001
0.0001
0.000001
0.00001
•
•
•
•
01
/1
0/
20
06
01
/0
1/
20
07
01
/0
4/
20
07
01
/0
7/
20
07
01
/1
0/
20
07
01
/0
1/
20
08
01
/0
4/
20
08
01
/0
7/
20
08
01
/1
0/
20
08
01
/0
1/
20
09
01
/0
4/
20
09
01
/0
7/
20
09
01
/1
0/
20
09
0.0001
01
/1
0
01 /20
/0 03
1
01 /20
/0 04
4
01 /20
/0 04
7
01 /20
/1 04
0
01 /20
/0 04
1
01 /20
/0 05
4
01 /20
/0 05
7
01 /20
/1 05
0
01 /20
/0 05
1
01 /20
/0 06
4
01 /20
/0 06
7
01 /20
/1 06
0
01 /20
/0 06
1
01 /20
/0 07
4
01 /20
/0 07
7
01 /20
/1 07
0
01 /20
/0 07
1
01 /20
/0 08
4
01 /20
/0 08
7
01 /20
/1 08
0
01 /20
/0 08
1
01 /20
/0 09
4
01 /20
/0 09
7
01 /20
/1 09
0/
20
09
0.00001
6/9 CML patients had a major molecular response (>3
log reduction from diagnosis)
1/9 only recently diagnosed
1/9 No follow up data, presentation sample had high ratio
(?blast crisis)
BCR/ABL +ve ALL received BMT, with no response
0.000001
0.00001
Responsive patient
• Patient treatment now changed to dasatinib
01
/0
9/
20
09
0.0001
01
/0
6/
20
09
0.00001
01
/0
3/
20
09
0.0001
01
/1
2/
20
08
0.001
01
/0
9/
20
08
0.1
Ratio
on
log
scale
01
/0
6/
20
08
1
01
/0
3/
20
08
Ratio
on
log
scale
01
/1
2/
20
07
Patient A
01
/0
9/
20
07
01
/1
0
01 /20
/0 03
1
01 /20
/0 04
4
01 /20
/0 04
7
01 /20
/1 04
0
01 /20
/0 04
1
01 /20
/0 05
4
01 /20
/0 05
7
01 /20
/1 05
0
01 /20
/0 05
1
01 /20
/0 06
4
01 /20
/0 06
7
01 /20
/1 06
0
01 /20
/0 06
1
01 /20
/0 07
4
01 /20
/0 07
7
01 /20
/1 07
0
01 /20
/0 07
1
01 /20
/0 08
4
01 /20
/0 08
7
01 /20
/1 08
0
01 /20
/0 08
1
01 /20
/0 09
4
01 /20
/0 09
7
01 /20
/1 09
0/
20
09
Chris Bowles WMRGL
Response to imatinib
• 1/9 RQ-PCR showed not responding to imatinib
Patient E
10
1
0.01
0.1
0.01
0.001
Unresponsive patient
• Previously unknown level of treatment response
Chris Bowles WMRGL
Conclusions
• Characterised variants
• Identified additional bands
• Introduced RQ-PCR for rare variant CML
patients
• Effective clinical intervention
• Patients with other rare variants treated on a
case by case basis.
Chris Bowles WMRGL
Acknowledgments
•
•
•
•
•
Jo Mason
Mike Griffiths
Susanna Akiki
Anna Yeung
Sarah Whelton