imatinib resistance,4 or as a front-line therapy.5,6. As BCR-ABL1 is the ... patients, low levels of residual disease persist below the sen- ... BCR-ABL1 results are usually reported relative to the expres- ... Scientist, Cancer. Molecular ..... tion of minimal residual disease in chronic myeloid leukaemia using nested quantitative ...
MYELOID DISORDERS IN PRACTICE 2011; Vol 5 No 2
Molecular monitoring in chronic myeloid leukaemia Chronic myeloid leukaemia (CML) is a clonal exon 14 of BCR resulting in the production of eidisease of haematopoietic stem cells charac- ther e13a2 or e14a2 BCR-ABL1 transcripts giving terised by a reciprocal translocation between rise to a p210 protein. In the remaining 5% of chromosomes 9 and 22 that results in the cases, variant BCR-ABL1 fusion transcripts can be Philadelphia (Ph) chromosome. This translo- identified that usually involve either fusion of alcation fuses sequences of the BCR gene on chro- ternative BCR or ABL1 exons – for example, e1a2, mosome 22 to the ABL1 gene on chromosome e6a2, e8a2, e19a2 or e13a3 – or, more rarely, in9 to create the BCR-ABL1 oncogene with the re- tronic insertions at the fusion site or breakpoints sultant protein displaying enhanced tyrosine within exons. Characterisation of the diagnostic kinase activity that propagates the CML phe- BCR-ABL1 transcript type is achieved by either sinnotype via numerous deregulated signalling gleplex or multiplex RT-PCR,8–10 and is important pathways involved in proliferation, apoptosis, because the molecular breakpoint type may be associated with certain phenotypic and clinical feacellular adhesion and genomic stability.1 CML has come to be regarded as the paradigm tures. This is also necessary for selection of the for malignancies defined by a unique molecular appropriate primer combinations and probes for event that is targeted by specific tyrosine kinase monitoring residual disease. inhibitor (TKI) therapy; in the first instance by imatinib,2,3 and, subsequently, by second-genera- Molecular monitoring tion TKIs dasatinib and nilotinib in the setting of Historically, cytogenetic analysis has been the imatinib resistance,4 or as a front-line therapy.5,6 gold standard in monitoring treatment response As BCR-ABL1 is the specific gein CML with evaluation of the A conclusive diagnosis netic marker of CML, molecular percentage of residual Ph cells in testing is required in three par- of CML requires either the bone marrow required at ticular situations: at diagnosis to cytogenetic analysis presentation, three months and detect and characterise the mosix months, then every six or molecular testing lecular breakpoint, during treatmonths until a complete cytogement to serially quantify BCR-ABL1 transcript netic response (CCyR) is achieved.11 Evaluation at levels and also for identification of ABL1 kinase yearly intervals post CCyR ensures the detection domain (KD) mutations associated with acquired of a re-emerging Ph clone or the presence of chroresistance and loss of TKI response. mosome abnormalities in a Ph-negative clone. However, in the majority of TKI-treated patients, Molecular diagnosis low levels of residual disease persist below the senA conclusive diagnosis of CML requires either cy- sitivity of cytogenetic analysis, necessitating quantogenetic analysis or molecular testing to identify titative molecular techniques such as real-time either the t(9;22)(q34;q11) and/or the BCR-ABL1 quantitative PCR (RQ-PCR) to evaluate response fusion gene. Conventional cytogenetic techniques and confirm various milestones. are able to demonstrate the Ph chromosome in 90– Although a number of RQ-PCR methodologies 95% of patients, and any other gross chromosomal and platforms exist, all require the isolation of RNA abnormalities if present. Remaining patients may from peripheral blood or bone marrow, reverseharbour a variant translocation that involves three transcription of the RNA into cDNA and subseor more chromosomes or a cryptic translocation quent specific amplification and detection of detectable either by fluorescence in situ hybridisa- BCR-ABL1 and control gene transcripts. BCR-ABL1 tion (FISH) or reverse-transcriptase polymerase results are usually reported relative to the expreschain reaction (RT-PCR). FISH is also able to detect sion of the control gene that is amplified in parallel concomitant deletions of either chromosome 9 to compensate for variations in RNA quantity and and/or 22 that may be of prognostic relevance.7 quality and to calculate the sensitivity of each The molecular consequence of the Ph chromo- measurement. Selection of the appropriate control some is the fusion of 3’ segment of ABL1 to the 5’ gene is essential as similar amplification efficienpart of the BCR gene. In the majority of CML pa- cies, expression levels and stabilities are required to tients ABL1 exon a2 is joined to either exon 13 or those of the abnormally expressed BCR-ABL1.12
Sarah McCarron BSc MSc Senior Clinical Scientist Stephen Langabeer BSc MSc PhD
Principal Clinical Scientist, Cancer Molecular Diagnostics, St James’s Hospital, Dublin, Ireland
MYELOID DISORDERS IN PRACTICE 2011; Vol 5 No 2
The development of a common protocol for BCRABL1 RQ-PCR that included primer/probe combinations and the use of plasmids to construct standard curves allowed a degree of consensus for residual disease monitoring in CML.13 However, several aspects of the testing process such as sample type, sample age, isolation of the appropriate cell population, RNA extraction method, cDNA synthesis, RQ-PCR methodology and platform, construction of standard curves, setting of threshold cycles, use of appropriate controls, data interpretation and reporting of results are all potential sources of variation and require rigorous attention.14 The International Randomized Study of Interferon and STI571 (IRIS) trial proposed a crucial and predictive level of residual disease known as a major molecular response (MMR).15 MMR was defined as a BCR-ABL1 transcript ratio of ≤0.1%, equivalent to a three log reduction from a standardised, baseline level. Subsequent follow-up analysis has confirmed that attainment of this level of residual disease is highly predictive of progression-free survival.3 Such is the utility of BCR-ABL1 RQ-PCR that routine molecular monitoring is now an integral part of CML management and advocated in all patients who have achieved CCyR. Achievement of an MMR at 18 months of imatinib treatment is useful to distinguish between those patients with an optimal or suboptimal overall response who may benefit from an alternative therapy.11 The clinical importance of attainment of MMR emphasises the requirement for accurate molecular analysis in determining patient response to treatment. However, considerable diversity still exists in molecular monitoring techniques and how results are reported, making consistent interpretation of individual patient responses and comparison of data from multicentre clinical trials of newly developed therapeutic agents difficult to ascertain.16 To address these issues, a process for accurate alignment of BCR-ABL1 transcript levels to an international scale (IS) for reporting BCR-ABL1 results has been adopted.17 Harmonisation of RQ-
Key points ● Characterisation of the BCR-ABL1 breakpoint is essential at diagnosis of chronic myeloid leukaemia. ● Real-time quantitative polymerase chain reaction of BCR-ABL1 transcripts is becoming the gold standard approach for monitoring treatment response. ● Identification of ABL1 kinase domain mutations underlying imatinib resistance is of value in predicting efficacy of subsequent second-generation tyrosine kinase inhibitor therapy.
DEPT. OF CLINICAL CYTOGENETICS, ADDENBROOKES HOSPITAL/SCIENCE PHOTO LIBRARY
At diagnosis of chronic myeloid leukaemia, molecular testing can help to identify the BCR-ABL1 rearrangement that is the consequence of the Philadelphia chromosome
PCR methods, to allow an accurate and reproducible IS measurement of an MMR of ≤0.1%, requires sample exchange with an international or national reference laboratory. A laboratory-specific conversion factor (CF) is generated, validated and should be reassessed with any subsequent methodological alterations. The assignment and implementation of laboratory-specific CF requires stringent assay performance characteristics relating to bias and precision,18 but this process is also timeconsuming, complex and expensive. To further propagate harmonised BCR-ABL1 measurement and result reporting on the IS, a reference standard material should ideally be included. Such a material that is directly linked to the IS has recently been established, potentially enabling commercially available, calibrated secondary standards.19
Mutation detection Although the introduction of imatinib has significantly improved overall outcome in CML patients, a significant proportion of patients have primary refractory disease or relapse after achieving an initial good response. The causes of treatment failure are numerous and include non-adherence, poor absorption, reactivity with other medications and reduced plasma protein binding. Resistance may be manifested by reduced influx or increased drug efflux, BCR-ABL1 gene amplification, stem cell quiescence, BCR-ABL- independent mechanisms or by acquired mutations in the ABL1 KD that reduce or inhibit imatinib binding.20 Acquired resistance usually manifests as loss of cytogenetic or molecular response seen as a sustained increase in BCRABL1 transcript level. In these circumstances, ABL1 KD mutation testing is strongly indicated to make a rational choice of therapy such as dose escalation of imatinib, alternative TKI therapy, allogeneic stem cell transplantation (ASCT) or an experimental treatment in a clinical trial. Techniques to identify ABL1 KD mutations include direct sequencing, denaturing high-performance liquid chromatography, pyrosequencing and allele-specific PCR. All
MYELOID DISORDERS IN PRACTICE 2011; Vol 5 No 2
have different sensitivities that will affect detection frequency, but as the impact of low level mutations is uncertain, guidelines recommend the use of direct sequencing for ABL1 KD analysis.21,22 Over 70 different KD mutations have been described; many are rare and their effects may be overcome by escalation of imatinib dose or switching to dasatinib or nilotinib. Mutations in seven codons account for 80–90% of imatinib-resistant mutations and are associated with a differential response to second-generation TKIs. The T315I mutation is associated with pan-TKI resistance, with ASCT or investigational drugs the alternative therapeutic option. V299L, T315A and F317L/V/I/C mutations are more responsive to nilotinib, whereas Y253H, E255K/V and F359V/C/I are more responsive to dasatinib treatment.22
Future developments As residual disease below MMR is observed in a significant proportion of patients on long-term imatinib, on second-generation TKIs and in some cases post ASCT, quantitation below this level is likely to be of clinical relevance.23 Detection and monitoring by RQ-PCR, therefore, requires definition of a complete molecular response (CMR). CMR can be defined as a 4.0, 4.5, or 5.0 log reduction in transcript level, or even undetectable BCR-ABL1 transcripts, but is highly dependent on sampling and the RQ-PCR methodology in determining appropriate control gene levels. This may be particularly relevant in discontinuation studies,24 where only patients with stable CMR are considered for cessation. A recently described method of determining residual disease is nested DNA quantitative PCR, which requires characterisation of the patient-specific, genomic BCR-ABL1 breakpoint.25 This individualised approach is able to detect BCR-ABL1 levels below the sensitivity of RQ-PCR. A further advance with the potential to standardise the quantitation of BCR-ABL1 transcripts would be the introduction of automated, rapid turnaround platforms that could also eliminate the discrepancies of the aforementioned pre-analytical variables. Such systems are already available and may provide an alternative to a manual methodology.26 The occurrence of the disease-characteristic BCR-ABL1 oncogene has driven the development of sensitive methodologies to monitor response to therapy in CML. With increasing knowledge that early molecular responses with TKIs are linked to progression-free survival,27,28 and with the development of an array of novel agents for imatinibresistant disease that will require demonstration of efficacy,29 it is likely that molecular monitoring will play an increasingly important role in the management of CML patients ■
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Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423–1432. 16. Cross NC. Standardisation of molecular monitoring for chronic myeloid leukaemia. Best Pract Res Clin Haematol 2009; 22: 355–365. 17. Hughes T, Deininger M, Hochhaus A et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCRABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37. 18. Branford S, Fletcher L, Cross NC et al. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials. Blood 2008; 112: 3330–3338. 19. White HE, Matejtschuk P, Rigsby P et al. 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