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Identification and RQ-PCR monitoring of CML patients with rare variant BCR-ABL transcripts Chris Bowles West Midlands Regional Genetics Laboratory.

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Presentation on theme: "Identification and RQ-PCR monitoring of CML patients with rare variant BCR-ABL transcripts Chris Bowles West Midlands Regional Genetics Laboratory."— Presentation transcript:

1 Identification and RQ-PCR monitoring of CML patients with rare variant BCR-ABL transcripts Chris Bowles West Midlands Regional Genetics Laboratory

2 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

3 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

4 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 14 Exon 2 Exon 3 Exon 2 Exon 3 BCRABL Each patient has unique genomic breakpoint Use RNA to allow streamlined monitoring

5 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-PCRRQ-PCR

6 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

7 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

8 RT-PCR 243bp 168bp ???? Control Patient 1 Patient 2Patient 3 Patient 4 Patient 5 Negative Marker Chris Bowles WMRGL 3 patients with 243bp band 6 patients with 168bp band + extra band

9 Sequencing of rare variants Sequence 243bp sized band – e14a3 Sequence 168bp sized band – e13a3 Variation of fusion point between patients BCR exon 14ABL exon 3 BCR exon 13ABL exon 3 BCR intron 13ABL intron 2 Chris Bowles WMRGL Sequence additional bands – fusions of BCR intron 13 to ABL intron 2 e14a3e13a3

10 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

11 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

12 Other rare variants BCR exon 13ABL 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

13 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

14 Validation Typical BCR/ABL patient diagnostic mean ratio1.3 Rare variant BCR/ABL diagnostic mean ratio1.4 3.Comparison of diagnostic ratio values Typical BCR/ABL fusion ratio are similar for all diagnosis samples Patient A CytogeneticsRT-PCRRQ-PCR Patient B CytogeneticsRT-PCRRQ-PCR Diagnosis100% MNot done Diagnosis100% MSS +ve1.11376 3 months0% MNot done 3 months84% MSS +ve1.34918 6 months11% MFailed0.023086 months0%wIF, 0%gIFSS +ve0.00555 9 months0% MSS +ve0.000949 monthsFailedN+ve0.00131 12 monthsNot doneNegative012 monthsNot doneN +ve0.00124 15 monthsNot doneNegative015 monthsNot doneN +ve0 18 monthsNot doneNegative018 monthsNot doneN +ve0 2.Comparison with other monitoring methods Chris Bowles WMRGL

15 Retrospective patient monitoring Archive of patient RNA throughout disease Test using new assay Chris Bowles WMRGL 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

16 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

17 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

18 Acknowledgments Jo Mason Mike Griffiths Susanna Akiki Anna Yeung Sarah Whelton Chris Bowles WMRGL

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