Identification and RQ-PCR monitoring of CML patients with rare variant BCR-ABL

transcripts

Chris BowlesWest Midlands Regional

Genetics Laboratory

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.

Chris Bowles WMRGL

Normal CML

Genetics ofCML

• 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)

Chris Bowles WMRGL

Exon 13

Exon 13

Exon 14 Exon 2 Exon 3

Exon 2 Exon 3

BCR ABL

•Each patient has unique genomic breakpoint

•Use RNA to allow streamlined monitoring

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

Chris Bowles WMRGL

RT-PCR RQ-PCR

• 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

Rare Variants

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

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168bp

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Chris Bowles WMRGL

•3 patients with 243bp band

•6 patients with 168bp band + extra band

Sequencing of rare variants

•Sequence 243bp sized band – e14a3

•Sequence 168bp sized band – e13a3

Variation of fusion point between patients

BCR exon 14 ABL exon 3 BCR exon 13 ABL exon 3

BCR intron 13 ABL intron 2

Chris Bowles WMRGL

•Sequence additional bands – fusions of BCR intron 13 to ABL

intron 2

e14a3 e13a3

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

• 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

e13a2

e14a2

Chris Bowles WMRGL

Other rare variants

BCR exon 13 ABL exon 2

•One additional rare variant – 290bp

•Sequencing revealed truncated BCR exon 13 with insertion of 7 bases

•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

PCR Efficiency

ABL = 93% PCR efficiency

Rare variants = 90% PCR efficiency

1.Comparison of PCR efficiency

• Can determine PCR efficiency using RQ-PCR

• Accurate high and low quantification

• Similar for comparison between genes

Chris Bowles WMRGL

Validation

Typical BCR/ABL patient diagnostic mean ratio 1.3

Rare variant BCR/ABL diagnostic mean ratio 1.4

3.Comparison of diagnostic ratio values

• Typical BCR/ABL fusion ratio are similar for all diagnosis samples

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

2.Comparison with other monitoring methods

Chris Bowles WMRGL

Retrospective patient monitoring

• Archive of patient RNA throughout disease• Test using new assay

Chris Bowles WMRGL

Patient A

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Patient B

0.00001

0.0001

0.001

0.01

0.1

1

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Ratioonlog

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•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

Response to imatinib

• 1/9 RQ-PCR showed not responding to imatinib

•Previously unknown level of treatment response

•Patient treatment now changed to dasatinib

Chris Bowles WMRGL

Responsive patient Unresponsive patient

Patient A

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Patient E

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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

Chris Bowles WMRGL