Molecular Detection of BCR/ABL1 for the Diagnosis and Monitoring of CML

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1 Molecular Detection of BCR/ABL1 for the Diagnosis and Monitoring of CML Imran Mirza, MD, MS, FRCPC Pathology & Laboratory Medicine Institute Sheikh Khalifa Medical City, Abu Dhabi, UAE. No relevant conflict of interest.

2 Objectives To review the molecular biology of BCR/ABL1 fusion in CML To describe the various methods used in the diagnosis and monitoring of CML To provide a synopsis of the National Comprehensive Care Network s guidelines (v2.2011) for CML disease monitoring

3 BCR/ABL1

4 CML The Disease Stem cell disease with increased myelopoiesis Annual incidence 1.6/100,000 adults ~15 20% of all adult leukemias Median age at diagnosis: 65 years No geographic or familial predisposition Thriphasic disease: Chronic phase (3-5 years) Accelerated phase (gradual loss of maturation) Blast phase (70% myeloid, 30% lymphoid)

5 CML Pathophysiology Kantarjian H, et al. Blood. 1993; 82:691 Quintás-Cardama A, Cortes J. Blood. 2009; 113:1619

6 CML Philadelphia Chromosome 46,XX,t(9;22)(q34;q11)[20]

7 CML BCR/ABL1 Molecular Fusion - Common e1 e13 e14 e19 M-bcr p210 a1 a2 a3 a11 ABL1 Translocation, transcription, and splicing of mrna BCR DNA e1 e1 e13a2 e14a2 a11 a11 p210 (e13/a2) RNA p210 (e14/a2) 98%

8 CML BCR/ABL1 Molecular Fusions - Rare e1 e6 e13 e14 e19 BCR e1 e1 e1 m-bcr p190 M-bcr p210 a1 a2 a3 a11 e6a2 n-bcr p190 e1 a2 a11 e13a3 e14a3 m-bcr p230 Translocation, transcription, and splicing of mrna e19a2 a11 a11 a11 a11 ABL1 p190 (e1/a2) p190 (e6/a2) p210 (e13/a2) p210 (e14/a2) p230 (e19/a2) 1%

9 Mechanism of Action BCR/ABL1 Alvarez R. Sem Hematol. 2007; 44:S4-S14

10 CML Laboratory Tests for t(9;22) = BCR/ABL1 1. Karyotype (cytogenetics), 20 metaphases 2. Fluorescent in situ hybridization (FISH) 200 cells DNA 3. Qualitative reverse transcriptase PCR (RT-PCR) and Quantitative reverse transcriptase polymerase chain reaction (QPCR) RNA

11 CML Diagnosis CML? CML Cytogenetics BM ~95% +? t(9;22)? acml? CMML? MPN-U? leukemoid ~5% - Molecular Genetics Avoid term Ph-neg CML CML ~2.5% +? BCR/ABL1 ~2.5% - X CML

12 CML RT-PCR in Diagnosis & QPCR in Monitoring RNA RT cdna BCR ABL p210 (e13/a2) p210 (e14/a2) p190 (e1/a2) p230 (e19/a2)

13 CML QPCR in Monitoring BCR - ABL1 Hydrolysis Probe (Taqman) Fluorescence during amplification Hybridization Probe (FRET) + Fluorescence during annealing

14 Real-Time QPCR Quantifies Target DNA Amount of PCR Product 50,000 copies of target DNA 5, Threshold Cycle Number

15 QPCR Quantifies Target DNA Extrapolation from Standard Curve 25 copies of Target DNA Cycle Threshold (Ct) ,000 50,000 Target DNA copy number

16 QPCR Monitoring & Reporting Current State Standardized baseline Median level from 30 CML samples at diagnosis Result As percent ratio of BCR/ABL1 to ABL1, BCR, GUSB Log reduction compared to the standardized baseline Any log-fold change compared to previous result

17 QPCR Monitoring & Reporting Reference Panel To provide accurate and reproducible results aligned to the first WHO International Genetic Reference Panel for quantification of p210 transcripts. Reference Panel for calculation of the Conversion Factor (CF) specific for each laboratory and result reporting on the International Scale (IS). To report a standardized value for the quantitative BCR/ABL1 testing using QPCR. White HE, et al. Blood. 2010; 116

18 CML Diagnosis & Monitoring Modality Sensitivity CBC ~10% [~10-1 ] Cytogenetics ~5% [~10-2 ] FISH ~0.5% [~10-3 ] Quantitative PCR* ~0.001% [~10-5 ] *non-nested; can reach 10-8 with nested RT-PCR

19 CML Therapy Sensitivity of Monitoring Methods / QPCR Radich JP. Blood. 2009; 114:3376

20 CML Comparison of Analytical Methods Parameter Cytogenetics FISH RT-PCR/QPCR Sensitivity, % tumor 5% 1% - 5% Metaphases required Yes No No Specimen BM PB usable PB usable Equivalence of PB and BM results NA Yes Yes Specificity Highest High Low False positive No Yes, cell overlap Yes, contamination False negative Yes, cryptic & 3 way translocations Advantage Detection of other chromosomal abnormalities Extremely rare Yes, a3 and e19a2 - Determine the breakpoints of fusion genes

21 CML Treatment Two major forms of therapy Initial therapy of choice TKI Excellent long term outcome Minimal toxicity 1 Rx goal BCR/ABL1 reduction SCT No longer 1 st line Rx 2 Only treatment that cures CML 3 Major toxicity and mortality 4 BCR/ABL1 negativity 1. cytopenias (~40%), cardiotoxicity,?mutagenicity [Ph(-) clones] 2. Indicated when [i] very young, [ii] TKI failure, [iii] AP and BC yr survival ~65% % mortality even when low risk

22 Tyrosine Kinase Inhibitors (TKI) First Generation (FDA Approved) Imatinib mesylate Second Generation (FDA Approved) Nilotinib Dasatinib Third Generation (Investigational) Bosutinib Ponatinib

23 TKI Mechanism of Action of Imatinib Savage D. N Eng J Med. 2002; 346:683

24 TKI Therapy Response Levels Level of Response Complete hematologic response Minor cytogenetic response Partial cytogenetic response Complete cytogenetic response Major molecular response Complete molecular remission Definition Normal CBC/differential and spleen 35%-95% Ph-positive metaphases 1%-34% Ph-positive metaphases 0% Ph-positive metaphases 3-log reduction of BCR/ABL1 mrna BCR/ABL1 mrna not detected

25 TKI Therapy Response Timeframe Time after diagnosis Failure Suboptimal 3 months No HR < CHR 6 months <CHR No cytogenetic response < PCyR 12 months < PCyR < CCyR 18 months < CCyR < MMR

26 CML TKI Therapy IRIS Trial At 8 years, the estimated overall survival was 85%; when only CML related deaths were considered, estimated overall survival was 93%. Druker BJ, et al. NEJM. 2006; 335:2408 Deininger M, et al. ASH Abstract 1126

27 TKI Monitoring Hematologic & Genetic Testing Intervals CBC (PB) Every 2 weeks CHR Every 3 months Cytogenetics (BM) Every 6 months yearly CCyR FISH (PB) Every 2-3 months?yearly Molecular (PB or BM) CCyR QPCR every 3 months QPCR every 3 months Qualitative RT-PCR MMR Ou J, et al. Am J Hematol. 2008; 83:296

28 Number of leukemic cells BCR/ABL1 Ratio (IS) TKI Therapy Monitoring International Scale (IS) Diagnosis or Hematologic Relapse Complete Cytogenetic Response Major Molecular Response Complete Molecular Response

29 CML Preanalytical Issues of PCR Testing PB or BM, not to interchange between specimen types 5-10 ml of PB, WBC count important (>1-2 x 10 7 /L) EDTA samples to be submitted on wet ice or at 4 o C CLSI Guideline MM13-A, RNA should be extracted within 4 hours of sample procurement CLSI Guideline MM13-A, Disclaimer if >4 hours in transit, but on ice Cancellation - if >8 hours in transit OR not on ice

30 CML Sample Tempus Blood RNA Tubes Available from Applied Biosystems; used at SKMC The tubes are designed for immediate lysis of blood cells and stabilization of RNA in a single step Freeze global gene expression profiles for up to five days at room temperature, seven days at 4 C

31 CML Analytical Issues of PCR Testing Avoid Ficoll to enrich mononculear cells RNA extraction: all methods acceptable RT enzyme: Superscript or MMLV RT primers: Random hexamers Probe design: hydrolysis or hybridization Instrument: ABI, Light Cycler, etc. Calibration standards: serial dilutions, plasmid DNA QC Sample: High & low, should mimic primary material Ou J, et al. Am J Hematol. 2008; 83:296

32 TKI Mechanisms of Resistance Apperley J. Lancet Oncol. 2007; 8:1018 T315I Y253F/H E255D/K/R/V M351T G250A/E F359C/L/V H396P/R

33 TKI Mutational Analysis Prior to treatment: No During treatment: failure, suboptimal response, sustained increase of BCR/ABL1 transcript level Point mutations occur in <50% of CML-CP patients with imatinib failure Method: Direct sequencing: sensitivity 10% - 25% DHPLC: sensitivity 1% - 10% Small mutated clone is not clinically relevant European Leukemia Net/NCCN Guidelines

34 CML Summary Molecular diagnosis and monitoring of response to CML treatment with TKI is the standard of care Clinicians need to understand and consistently apply molecular monitoring for better patient care Efforts to report QPCR results according to the International Scale using WHO reference standards are ongoing