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Ana Terron-Kwiatkowski, MRCPath part I course, London 2010

Using the example of chronic myeloid leukaemia, describe the genetic mechanisms underlying development of the disease and how molecular testing can be used to assist with clinical management. Ana Terron-Kwiatkowski, MRCPath part I course, London 2010.

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Ana Terron-Kwiatkowski, MRCPath part I course, London 2010

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  1. Using the example of chronic myeloid leukaemia, describe the genetic mechanisms underlying development of the disease and how molecular testing can be used to assist with clinical management Ana Terron-Kwiatkowski, MRCPath part I course, London 2010

  2. CML: genetic mechanisms, clinical management and molecular testing CML t(9;22)  BCR-ABL  TK activity CP to AP evolution: - differentiation arrest - genomic instability - telomere shortening - loss of tumour supressor function - genes involved in disease progression Clinical management - Imatinib: mechanism of action resistance monitoring response: cytogenetic remission MRD - qRT-PCR - Alternatives: 2nd generation TK inhibitors allogeneic stem-cell transplantation interferon-

  3. Chronic myeloid leukaemia: molecular pathogenesis • Rare disease: incidence 1-2 cases/100,000 people every year • Disease of haemopoietic stem cells: excessive granulocytosis • Due to reciprocal translocation t(9;22)(q34;q11) - Philadelphia chromosome - fusion gene BCR-ABL:  tyrosine kinase activity • CML two phases: - Chronic phase (CP) : mature granulocytes but  myeloid progenitor cells in blood, < 2% blasts in peripheral blood - Accelerated phase (AP) followed by blast crisis (BC) - haematopoietic differentiation arrest  inmature blasts accumulate in bone marrow (BM), >20% blasts in blood

  4. CML disease progression CEBP Leu-zipper transcription factor Role in granulopoiesis GCSFR (myeloid cells) ID1 differentiation arrest  • Supression translation • HNRNPE2 stability (epigenetic mechanism) BCR-ABL activation nucleus  •  MYC • CCND1 (CD34+ cells in AP) self-renewal GMPs LEF1 TCF transcription factors ß catenin interacts with Activating mutations in epithelial cancer: APC, CRC, ovarian, NSC lung

  5. CML disease progression - genomic instability • The mechanisms of surveying the genome for DNA damage and repairing lesions are compromised in CML (similar to other cancers) • Non-random chromosomal abnormalities in CML: • trisomies 8, 19, 21; additional Ph chromosome; iso 17; monosomy 7 • - markers of disease progression - Failure of genome surveillance Inappropriate DNA replication:  deletions/ translocations BRCA1 coordinates DNA repair activity ATM ATR nuclear PK DNA damage ‘sensors’ BCR-ABL In nucleus Binding and downregulation + CHK1-P intra-S-phase cell-cycle checkpoint

  6. CML disease progression - genomic instability DNA-PKcs: key protein in NHEJ DSBs repair  in CD34+ CML deficient in high-grade bladder carcinomas SNPs in KU70+XRCC4 associated with  risk breast cancer TK activity post-transcriptional ubiquitin-proteasomal pathway Deficiency in DNA repair regulates BCR-ABL RAD51 transcription, activation, degradation + STAT5  upregulation RAD51 + caspase 3  blocks RAD51 degradation TK activity  HR DNA repair - rapid but lack of fidelity deficient HR in PALB2 (partner BRCA2) in breast and prostate cancer Deficiency in NER - repair of UV induced DNA lesions Advanced phase CML- resistance to cytotoxic drugs that induce lesions ~ UV Risk factor in early onset lung cancer also in Xeroderma pigmentosa  Expression of DNA polymerase ß involved in BER  low fidelity DNA-repair  genomic instability (mutagenesis in BCR-ABL)

  7. CML disease progression Telomere shortening solid tumours and leukaemias  genomic instability and disease progression (CML: CP  AP)  hTERT in cancers  immortalization of malignant cells ? cause of disease evolution in cancer or ? caused by  turnover of malignant cells BCR-ABL (potent oncogene) post-transcriptional upregulation SET - tumour supressor phosphatase - overexpressed in solid tumours and leukaemias inactivation de-P SH1 Loss of tumour supressor function + PP2A tumour supressor in CML MAPK STAT5 Atk

  8. CML disease progression candidate genes • None of the available treatments have a significant impact on the control or reversal of advanced disease • Microarray technology: identify good prognostic markers of disease progression in CML • Gene expression profiling studies identified candidate genes differentially regulated in CML (CP/AP phases) - Deregulated: transcription factors AML1, AF1Q, ETS2, LYL1, JUNB, FOS genes associated with other malignancies WNT-ß-catenin pathway (activated in CRC) - Upregulated: ABL-related gene (ABL2 or ARG) preferentially expressed antigen of melanoma (PRAME)

  9. CML disease progression candidate genes • Heterogeneity of disease progression - common in human cancers – - All CML patients have equal chance to disease progression - CP to AP disease progression due to deleterious mutations in essential genes - - Individual susceptibility to disease progression determined by differences in gene expression at presentation of CP • Expression of some genes correlated with patient survival

  10. CML therapeutic strategies Specific kinase inhibitors: Imatinib blocks binding of ATP to BCR-ABL - Overall survival with imatinib at 5 years is 89% - 93% imatinib-treated patients remain free from disease progression - 87% treated patients achieve complete cytogenetic response but majority have levels of BCR-ABL detectable by RT-PCR  CML as long term controllable illness - Side effects mild-moderate. Well tolerated, oral therapy Imatinib also inhibits PDGFR and KIT tyrosine kinases (GIST) can be used in BCR-ABL positive ALL and in other translocations

  11. CML therapeutic strategies Mechanisms of imatinib relapse: - Mutations in ABL kinase domain prevent blocking by imatinib (i.e.T315I) - BCR-ABL genomic amplification - Mechanisms independent of BCR-ABL: activation of FRAP1 (mTOR/Akt) pathway, loss of functional p53 SRC activation, clonal changes resulting in aneuploidy Second generation of ABL kinase inhibitors: nilotinib and dasatinib - active against most imatinib-resistant mutants except T315I

  12. CML therapeutic strategies • Allogeneic stem-cell transplantation • - 50% survival at 18 years • - Low % relapses up to 21 years after transplantation • - Disadvantage: mortality and morbidity of procedure 10-70% • - Recommended for high-risk patients with low transplantation risk • Preferred for children with HLA-identical sibling donors • Interferon-alfa • - Overall survival  10 years: 20-53% • - 50% complete cytogenetic remission • Adverse effects • Immunological therapy: induction of T-cell response

  13. CML treatment monitoring response • Response to IM varies considerably in speed and degree among patients • Majority of patients achieve complete HR by 6 months, 65% CCyR (<35% Ph-positive marrow metaphases) after 1 year therapy • A small proportion will eventually progress to advanced-phase disease • Hematological response: every 2 weeks until remission then every 3 months • Cytogenetic response: BM every 6 months until complete response, then every 12 months. Routine cytogenetics to detect new cytogenetic aberrations in Ph-negative cells • Molecular response - checked every 3 months to detect treatment failure or relapse-

  14. Detection of minimal residual disease(MRD) by quantitative RT-PCR of BCR-ABL qRT-PCR by real-time PCR using specific primers and hydrolysis probes for BCR-ABL and control gene (ABL). Ct values proportional to mRNA levels. Quantification using standard curves.

  15. Detection of MRD by qRT-PCR of BCR-ABL Results - ratio of BCR-ABL/control gene transcript numbers on log10 scale x100% (normalization of results from different laboratories) • - log reduction of a standardized baseline • 2-log reduction considered Ph-negative • 3-log reduction (0.1%) considered major molecular response (MMR) • 4-5-log reduction (transcripts not detectable)  complete molecular response (CMR) only achieved in minority of patients • Interpretation • MMR achieved 12-18 months associated with long-term clinical response • For patients in MMR: a 2x mRNA increase may warrant change in therapy • MRD > 2-10% during 1st year imatinib treatment - primary resistance → Monitoring for KD mutations (direct sequencing, pyrosequencing, scanning methods) • 2x BCR-ABL mRNA increase in patients in CCyR → at risk of progressing to AP

  16. References • Goldman J (2007) Blood, 110, 2828-2837 • Hehlmann R et al (2007) Lancet, 370,342-350 • Macdonald D and Cross N (2007), Pathobiology, 74, 81-88 • Melo JV and Barnes D (2007) Nat Rev Cancer, 7, 441-453 • Druker B (2008) Blood, 112, 4808-4817 • Foroni et al (2009) Am J Hematol 84:517-22

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