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

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

2. 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

3. Chris Bowles WMRGL 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

4. Chris Bowles WMRGL Genetics of CML • 98% of time exon 13 or 14 of BCR fuses with exon 2 of ABL (e13a2/e14a2) BCR ABL Exon 3 Exon 14 Exon 13 Exon 2 Exon 2 Exon 3 Exon 13 • Each patient has unique genomic breakpoint • Use RNA to allow streamlined monitoring

5. 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

6. 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

7. 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

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

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

10. 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?

11. 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

12. Chris Bowles WMRGL BCR exon 13 ABL exon 2 Other rare variants • 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

13. Chris Bowles WMRGL Validation • Normally ensure quantitative accuracy using plasmid DNA • PCR efficiency, different monitoring methods, diagnostic ratios • 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

14. Chris Bowles WMRGL Validation • Comparison with other monitoring methods • Comparison of diagnostic ratio values • Typical BCR/ABL fusion ratio are similar for all diagnosis samples

15. Chris Bowles WMRGL Retrospective patient monitoring • Archive of patient RNA throughout disease • Test using new assay • 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. Chris Bowles WMRGL Response to imatinib • 1/9 RQ-PCR showed not responding to imatinib Responsive patient Unresponsive patient • Previously unknown level of treatment response • Patient treatment now changed to dasatinib

17. 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.

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