An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.

Summary Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph+) leukemia, including the recalcitrant BCR-ABL1T315I mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph+ leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

IntroductionMany cases of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by an activating point mutation of JAK2 (JAK2 V617F ). It has been generally accepted that JAK2 V617F -positive cells outpace normal hematopoietic cells as a result of constitutively active growth factor signaling 1 ; however, failure of JAK2 V617F to confer a significant competitive advantage over normal hematopoiesis in 2 independent knock-in MPN models 2,3 suggests that additional factors may be required to promote expansion of JAK2 V617F -positive cells in patients.As MPN patients overproduce certain proinflammatory cytokines known to suppress normal hematopoiesis, 4 it is conceivable that JAK2 V617F may protect mutant stem cells and progenitors from the apoptotic cues induced by these cytokines.In this context, we recently observed that TNF␣ levels are elevated in mice with retrovirally induced JAK2 V617F MPN. 5 The physiologic effects of TNF␣ are complex and cell type-dependent, ranging from stimulation of proliferation to induction of apoptosis. 6 TNF␣ negatively regulates the expansion and self-renewal of pluripotent hematopoietic stem cells (HSCs) 7,8 and has inhibitory effects on normal as well as some leukemic human hematopoietic progenitor cells. [9][10][11] TNF␣'s involvement in the evolution of leukemia is not without precedent. Studies in Fanconi anemia (FA) have implicated TNF␣ hypersensitivity as a central mechanism of clonal evolution and progression to acute myeloid leukemia. In the FA Complementation Group C murine model (Fancc Ϫ/Ϫ ) TNF␣ induces bone marrow failure 12 and can promote the evolution of somatically mutated TNF␣-resistant preleukemic stem cell clones. 13 Taking into account TNF␣'s role in clonal evolution and that elevated TNF␣ levels are present in human MPN we hypothesized that JAK2 V617F induces TNF␣ expression and simultaneously confers TNF␣ resistance to MPN progenitor cells. Methods Isolation and culture of primary cellsBlood mononuclear cells (MNCs) were obtained from peripheral blood samples of patients with polycythemia vera (PV) and essential thromobocythemia (ET), myelofibrosis (MF), or normal volunteers. CD34 ϩ cells were obtained from bone marrow of normal, PV and ET patients or peripheral blood of MF patients. All patients gave their informed consent in accordance with the Declaration of Helsinki to participate in the study, which was approved by the Institutional Review Boards of Oregon Health & Science University (OHSU), Portland Veterans Affairs Medical Center, Cornell University, and Freiburg University.Submitted April 13, 2011; accepted August 7, 2011. Prepublished online as Blood First Edition paper, August 22, 2011; DOI 10.1182 DOI 10. /blood-2011 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''adverti...

Serial quantitation of BCR-ABL IntroductionReverse-transcription real-time quantitative polymerase chain reaction (RQ-PCR) is used routinely to quantify levels of BCR-ABL mRNA in peripheral blood and bone marrow samples from chronic myelogenous leukemia (CML) patients undergoing therapy. The technique can accurately determine response to treatment and is particularly valuable for patients who have achieved a complete cytogenetic response. The National Comprehensive Cancer Network (NCCN) 1 and the European LeukemiaNet (ELN) 2 recommend similar monitoring schedules for patients treated with imatinib and the ELN defines an optimal response as the attainment of a major molecular response (MMR) after 18 months of therapy. Monitoring of BCR-ABL mRNA levels is also useful for gauging therapeutic response for patients with Philadelphia chromosomepositive acute lymphoblastic leukemia (Ph ϩ ALL). The CML meeting at the National Institutes of Health in Bethesda in October 2005 made several recommendations for the harmonization of minimal residual disease (MRD) assessment and proposed an international scale (IS) for BCR-ABL RQ-PCR measurements. 8 Importantly, the IS is essentially identical to that used in the International Randomized Study of Interferon and STI571 (IRIS) study, 9 with the IRIS standardized baseline defined as 100% BCR-ABL IS and MMR (3-log reduction relative to the standardized baseline) defined as 0.1% BCR-ABL IS . The original standards used for the IRIS trial are no longer available, however traceability to the IRIS scale is provided by the extensive quality control data generated by the Adelaide laboratory over a period of several years. 10,11 To enable testing centers to gain access to the IS, the Adelaide laboratory initiated a process to develop and validate laboratoryspecific conversion factors (CFs) that can be used to convert local values to IS values. 11 The strength of this approach is that testing centers can continue to use their existing assay conditions and continue to express results according to local preferences in addition to expressing results on the IS. The concept of the IS is analogous to established procedures for other quantitative assays, for example the International Normalized Ratio (INR) for prothrombin time. 12 Many laboratories with validated CFs have established themselves as national or regional reference laboratories and are in the process of propagating CFs to local centers. 13 While this process has generally worked well, it is apparent that the establishment of CFs is time-consuming, complex, expensive, and open to only a limited number of laboratories at any given time. Furthermore, it is unclear how frequently any individual CF will need to be revalidated. We sought therefore to develop an alternative means for testing laboratories to access the IS by developing calibrated, accredited primary reference reagents for BCR-ABL RQ-PCR analysis. StrategyIdeally, the formulation for primary reference reagents should be as close as possible to the usual analyte, should cove...

Although most patients with chronic myeloid leukemia (CML) treated with imatinib mesylate achieve a complete cytogenetic response (CCR), some patients will relapse. To determine the potential of real-time quantitative BCR-ABL reverse transcriptase-polymerase chain reaction (RT-PCR) to predict the duration of continued CCR, we monitored 85 patients treated with imatinib mesylate who achieved a CCR. With a median follow-up of 13 months after CCR (29 months after imatinib mesylate; median 6 RQ-PCR assays), 23 patients (27%) had disease progression (predominantly loss of CCR). Compared with the median baseline level of BCR-ABL mRNA, 42% of patients achieved at least a 2-log molecular response at the time of first reaching CCR. Failure to achieve a 2-log response at the time of CCR was an independent predictive marker of subsequent progressionfree survival (hazard ratio ‫؍‬ 5.8; 95% CI, 1.7-20; P ‫؍‬ .005). After CCR, BCR-ABL mRNA levels progressively declined for at least the next 15 months, and 42 patients (49%) ultimately achieved at least a 3-log reduction in BCR-ABL mRNA. Patients failing to achieve this 3-log response, at any time during therapy, had significantly shorter progression-free survival (hazard ratio ‫؍‬ 8.1; 95% CI, 3.1-22; P < .001). The achievement of either a 2-log molecular response at the time of CCR or a 3-log response anytime thereafter is a significant and independent prognostic marker of subsequent progressionfree survival. (Blood. 2006;107:4250-4256)