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

Abstract

The introduction in 1998 of imatinib mesylate (IM) revolutionized management of patients with chronic myeloid leukemia (CML) and the second generation of tyrosine kinase inhibitors may prove superior to IM. Real-time quantitative polymerase chain reaction (RQ-PCR) provides an accurate measure of the total leukemiacell mass and the degree to which BCR-ABL transcripts are reduced by therapy correlates with progression-free survival. Because a rising level of BCR-ABL is an early indication of loss of response and thus the need to reassess therapeutic strategy, regular molecular monitoring of individual patients is clearly desirable. Here we summarize the results of a consensus meeting that took place at the National Institutes of Health (NIH) in Bethesda in October 2005. We make suggestions for (1) harmonizing the differing methodologies for measuring BCR-ABL transcripts in patients with CML undergoing treatment and using a conversion factor whereby individual laboratories can express BCR-ABL transcript levels on an internationally agreed scale; (2) using serial RQ-PCR results rather than bone marrow cytogenetics or fluorescence in situ hybridization (FISH) for the BCR-ABL gene to monitor individual patients responding to treatment; and (3) detecting and reporting Philadelphia (Ph) chromosome-positive subpopulations bearing BCR-ABL kinase domain mutations. We recognize that our recommendations are provisional and will require revision as new evidence emerges.

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.-,,,,,-

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-PCR 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. 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.