BCR-ABL1 kinase domain mutations: Methodology and clinical evaluation

Transcription

1 AJH Educational Material Test of the Month BCR-ABL1 kinase domain mutations: Methodology and clinical evaluation Mary Alikian, 1 Gareth Gerrard, 1 Papagudi G. Subramanian, 2 Katherine Mudge, 1 Pierre Foskett, 1 Jamshid Sorouri Khorashad, 3 Ai Chiin Lim, 4 David Marin, 1 Dragana Milojkovic, 1 Alistair Reid, 1 Katy Rezvani, 1 John Goldman, 1 Jane Apperley, 1 and Letizia Foroni 1 * The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR-ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a manual" style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D-HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method. Am. J. Hematol. 87: , VC 2011 Wiley Periodicals, Inc. Background Chronic myeloid leukemia Development of chronic myeloid leukemia (CML), the best characterized of myeloproliferative disorders, is associated in over 95% of affected patients with the production of the fusion oncogene BCR-ABL1, the result of the t(9;22)(9q34.1)(22q11.2) chromosomal translocation. The chimeric protein carries a constitutive tyrosine kinase activity with associated activation of downstream mitogenic, proliferative, and anti-apoptotic pathways. Since the early 2000s, tyrosine kinase inhibitors (TKIs), the first of which was imatinib, are the established therapy, with the tyrosine kinase domain (KD) of BCR-ABL1 as the primary target. In brief, the TKIs compete at the BCR-ABL1 ATP binding pocket preventing kinase phosphorylation and thereby shutting down the down-stream signaling pathways [1,2]. A variety of second (dasatinib, nilotinib, and bosutinib) and third generation (ponatinib) tyrosine kinase inhibitors (2GTKIs and 3GTKIs) have been developed with increased pharmacokinetic and clinical efficacy [3,4]. Therapy milestones (complete cytogenetic and major molecular remission) (CCyR; MMR) are achieved sooner with 2GTKIs [3 5] as front line therapy while 3GTKIs have just entered clinical trials for patients resistant or intolerant to other treatments. However, their impact on overall survival (OS) and eventfree survival (EFS) appears not to differ significantly from that achieved with imatinib but longer follow-up is required to evaluate their full clinical impact [6]. It is also well established that 50% of patients receiving 2GTKI after imatinib fail to achieve CCyR [7]. In these challenging cases, bone marrow transplantation may remain an alternative treatment. Resistance to TKI Despite the unprecedented clinical success, a proportion of patients exhibit primary resistance to TKIs whilst others become refractory following an initial response (secondary resistance). While primary resistance is often of unknown etiology, secondary resistance is usually associated with ABL1 tyrosine KD mutations which in advanced phase may be coupled with a progression to BCR-ABL1 independence [5,8 10]. Kinase domain (KD) mutations Acquisition of mutations within the BCR-ABL1 KD is observed in 30 50% of chronic phase patients who develop VC 2011 Wiley Periodicals, Inc. secondary resistance to imatinib [11 19] with an increased frequency of these mutations observed in accelerated phase (AP) and blast crisis (BC) patients [13 20]. Compared to CML, the frequency of these mutations is much higher in patients with Ph positive acute lymphoblastic leukemia (ALL) at the time of relapse (80 90%) [13,21,22]. About 100 different mutations involving different amino acids have been reported in the BCR-ABL1 KD [9,11 13,23 41]. Only subset of these (G250E, Y253H, E255K/V, V299L, T315I, F317L/I, F359V/I/C, H396R, E450G/V, E459K) are associated with TKI failure [9,13,42,43]. It is not yet established whether the mutated clones predate treatment with TKI (as a result of genomic instability) [44 46] and then gain a growth advantage with treatment, on a similar model to antibiotic resistance [9,47 52] or whether they develop de novo during TKI therapy [13]. It is still an open debate whether tyrosine kinase domain (TKD) mutations represent the primary cause of resistance [14,15,17,53,54] or whether they may simply be an indicator of underlying cytogenetic or genomic instability. Yet, it is advisable to carry out mutation analysis prior to any switch in treatment so that the correct TKI is used [9,10,12,17,20,55 59]. Additional Supporting Information may be found in the online version of this article. 1 Imperial Molecular Pathology Laboratory, Imperial College NHS Trust and Academic Science Centre, Hammersmith Hospital, London W12 OHS, United Kingdom; 2 Haematopathology Laboratory, Tata Memorial Hospital, Parel, Mumbai , India; 3 Deininger Laboratory, Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, Utah; 4 The Institute of Cancer Research, Medicine, Sutton, Surrey, SM2 5NG, United Kingdom Conflict of interest: Nothing to report. Contract grant sponsor: Leukaemia and Lymphoma Research (LLR); Contract grant number: *Correspondence to: Letizia Foroni, Imperial Molecular Pathology Laboratory, Imperial college NHS trust and academic science centre, Hammersmith Hospital, London W12 0HS. Tel.: 144-(0) Fax: 144(0) l.foroni@imperial.ac.uk Received for publication 15 November 2011; Revised 24 November 2011; Accepted 1 December 2011 Am. J. Hematol. 87: , Published online 17 December 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: /ajh American Journal of Hematology 298

2 In terms of KD mutation and TKI resistance, currently approved TKIs differ in two aspects; (1) the spectrum of KD mutations they inhibit and (2) the type of de novo KD mutations they induce throughout therapy. Where the relation between KD mutations and imatinib resistance is well documented, there is very little data available about new mutations arising through 2GTKI therapy following imatinib [60 62] with some already arguing that a patient is more likely to have a mutation causing resistance to a 2GTKI if they had a mutation on imatinib [33,63] and no data available about those arising during first line 2GTKI therapy. As second line therapy, it has been shown that dasatinib is usually selective for the V299L, T317L/I and T315A, M351T mutations [8,10,13,58,64,65], whereas nilotinib exerts selection for p-loop mutations such as F359V/C and F311L [8,10,13,33,63,64,66]. In addition, single or concomitant KD mutations have been detected in relapsed patients sequentially treated with imatinib and 2GTKI [65]. Mutations such as T315I show resistance to all types of TK inhibitors [8,10,67,68] which remain an obstacle facing these otherwise successful therapies. Novel promising 3GTKIs (ponatinib, aurora kinase inhibitors, AP24534, etc.) and other agents that inhibit the growth of CML cells in a BCR-ABL1 independent manner (omacetaxine, HDAC inhibitors, heat shock protein inhibitors, inhibitors of the Hedeghog pathway, etc.) with anti T315I activity are already under way in different phases of different clinical trials. TKD alterations other than point mutations Additional molecular mechanisms have been described as possible mechanisms of resistance and include alternative splicing [13,38,69], spliced products with the entire loss of exon 4, 7, 8 [13], InDels [13,69], 35bp intronic insertion at the exon 8/9 junction [13,70 72,85], 42bp Intronic pseudo-exon in-frame insertion [73], and duplications [13]. These possibilities have not been fully investigated and validated and their clinical significance is currently unknown. Detecting kinase domain mutations There is a range of methodologies for TKD mutation analysis. However, several factors affect the choice of method including sensitivity and specificity, the ability to detect novel alterations versus known alterations, quantification and last but not least, turnaround time and costs. In this article, we review the methods employed in our laboratory and list other methods when appropriate. For each methodology, we provide detailed protocols presented in a manual-like style for easy interpretation and application in any laboratory intending to set up these tests, discuss advantages and disadvantages as well as a brief guide to the interpretation of results and clinical applications. Materials and Methods In this section, we describe general guidelines while detailed protocols for each of the methods are available in the Supporting Information accessible online. Methodologies. General guidelines. Sample processing and RNA extraction. RNA preparation is an extremely important step prior to any procedure. For a detailed protocol see a recent previous publication [74]. Hazards, health, and safety precautions. All samples are potentially hazardous and carry the risk of infection. Gloves and lab coats should be worn when handling potentially hazardous samples and reagents. All spillage and breakage must be handled in accordance with the department s decontamination of spillage policy. Prevention of cross contamination. Physical separation of working areas is absolutely essential to prevent cross contamination between pre- and post-polymerase chain reaction (PCR). In particular, when using nested/two round PCR amplification it is very important to carry the work within a type I cabinet and maintain contamination-free technique throughout. The use of pre-sterilized filter tips and plastic-ware is recommended. In our hands we find that there is no need to autoclave test of the month Figure 1. Direct sequencing and pyrosequencing strategy and primers positions. Schematic representation of the BCR-ABL1 fusion gene and its different transcripts showing primers position, size, and location of each amplicon depending on fusion-transcript type and based on 1st and nested/2nd round amplification. (a) e13a2 and e14a2 (major BCR-ABL1 transcripts also known as b2a2/b3a2; p210 protein). Forward primer BCR/Ex13-F and reverse primer ABL1/Ex10-R1 are used to amplify the major fusion transcripts. (b) e1a2 (minor BCR-ABL1 transcript; p190 protein). Forward primer BCR/Ex1-F and reverse primer ABL1/Ex10-R1 are used to amplify the minor fusion transcript. As shown, the reverse primer is common among both transcript types; however, the forward primer varies based on the transcript type. (c) BCR-ABL1 KD: nested primers position and primers used for direct sequencing are illustrated. Internal primers for 2nd PCR direct sequencing are ABL1/Ex4-F and ABL1/Ex10-R2. (d) (Referred to in the Supporting Information) Cycle sequencing is performed using four overlapping primers to ensure the KD is fully screened at least twice so that each base pair substitution observed is confirmed via two independent sequence traces. Primers are: ABL1/Ex4-F, ABL1/ Ex10-R2, ABL1/Ex6-F, ABL1/Ex6,7-R (e) BCR-ABL1 KD: primers and their localization used to amplify this domain for pyrosequencing are shown. Internal primers for 2nd PCR pyrosequencing are P-loop-F and 315-R [biotinylated; btn]. The assays and the SNP primers are in the forward direction, therefore the reverse primer has to be biotinylated on the 5 0 primer end so that it is captured via the Streptavidin coated sepharose beads and made available to the SNP primers as a template during pyrosequencing. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] plastic-ware as long as they are handled while wearing gloves and they are stored in sealed bags or boxes. It is paramount that pipettes are regularly serviced and cleaned as pipettes and tips are common source of contamination. It is important to set up all master mix reactions prior to the handling of patients cdna material and use a set of pipettes specifically assigned to the handling of reagents pre-pcr. Post-PCR analysis (gel electrophoresis and/or setting dilutions or other manipulation of samples) must always be carried out in a different area/room to avoid cross contamination. It is helpful to distinguish each set of PCR (first round, second round, sequencing, pyrosequencing, etc) with different labels or colored tubes. American Journal of Hematology 299

3 TABLE I. Comparison of Methods for BCR-ABL1 Detection Method Sensitivity Advantages Disadvantages Ref. Direct sequencing 15 25% Mutation characterization Lowest sensitivity [13,75] Semi quantitative Bidirectional conformation of mutations Pyrosequencing 5% High sensitivity Short read length [9,17,76] High specificity Prior mutations knowledge is required Quantitative Labor intensive Internal quality and negative controls DHPLC (WAVE) 1% High throughput Unable to characterize the mutations. [9,25,26,37,38,53,77] Cost effective Wild Type DNA Spiking required. Good for large scale studies Prone for contamination due to the additional steps required after the PCR amplification. Requires several PCR amplifications to obtain amplicons of ideal size. Occurrence of non-specific peaks making data interpretation difficult. HRM 5 10% High throughput. Unable to characterize the mutations. [83,84] Cost effective. Optimal function requires small amplicon size which implies several (4 5) PCR reactions. Spikes not required. No additional steps required after the PCR step. Ideal for large scale screening. ASO-PCR % Highly sensitive. Sensitivity could be compromised by closely located mutations. [9,24,38,42,48,78 82,84,87] Quantitative. Requires prior knowledge of mutations. Easy to perform. False positives issue. Labor intensive if screening for multiple mutations Nanofluidics Absolute Absolute digital quantification Very expensive [38,43] Fluidigm BioMark nanofluidic digital array. Real time data. Not much data is yet available. This has proven, in our hands, to be very helpful in organizing the work flow and preventing tube mix up. We have recently moved to use QIAgility (Qiagen) which enables rapid and efficient reaction setup in a wide range of formats and eliminates the tedious manual steps that can be prone to human error. Qualitative assessment of samples prior to mutation analysis. BCR- ABL1 mutation analysis should be carried out after RT-qPCR has been qualitatively and quantitatively assessed. It is also important to establish the correct type of fusion transcript, (qualitative test) (i.e., major, minor, or rare variants). In our laboratory, any sample with less than 10,000 ABL1 molecules (quantitative test) is generally rejected, reextracted, and retested [74]. Also, a minimum of 50 BCR-ABL1 molecules by RT-qPCR are required to perform a mutation analysis test on any sample (25 molecules for direct sequencing analysis and 50 molecules for pyrosequencing, see below). Lower number of transcripts will lead to inadequate PCR amplification for sequencing. The volume of cdna required per reaction is 2.5 ll (containing a pre-determined minimum of 10,000 ABL1 molecules as a measure of cdna quality [74]. It is also important to remember that sensitivity varies for the different techniques and therefore negative results may be due to a mutation present at a level below the threshold of sensitivity of the technique applied, i.e, for pyrosequencing: 5%; direct sequencing: 20%, etc. This will be discussed in more details below. Positive and negative quality control. To comply with standard operating procedure, tests need to be carried out using positive and negative controls for each technique. Negative controls are referred to in the following text as non-template control (NTC). During the second round amplification an aliquot from the first round NTC is also used in addition to a separate NTC for the second round reaction. Positive controls can be used to assure that each technique maintains its sensitivity, but extreme care must be applied to avoid cross contamination. PCR master mixes. PCR master mixes are prepared for each of the different tests. It is advisable to set up master mixes for (small to medium size laboratories) up to 100 or 1,000 reactions (large throughput laboratories). Master mixes will contain all reagents (including forwards and reverse primers) except Taq polymerase. For instance, we may prepare master mixes for 1,000 reactions (in 50 ml conical tubes) and then store aliquots sufficient for reactions at 2208C. This will reduce pipetting errors and inter-experiments variations. Just prior to use, Taq polymerase enzyme is added as required based on the number of reactions needed in each experiment. Preparation of master mixes for different tests is described below for each technique. Direct sequencing. In most laboratories providing tests for the identification of TKD mutations, direct sequencing (by Sanger method) remains the most widely used and recommended technique. However, with an established 15 25% sensitivity, low level mutations can easily be missed. Since the role of very low level mutations (below 20%) is yet to be proven to be of clinical significance in predicting resistance and disease progression, this limitation might not be such a disadvantage [13,75]. The strategy used involves the amplification of the ABL1 KD derived from the fusion BCR-ABL1 allele and must exclude amplification of the normal ABL1 allele. Consequently, the procedure involves a first round amplification starting with the BCR-ABL1 fusion gene (from BCR exon 2toABL1 exon 10) followed by a 2nd round/nested PCR amplification across the TK domain (ABL1 exon 4 to exon 10) (Fig. 1a,b for the major and minor BCR-ABL1 fusion type). It is important to establish the fusion subtype for each patient, in order to use the appropriate primer set [74]. In details, for the major BCR-ABL1 transcript type (referred as b2a2 and b3a2 or e13a2 and e14a2), the first round PCR amplifies from exon13 of the BCR gene to exon 10 of the ABL1 gene (with an amplicon approximately 1,600 bp in size) using primers BCR/Ex13-F and ABL1/Ex10-R1. For other transcript types, (e1a2, e6a2, e8a2, or e19a2), the appropriate forward primers located in the relevant BCR exon must be used. The resulting amplicon template is then subjected to a second round of PCR amplification using internal primers ABL1/Ex4-F and ABL1/ Ex10-R2 (Fig. 1c) to generate a fragment of approximately 863 bp corresponding to the ABL1 KD (exon 4 to exon 10). To verify that the two round amplifications have yielded the correct size product, an aliquot of the 2nd round PCR is subjected to an agarose gel electrophoresis (also to estimate the amount of product obtained) followed by direct sequencing using the Big-Dye V3.1 chemistry (Life Technology, Ltd.) (detailed description is provided in the Supporting Information). 300 American Journal of Hematology

4 Figure 2. D-HPLC strategy and primers positions. A schematic representation of the BCR-ABL1 fusion gene and its different transcripts showing the primers and their locations used to amplify each transcript type during the 1st WAVE PCR reaction and the internal primers and their localization used to amplify the KD within the fusion gene. (a) A schematic representation of the major BCR-ABL1 transcripts e13a2 and e14a2. Forward primer BCR/Ex12-13F and reverse primer ABL1/Ex10-R3 are used to amplify the major transcripts. (b) A schematic representation of the minor BCR-ABL1 transcript e1a2. Forward primer BCR/Ex1-F and reverse primer ABL1/Ex10-R3 are used to amplify the minor transcript. As shown, the reverse primer is common among both transcript types; however, the forward primer varies based on the transcript type. (c) A schematic representation of the BCR-ABL1 KD showing the internal primers and their localization used to amplify this domain. Internal primers for 2nd WAVE PCR Direct sequencing are A-F/A-R (codons ); B-F/B-R (codons ); C-F/C-R (codons ). The use of three sets of internal primers ensures that the entire BCR-ABL KD is divided into three partially overlapping fragments of optimal length for D-HPLC analysis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] test of the month Pyrosequencing. Pyrosequencing is based on sequencing by synthesis and provides information about the status of the nucleotide of interest in real-time. Its unique capacity to measure multi-allelic mutations and allele representation in mixed cell population, in addition to its 5% sensitivity gives it an advantage to quantify the KD mutations occurring in a heterogeneous population of leukemic cells. Another advantage is that it is highly specific and provides built-in quality control and negative control per run. However, prior knowledge of the mutation under investigation is required [9,17,76]. As discussed in the Direct sequencing section, successful amplification of the KD within the BCR-ABL1 fusion gene requires the exclusion of the normal ABL1 allele from the analysis. This is achieved by adopting a nested/2 rounds PCR approach (described earlier). It is important to know the fusion subtype for each patient because different fusion subtypes require the use of different forward primer located in the relevant BCR exon. See Table I in the Supporting Information for primers. The resulting amplicon template is then amplified using the internal primers, P-loop-F and 315-R [btn] (the reverse primer is biotinylated) to generate an amplicon of about 280 bp length that covers the six most clinically relevant mutations we analyze by pyrosequencing. A 6 ll aliquot of the PCR products is subjected to electrophoresis on a 2.0% agarose gel alongside a 1 kb DNA ladder to verify the size of the produced product. The PCR products are then used for pyrosequencing to quantify the mutations. Each patient sample is quantified in duplicates for all six relevant mutations. Single quantifications are not acceptable. It is absolutely vital for the internal reverse primer to be biotinylated at the terminal 5 0 position, because pyrosequencing is dependent upon capturing the single resultant biotin-labeled strand of the PCR products via streptavidin coated sepharose beads and making them available as a template for the sequencing by the (SNP) primer. Given that the reverse primer is biotinylated, the SNP primers have to be in the forward position so that they can anneal to the reverse template. All pyrosequencing assays in current use were designed in the forward (sense) direction, meaning the SNP primers are in the forward direction and the internal reverse primer (315-R [btn]) is biotinylated. A schematic representation of the primers and their locations is illustrated in Fig. 1e. Denaturing High Performance Liquid Chromatography (D-HPLC; Transgenomic WAVE) Denaturing ion-pair reverse phase liquid chromatography (D-HPLC or WAVE) relies upon the differential retention kinetics of heteroduplex and homoduplex DNA species within a cartridge matrix designed to separate DNA fragments according to charge density against an electrolyte gradient. Among its many advantages and applications, D- HPLC is a method that facilitates the high throughput screening for unknown genetic variations without the need for sequencing, at a lower cost and with a sensitivity of approximately 1%. One drawback is that it is unable to characterize any found mutation. While it might be a useful method for large scale screening for research, we believe that due to its mechanical complexity, non-specificity and difficulties experienced in data interpretation, its application in the diagnostic setting may not be ideal when compared to other methods [9,25 27,37,38,53,77]. As for other methodologies, in order to increase the sensitivity and specificity of the BCR-ABL1 KD amplification, a nested PCR approach is adopted (protocol adapted from Ref. 26 to ensure that the non-rearranged ABL1 allele is excluded from the analysis. The first round PCR amplifies the fusion BCR-ABL1 gene using different sets of primers based on the transcript type. The primers P210-F (positioned in exons 12/ 13 of the BCR gene, or it may vary if variant fusion transcripts are involved) and ABL-R (positioned in the exon 10 of the ABL1 gene) are used to amplify the major transcript type, whereas the primers p190-f (positioned in exon 1 of the BCR gene for the fusion transcript e1a2) and ABL1-R are used to amplify the minor transcript type Fig. 2a,b. The second round nested PCR consists of three separate PCR reactions using three different sets of internal primers to generate three partially overlapping fragments of optimal length for DHPLC analysis: ABL1-A (393bp: codons ); ABL1-B (482bp: codons ); ABL1-C (465bp: codons ) (Fig. 2c). Amplicons are checked by gel electrophoresis on a 2% agarose gel. PCR products are then directly analyzed using a Wave 4500HT D-HPLC (Transgenomic). Aliquots of ll (depending on the intensity of the bands visually assessed on the gel electrophoresis) are preheated for 10 min at 968C and then gradually re-annealed for 10 min at room temperature to allow heteroduplex formation and eluted into a DNASep HT column (Transgenomic) at two different temperatures per fragment (details are described in the protocol below). DNA is eluted from the column by a linear acetonitrile gradient in 0.1 mm triethylamine acetate buffer (TEAA; Transgenomic) at a constant flow rate (1.5 ml/min). Gradient is made by mixing buffer A (0.1 mm TEAA) and buffer B (0.1 mm TEAA, 25% Acetonitrile). Eluted amplicons are detected by 260 nm UV absorbance, and these events are visualized as a peak on the chromatogram. The heteroduplex species are eluted faster than the homoduplexes because the base-pair mismatch between the mutated and wild-type alleles causes a conformational change that lowers the charge density across the duplex, leading to a weakened association with the cartridge matrix. Under ideal conditions, the differential elution times of the four potential duplex species leads to the characteristic wave" pattern on the chromatogram. A wild type sample is included as a normal control. The chromatogram from each tested patient is overlaid with the wild type profile, and samples with extra peak(s) or abnormalities in the elution profiles are scored as positive. American Journal of Hematology 301

5 TABLE II. Clinical Relevance of Mutations in CML Patients Indications for mutation analysis Trigger point Comment Recommendation At diagnosis Primary resistance defined as failure according to the ELN criteria Sub-optimal response as defined by ELN Progression to advanced phase Loss of hematological, cytogenetic, or major molecular responses Increase in BCR-ABL transcript numbers whilst maintaining MMR Without the selective pressure of a TKI, it is extremely unusual to find a mutation at diagnosis by conventional methods. Some cases have been reported in which mutations at diagnosis were identified via a specific and highly sensitive PCR [87]. Mutations were only identified in patients presenting with advanced phase disease Failure of imatinib as defined by the ELN criteria is an indication for a change of management, most usually to an alternative TKI. Although mutations are rare in this population, particularly those who meet the criteria for failure after short exposures to TKI, the finding of a mutation will guide the choice of therapy, particularly in those with T315I mutations who might be eligible for stem cell transplantation The state of sub-optimal response describes a number of different response scenarios that most probably do not reflect similar prognoses. Although potentially consistent with prolonged survival, sub-optimal responses are of less clinical value now that 2GTKI are readily available, as most patients and their physicians will opt for a change of drug in an attempt to induce deeper responses as soon as possible. Whenever a change of therapy is considered, mutation analysis should be performed. One area of controversy is the value of mutation analyses in patients who are in CCyR but not in MMR. Although the incidence of mutations in this group is low, once found they are associated with an increased risk of loss of CCyR (Khorashad, de Lavallade). It is unclear however whether finding a mutation in a patient in CCyR would be an indication for a change in treatment. It would be reasonable to quantify the level of the mutation and continue to monitor the patient. A rising level of transcripts and/or an increase in the proportion of the mutated clone might be an indication for a change of therapy. Mutations are most commonly found at a time of disease progression after a period of TKI therapy. More than 50% of patients will have mutations at the time of development or recurrence of blast transformation and this figure is even higher in Ph1 ALL previously treated with TKI (Apperley Lancet Part 1) Acquired (secondary) resistance to TKI is an indication to change therapy. If a further TKI is under consideration then the detection of a mutation might influence the choice of drug. Loss of major molecular response should be confirmed by at least one further RQ-PCR test on a subsequent sample. This is a highly controversial area in that one group have reported that a two fold increase in BCR- ABL transcript level predicted the finding of a mutation but these data have not been substantiated subsequently. The difficulty of using a fold increase is that the significance most probably depends on the absolute starting value. A more rational approach might be to perform a mutation analysis if there are three or more results showing a progressive, albeit slight, increase in levels and other explanations such a s poor compliance or temporary cessation of treatment, have been excluded only for patients presenting in acceleration or blast crisis Routine monitoring of patients beyond 18 months of start of therapy who are in CCyR but not MMR is not generally recommended unless facilities for quantification of the clone are available in selected cases In addition, a mixture of a wild type control and the patient s PCR products (in a 1:1 ratio) is also run to ensure that mutations present in =90% of BCR-ABL1 positive cells cannot escape DHPLC detection. In such cases, the single wave may be mistaken for a wild type in the absence of an internal real unmutated control (for instance a non-cml human cell line). Other Less Frequently Used Techniques We refer to specific references for each of the mutations detection systems described below which are less widely applied in our laboratory. However, some of them such as high resolution melt curve (HRM) analysis are progressively entering the general market and may have more wide application in the future. 1. Allele specific (ASO) PCR based genotyping technique [9,24,38,42,48,78 82,84,87]. This yechnique is widely used by some investigators but may be laborious and its increased sensitivity would identify mutations at extremely low level (<1% or even lower) the clinical significance of which is yet to be established. 2. High Resolution Melt Curve Analysis (HRM) [83,84]. This technique is powerful and relatively fast for high throughput. It is, however, very sensitive to the quality of DNA preparation and often gives rise to false negative tests. 3. Sub cloning and sequencing. Cloning is laborious and a great source of contamination 4. Double-gradient denaturing electrophoresis: in our view far too sensitive and double the problems already linked to a temperamental" procedure. 5. Fluorescence PCR and PNA clamping. 6. Restriction fragment length polymorphism based assay. Of limited applicability and prone to false positive or false negative results. 7. Nanofluidic platform [38,43]: Still extremely expensive for a wide application in a diagnostic laboratory. Mutation Analysis Strategy and Overview We believe that for diagnostic purposes the ABL1 KD needs to be screened to characterize any type of sequence variation and only the clinically relevant mutations need to be quantified. Therefore, applying direct sequencing as a mean for whole KD screening and pyrosequencing as a means for quantifying the clinically relevant mutations is a convenient and practical approach to adopt in a molecular diagnostic lab providing this service, while reserving DHPLC and/or HRM for a large scale screening for research. The use of methods with much higher sensitivity and quantitative ability such as the ASO techniques described in the above paragraph will become meaningful once the low level mutations prove to be of prognosticative value. Figure 4 in the Supporting Information provides an example of a T315I mutation detected in the same patient applying the three main methods used in our lab for KD mutation detection. Table I summarizes some of the overall advantages, disadvantages and provide relevant references for each technology. Clinical Relevance of Mutations in Patients with CML The clinical relevance of mutation analysis has been extensively explored in several recent manuscripts [4,7,31,34,74] and therefore we only include Table II to summarize few guidelines that clinicians may find useful in their practice both to guide when to request mutation analysis and to the interpretation of results. Acknowledgments Authors gratefully acknowledge the members of the minimal residual disease (MRD) team in the Department of Haematology for their scientific contribution. 302 American Journal of Hematology

6 References 1. Alderborn A, Kristofferson A, Hammerling U. Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res 2000;10: Apperley JF. Part I: Mechanisms of resistance to imatinib in chronic myeloid leukaemia. Lancet Oncol 2007;8: Apperley JF. Part II: Management of resistance to imatinib in chronic myeloid leukaemia. Lancet Oncol 2007;8: Baccarani M, Castagnetti F, Gugliotta G, et al. Response definitions and European Leukemianet Management recommendations. Best Pract Res Clin Haematol 2009;22: Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia: An update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009;27: Barthe C, Gharbi MJ, Lagarde V, et al. Mutation in the ATP-binding site of BCR-ABL in a patient with chronic myeloid leukaemia with increasing resistance to STI571. Br J Haematol 2002;119: Branford S, Hughes TP. Mutational analysis in chronic myeloid leukemia: When and what to do? Curr Opin Hematol 2011;18: Branford S, Melo JV, Hughes TP. Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure: Does the BCR-ABL mutation status really matter? Blood 2009;114: Branford S, Rudzki Z, Walsh S, et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99: Branford S, Rudzki Z, Walsh S, et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003;102: Chu S, Xu H, Shah NP, et al. Detection of BCR-ABL kinase mutations in CD341 cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment. Blood 2005;105: Cortes J, Jabbour E, Kantarjian H, et al. Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors. Blood 2007;110: Davies A, Jordanides NE, Giannoudis A, et al. Nilotinib concentration in cell lines and primary CD34(1) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters. Leukemia 2009;23: Deininger MW, Druker BJ. Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol Rev 2003;55: Deininger MW, McGreevey L, Willis S, et al. Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. Leukemia 2004;18: Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood 2008;112: Ernst T, Erben P, Muller MC, et al. Dynamics of BCR-ABL mutated clones prior to hematologic or cytogenetic resistance to imatinib. Haematologica 2008;93: Ernst T, Gruber FX, Pelz-Ackermann O, et al. A co-operative evaluation of different methods of detecting BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on second-line dasatinib or nilotinib therapy after failure of imatinib. Haematologica 2009;94: Foroni L, Wilson G, Gerrard G, et al. Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia. Br J Haematol 2011;153: Fullmer A, Kantarjian H, Cortes J, et al. Nilotinib for the treatment of Philadelphia-chromosome-positive chronic myeloid leukemia. Expert Opin Pharmacother 2010;11: Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293: Gruber FX, Lamark T, Anonli A, et al. Selecting and deselecting imatinib-resistant clones: Observations made by longitudinal, quantitative monitoring of mutated BCR-ABL. Leukemia 2005;19: Guilhot F, Apperley J, Kim DW, et al. Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase. Blood 2007;109: Hochhaus A, Kreil S, Corbin AS, et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002;16: Hofmann WK, Jones LC, Lemp NA, et al. Ph(1) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 2002;99: Hofmann WK, Komor M, Wassmann B, et al. Presence of the BCR-ABL mutation Glu255Lys prior to STI571 (imatinib) treatment in patients with Ph1 acute lymphoblastic leukemia. Blood 2003;102: Hughes T, Branford S. Molecular monitoring of BCR-ABL as a guide to clinical management in chronic myeloid leukaemia. Blood Rev 2006;20: 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 BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108: Hughes T, Saglio G, Branford S, et al. Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase. J Clin Oncol 2009;27: Irving JA, O Brien S, Lennard AL, et al. Use of denaturing HPLC for detection of mutations in the BCR-ABL kinase domain in patients resistant to Imatinib. Clin Chem 2004;50: Jabbour E, Branford S, Saglio G, et al. Practical advice for determining the role of BCR-ABL mutations in guiding tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia. Cancer 2011;117: Jabbour E, Kantarjian H, Jones D, et al. Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia 2006;20: Jiang X, Saw KM, Eaves A, et al. Instability of BCR-ABL gene in primary and cultured chronic myeloid leukemia stem cells. J Natl Cancer Inst 2007;99: Jones D, Kamel-Reid S, Bahler D, et al. Laboratory practice guidelines for detecting and reporting BCR-ABL drug resistance mutations in chronic myelogenous leukemia and acute lymphoblastic leukemia: A report of the Association for Molecular Pathology. J Mol Diagn 2009;11: Jones D, Thomas D, Yin CC, et al. Kinase domain point mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia emerge after therapy with BCR-ABL kinase inhibitors. Cancer 2008;113: Kang HY, Hwang JY, Kim SH, et al. Comparison of allele specific oligonucleotide-polymerase chain reaction and direct sequencing for high throughput screening of ABL kinase domain mutations in chronic myeloid leukemia resistant to imatinib. Haematologica 2006;91: Kantarjian H, Cortes J, Kim DW, et al. Phase 3 study of dasatinib 140 mg once daily versus 70 mg twice daily in patients with chronic myeloid leukemia in accelerated phase resistant or intolerant to imatinib: 15-month median follow-up. Blood 2009;113: Kantarjian H, Shah NP, Hochhaus A, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2010;362: Kantarjian HM, Giles F, Gattermann N, et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance. Blood 2007;110: Khorashad JS, Anand M, Marin D, et al. The presence of a BCR-ABL mutant allele in CML does not always explain clinical resistance to imatinib. Leukemia 2006;20: Khorashad JS, de Lavallade H, Apperley JF, et al. Finding of kinase domain mutations in patients with chronic phase chronic myeloid leukemia responding to imatinib may identify those at high risk of disease progression. J Clin Oncol 2008;26: Khorashad JS, Milojkovic D, Mehta P, et al. In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib. Blood 2008;111: Khorashad JS, Thelwell N, Milojkovic D, et al. A new rapid and sensitive assay for detecting the T315I BCR-ABL kinase domain mutation in chronic myeloid leukaemia. J Clin Pathol 2008;61: Kim TD, Turkmen S, Schwarz M, et al. Impact of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to nilotinib in Philadelphia chromosome-positive chronic myeloid leukemia. Haematologica 2010;95: Lahaye T, Riehm B, Berger U, et al. Response and resistance in 300 patients with BCR-ABL-positive leukemias treated with imatinib in a single center: A 4.5-year follow-up. Cancer 2005;103: Laudadio J, Deininger MW, Mauro MJ, et al. An intron-derived insertion/truncation mutation in the BCR-ABL kinase domain in chronic myeloid leukemia patients undergoing kinase inhibitor therapy. J Mol Diagn 2008;10: Manrique Arechavaleta G, Scholl V, Perez V, et al. Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors. Clin Exp Med 2011;11: Mughal TI, Radich JP, Van Etten RA, et al. Chronic myeloid leukemia 2011: Successes, challenges, and strategies-proceedings of the 5th annual BCR- ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms workshop. Am J Hematol 2011;86: Muller MC, Cortes JE, Kim DW, et al. Dasatinib treatment of chronic-phase chronic myeloid leukemia: Analysis of responses according to preexisting BCR-ABL mutations. Blood 2009;114: Nagar B, Bornmann WG, Pellicena P, et al. Crystal structures of the kinase domain of c-abl in complex with the small molecule inhibitors PD and imatinib (STI-571). Cancer Res 2002;62: Nicolini FE, Chabane K, Tigaud I, et al. BCR-ABL mutant kinetics in CML patients treated with dasatinib. Leuk Res 2007;31: Nicolini FE, Corm S, Le QH, et al. Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: A retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP). Leukemia 2006;20: Nicolini FE, Hayette S, Corm S, et al. Clinical outcome of 27 imatinib mesylate-resistant chronic myelogenous leukemia patients harboring a T315I BCR- ABL mutation. Haematologica 2007;92: American Journal of Hematology 303

7 54. O Hare T, Eide CA, Deininger MW. Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood 2007;110: O Hare T, Zabriskie MS, Eide CA, et al. The BCR-ABL35INS insertion/truncation mutant is kinase-inactive and does not contribute to tyrosine kinase inhibitor resistance in chronic myeloid leukemia. Blood 2011;118: Oehler VG, Qin J, Ramakrishnan R, et al. Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR. Leukemia 2009;23: Pelz-Ackermann O, Cross M, Pfeifer H, et al. Highly sensitive and quantitative detection of BCR-ABL kinase domain mutations by ligation PCR. Leukemia 2008;22: Pfeifer H, Wassmann B, Pavlova A, et al. Kinase domain mutations of BCR- ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph1 ALL). Blood 2007;110: Polakova KM, Lopotova T, Klamova H, et al. High-resolution melt curve analysis: Initial screening for mutations in BCR-ABL kinase domain. Leuk Res 2008;32: Quintas-Cardama A, Kantarjian H, Cortes J. Third-generation tyrosine kinase inhibitors and beyond. Semin Hematol 2010;47: Ray A, Cowan-Jacob SW, Manley PW, et al. Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study. Blood 2007;109: Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004;50: Roche-Lestienne C, Lai JL, Darre S, et al. A mutation conferring resistance to imatinib at the time of diagnosis of chronic myelogenous leukemia. N Engl J Med 2003;348: Roche-Lestienne C, Preudhomme C. Mutations in the ABL kinase domain pre-exist the onset of imatinib treatment. Semin Hematol 2003;40: Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, et al. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002;100: Roumiantsev S, Shah NP, Gorre ME, et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc Natl Acad Sci USA 2002;99: Saglio G, Kim DW, Issaragrisil S, et al. Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med 2010;362: Schnittger S, Bacher U, Dicker F, et al. Associations between imatinib resistance conferring mutations and Philadelphia positive clonal cytogenetic evolution in CML. Genes Chromosomes Cancer 2010;49: Shah NP, Kim DW, Kantarjian H, et al. Potent, transient inhibition of BCR- ABL with dasatinib 100 mg daily achieves rapid and durable cytogenetic responses and high transformation-free survival rates in chronic phase chronic myeloid leukemia patients with resistance, suboptimal response or intolerance to imatinib. Haematologica 2010;95: Shah NP, Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002;2: Shah NP, Skaggs BJ, Branford S, et al. Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency. J Clin Invest 2007;117: Sherbenou DW, Hantschel O, Turaga L, et al. Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia. Leukemia 2008;22: Sorel N, Bonnet ML, Guillier M, et al. Evidence of ABL-kinase domain mutations in highly purified primitive stem cell populations of patients with chronic myelogenous leukemia. Biochem Biophys Res Commun 2004;323: Sorel N, Mayeur-Rousse C, Deverriere S, et al. Comprehensive characterization of a novel intronic pseudo-exon inserted within an e14/a2 BCR-ABL rearrangement in a patient with chronic myeloid leukemia. J Mol Diagn 2010;12: Soverini S, Colarossi S, Gnani A, et al. Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: by the GIMEMA Working Party on Chronic Myeloid Leukemia. Clin Cancer Res 2006;12: Soverini S, Gnani A, Colarossi S, et al. Philadelphia-positive patients who already harbor imatinib-resistant Bcr-Abl kinase domain mutations have a higher likelihood of developing additional mutations associated with resistance to second- or third-line tyrosine kinase inhibitors. Blood 2009;114: Soverini S, Hochhaus A, Nicolini FE, et al. Bcr-Abl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: Recommendations from an expert panel on behalf of European LeukemiaNet. Blood, in press. 78. Soverini S, Martinelli G, Amabile M, et al. Denaturing-HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to Imatinib. Clin Chem 2004;50: Soverini S, Martinelli G, Colarossi S, et al. Second-line treatment with dasatinib in patients resistant to imatinib can select novel inhibitor-specific BCR- ABL mutants in Ph1 ALL. Lancet Oncol 2007;8: Soverini S, Martinelli G, Rosti G, et al. ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: A study by the GIMEMA Working Party on Chronic Myeloid Leukemia. J Clin Oncol 2005;23: Soverini S, Rosti G, Iacobucci I, et al. Choosing the best second-line tyrosine kinase inhibitor in imatinib-resistant chronic myeloid leukemia patients harboring Bcr-Abl kinase domain mutations: How reliable is the IC? Oncologist 2011;16: Soverini S, Vitale A, Poerio A, et al. Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis. Haematologica 2011;96: von Bubnoff N, Schneller F, Peschel C, et al. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: A prospective study. Lancet 2002;359: Wei Y, Hardling M, Olsson B, et al. Not all imatinib resistance in CML are BCR-ABL kinase domain mutations. Ann Hematol 2006;85: Quigley NB, Henley DC, Hubbard RA, et al. ABL kinase domain pseudoexon insertion is not uncommon in BCR-ABL transcripts. J Mol Diagn 2008;10: ; author reply Quintas-Cardama A, Cortes J. Chronic myeloid leukemia in the tyrosine kinase inhibitor era: What is the best therapy? Curr Oncol Rep 2009;11: Willis SG, Lange T, Demehri S, et al. High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: Correlation with clonal cytogenetic evolution but not response to therapy. Blood 2005;106: American Journal of Hematology