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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

Timothy Hughes, Michael Deininger, Andreas Hochhaus, Susan Branford, Jerald Radich, Jaspal Kaeda, Michele Baccarani, Jorge Cortes, Nicholas C. P. Cross, Brian J. Druker, Jean Gabert, David Grimwade, Rüdiger Hehlmann, Suzanne Kamel-Reid, Jeffrey H. Lipton, Janina Longtine, Giovanni Martinelli, Giuseppe Saglio, Simona Soverini, Wendy Stock and John M. Goldman

Article Figures & Data

Figures

  • Figure 1.

    Suggested method for sequential reporting of results of RQ-PCR assays. Interpretation (assuming adequate quality RNA): (1) BCR-ABL detectable at a level greater than 1.0%, which suggests that the patient has some or 100% Ph-positive marrow metaphases (the patient may still be responding to therapy or may be relapsing from a Ph-negative status); (2) BCR-ABL detectable at a level greater than 0.1%, which suggests that the patient has not achieved or has lost a major molecular response; (3) BCR-ABL detectable at or below the level of 0.1% indicates achievement of a major molecular response (as defined by the IRIS study); and (4) BCR-ABL is not detectable, meaning that the BCR-ABL level is below the level of sensitivity of the assay, which should be at least 0.01% on the international scale, a value equivalent to a 4-log reduction below baseline. The laboratory value for a given result can be converted to a value on the international scale by use of a conversion factor. This factor is based on the relationship of the laboratory specific value for an MMR to the value equivalent to an MMR as established in the IRIS trial, namely a 3-log reduction below an internationally agreed standardized baseline. The conversion factor will be specific for each laboratory but may be affected by any change in the technical aspects of the assay. If the quality of the RNA is poor, no useful conclusion can be drawn from the results of the test.

  • Figure 2.

    The relative frequency of BCR-ABL kinase domain mutations detected at 31 different positions in clinical specimens from 245 patients in whom mutations were detected (219 with CML and 26 with Ph-positive acute lymphoblastic leukemia). The numbering of amino acids is based on the Abl protein variant B (which includes ABL exon 1b but not exon 1a). The letters inside the circles denote the amino acid encoded by the corresponding mutated nucleotide. At some positions 2 or 3 possible mutant nucleotides encode different amino acids. The percentage of patients in the series with each mutation specified on the y axis is color-coded as shown in the box. Data collated from 20 published papers.19-22,24,25,48,52,56-66

  • Figure 3.

    Suggested headings for expressing results of Abl KD analysis. For example, resistance may be classified as Fully Sensitive (FS), Partially Resistant (PR), Fully Resistant (FR), or Unknown (UKN).

  • Figure 4.

    The detection of KD mutations associated with a rise in the BCR-ABL level. The graphs plot the BCR-ABL levels as measured by RQ-PCR76 in 2 late-chronic-phase patients treated in Australia who achieved a CCyR on imatinib 400 mg/daily. The BCR-ABL levels were calculated according to the proposed international scale (IS). The mutation analysis was performed using a direct sequencing technique.77 The mutation results are depicted as open circles when wild-type BCR-ABL was detected; the amount of shading within the circles indicates the relative size of the mutant sub-clone. Diamonds indicate datapoints. (A) After 18 months on imatinib, the patient had undetectable BCR-ABL that was followed by a rise of at least 5-fold. At that time the D276G mutation was detected. The patient remained on 400 mg imatinib and a CCyR was maintained at 27 months. Thereafter the patient progressed rapidly to lymphoid blast crisis. (B) This patient had a rise of 2.1-fold and the E453G mutation was detected prior to the rise. On the basis of the rising BCR-ABL level and the detection of the mutation the dose of imatinib was increased from 400 to 800 mg per day. The BCR-ABL level subsequently decreased and the CCyR was maintained.

Tables

  • Table 1.

    Example of the use of a conversion factor to convert BCR-ABL values obtained in a given laboratory to the international scale

    LaboratoryMMREq, %0.1%MMREq (%) = conversion factorFormula for conversion of a given result to the international scale (BCR-ABLL × CF = BCR-ABLIS)
    Adelaide 0.08 0.1/0.08 = 1.25 BCR-ABLL × 1.25
    Mannheim 0.12 0.1/0.12 = 0.83 BCR-ABLL × 0.83
    London 0.045 0.1/0.045 = 2.22 BCR-ABLL × 2.22
    • BCR-ABLL = BCR-ABL/control ratio expressed as a percentage in a given laboratory. MMREq = BCR-ABLL that is equivalent to a MMR as extablished in the IRIS trial.12 In order to convert a given local result to the international scale, it is necessary to use a conversion factor (CF). This is calculated as follows: CF = 0.1% divided by MMREq (since 0.1% is the agreed value for MMR on the international scale). Once a laboratory-specific conversion factor has been derived, it can be used to convert all local values to the international scale. (This calculation will be invalid if the reproducibility or linearity of the assay is poor, in which case the methodology will need to be optimized.)

  • Table 2.

    Technologies available for identifying and quantifying BCR-ABL KD mutations

    TechnologySensitivity, %SpecificityBias*AvailabilityReference
    Direct sequencing 15-25 ++ No +++ 19,21,22,24,59
    Subcloning and sequencing 9 +++ No ++ 20
    Denaturing high-performance liquid chromatography (D-HPLC) 0.1-10 ++ No ++ 66-68
    Pyrosequencing 5 ++ No + 55,69
    Double-gradient denaturing electrophoresis 5 ++ No + 57
    Fluorescence PCR and PNA clamping 0.2 ++ Yes + 58
    Allele-specific oligonucleotide PCR (ASO-PCR) 0.01 ++ Yes + 53,59,60
    • PNA indicates peptide nucleic acid.

    • * Bias indicates that the test is designed to detect specific mutations.