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Objective: Minimal residual disease assessment of BCR-ABL messenger ribonucleic acid levels is crucial in Philadelphia chromosome-positive acute lymphoblastic leukemia for prognosis and treatment planning. However, accurately quantifying minor BCR-ABL transcripts, which comprise 70% of Philadelphia chromosome-positive acute lymphoblastic leukemia cases, lacks a national-approved method., Methods: We developed the "Otsuka" minor BCR-ABLmessenger ribonucleic acid assay kit with exceptional precision (0.00151%). Minor BCR-ABL messenger ribonucleic acid levels were analyzed in 175 adults, 36 children with acute lymphoblastic leukemia and 25 healthy individuals to evaluate the kit's performance., Results: The "Otsuka" kit showed high concordance with a commonly used chimeric gene screening method, indicating reliable detection of positive cases. Quantitative results demonstrated a robust correlation with both a laboratory-developed test and a diagnostic research product. The "Otsuka" kit performs comparably or even surpass to conventional products, providing valuable insights into Philadelphia chromosome-positive acute lymphoblastic leukemia pathology., Conclusions: The 'Otsuka" minor BCR-ABL messenger ribonucleic acid assay kit exhibits excellent performance in quantifying minor BCR-ABL transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia patients. Our results align well with established screening methods and show a strong correlation with laboratory-developed tests and diagnostic research products. The "Otsuka" kit holds great promise as a valuable tool for understanding Philadelphia chromosome-positive acute lymphoblastic leukemia pathology and guiding effective treatment strategies., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Objective: The study aims to establish a perfect BCR-ABL (P210) internal quality control system and ensure the long-term stability and comparability of the detection results between laboratories and to popularize and apply it in the three hospitals. Methods: The Qilu Hospital of Shandong University (H1) prepared a set of the BCR-ABL (P210) quality control substances to establish and improve internal quality control system. We went to other three participating hospitals (H2, H3, and H4) to inspect quality control before the measurement. In addition, we mailed 25 sets of quality control substances to each of the hospital for detection. The slope and intercept of the standard curve of each hospital and the detection results were analyzed and statistically judged using the Levey-Jennings quality control chart combined with the Westgard multirule theory. Then, we made a quality control evaluation. Results: ①An internal quality control system for the BCR-ABL (P210) transcript levels monitoring was successfully established for the quality inspection before the measurement, statistical judgment during the measurement, and evaluation after the measurement. ② Both the slope and intercept of the standard curve of the four hospitals was under control. ③The multicenter quality control substance judgment results were as follows: for H1 hospital, two times of "1(2s)" warning were found in the middle-level quality control substance, which was judged as being under control; for H2 hospital, one time of "1(2s)" warning was found for each quality control substance, which was judged as being "2(2s)" out of control; for H3 hospital, its high-level quality control substance violated the "1(3s)" rule, and low-level quality control substance appeared "1(2s)" warning, which was judged as "1(3s)" out of control; and all quality control substances were under control in H4 hospital. ④The quality control evaluation and correction were as follows: two hospitals were under control, and the other two hospitals had an "out of control." We found out the reason for the out of control and corrected them. ⑤The comparisons of the original values of the multicenter quality control substance were as follows: there were statistical differences in the results of high-level quality control substance among the four hospitals, and no significant difference was found in the results of the medium-level and low-level quality control substance. ⑥The comparisons of the IS values of the multicenter quality control substance were as follows: the IS values of the three quality control substance in H2 and H3 hospitals were significantly higher than those of H1 hospital, and H2 hospital was significantly higher than H3 hospital. Conclusion: A perfect and stable internal quality control system for the BCR-ABL (P210) transcripts has been established, which can effectively ensure the accuracy and stability of the clinical detection results. This internal quality control system has been successfully popularized and applied in other hospitals.

Breakpoint cluster region-Abelson (BCR-ABL) negative myeloproliferative neoplasms (MPNs) are chronic myeloid neoplasms initiated by the acquisition of gene mutation(s) in a haematopoietic stem cell, leading to clonal expansion and over-production of blood cells and their progenitors. MPNs encompass a spectrum of disorders with overlapping but distinct molecular, laboratory and clinical features. This includes polycythaemia vera, essential thrombocythaemia and myelofibrosis. Dysregulation of the immune system is key to the pathology of MPNs, supporting clonal evolution, mediating symptoms and resulting in varying degrees of immunocompromise. Targeting immune dysfunction is an important treatment strategy. In the present review, we focus on the immune landscape in patients with MPNs - the role of inflammation in disease pathogenesis, susceptibility to infection and emerging strategies for therapeutic immune modulation. Further detailed work is required to delineate immune perturbation more precisely in MPNs to determine how and why vulnerability to infection differs between clinical subtypes and to better understand how inflammation results in a competitive advantage for the MPN clone. These studies may help shed light on new designs for disease-modifying therapies., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Tyrosine kinase inhibitors (TKI) can be safely discontinued in chronic phase chronic myeloid leukemia (CP-CML) patients who had achieved a sustained deep molecular response. Based on the results of discontinuation trials, recommendations regarding patient selection for a treatment-free remission (TFR) attempt had been proposed. The aims of this study were to evaluate the rate of patients eligible for TKI discontinuation and molecular recurrence-free survival (MRFS) after stop according to recommendations., Methods: Over a 10-year period, newly diagnosed CP-CML patients and treated with first-line TKI in the nine French participating centers were included. Eligibility to treatment discontinuation and MRFS were analyzed and compared according to selection criteria defined by recommendations and first-line treatments., Results: From January 2006 to December 2015, 398 patients were considered. Among them, 73% and 27% of patients received imatinib or either second or third generation tyrosine kinase inhibitors as frontline treatment, respectively. Considering the selection criteria defined by recommendations, up to 55% of the patients were selected as optimal candidates for treatment discontinuation. Overall 95/398 (24%) discontinued treatment. MRFS was 51.8% [95% CI 41.41-62.19] at 2 years and 43.8% [31.45-56.15] at 5 years. Patients receiving frontline second-generation TKI and fulfilling the eligibility criteria suggested by recommendations had the lowest probability of molecular relapse after TKI stop when compare to others., Conclusion: One third of CP-CML patients treated with TKI frontline fulfilled the selection criteria suggested by European LeukemiaNet TFR recommendations. Meeting selection criteria and second-generation TKI frontline were associated with the highest MRFS., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: To assess the prevalence of PHT in patients with BCR-ABL1-negative CMPN and to evaluate impact of PHT on survival during long-term follow-up., Patients and Methods: A total of 122 patients with BCR-ABL1-negative CMPN underwent transthoracic echocardiographic (TTE) evaluation at the beginning of study. Patients undergoing PHT on TTE examination were also evaluated by a pulmonologist. Patients were divided into 3 groups. Group A comprised patients with CMPN-related PHT; group B, patients with no PHT; and group C, patients with PHT due to secondary causes. Patients were evaluated again every 3 to 6 months., Results: PHT was detected in 33 (27%) of 122 patients. Eight (6.5%) had CMPN-related PHT and the remaining 25 (20.5%) had non-CMPN-related PHT. Positivity for JAK2 V617F mutation in the study population was 72.9%. Groups were similar with respect to hematologic parameters and gender. Follow-up times were as follows: median (range) time from diagnosis to TTE and study end were 34 (1-158) months and 107 (16-251) months, respectively, and from TTE to study end was 88 (7-110) months. No significant differences found among the groups in terms of median time from diagnosis to TTE, follow-up, and overall survival., Conclusion: BCR-ABL1-negative CMPN patients had a lower prevalence of PHT compared to earlier studies. There was no statistically significant difference in median overall survival between patients with or without PHT. This may be because patients with PHT were asymptomatic and PHT was mild. The impact of PHT on survival was negligible., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic myeloid leukaemia (CML) provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Imatinib mesylate (IM), a BCR-ABL1 targeted tyrosine kinase inhibitor (TKI) drug, is the first line gold standard drug for CML treatment. Conventional cytogenetic analysis (CCA) can identify the standard and variant Philadelphia (Ph) chromosome, and any additional complex chromosome abnormalities at diagnosis as well as during treatment course. Fluorescence in situ hybridization (FISH) is especially important for cells of CML patients with inadequate or inferior quality metaphases or those with variant Ph translocations. CCA in conjunction with FISH can serve as powerful tools in all phases of CML including the diagnosis, prognosis, risk stratification and monitoring of cytogenetic responses to treatment. Molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) is used for the detection of BCR-ABL1 transcripts at diagnosis whereas quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) is used at the time of diagnosis as well as during TKI therapy for the quantitation of BCR-ABL1 transcripts to evaluate the molecular response and minimal residual disease (MRD). Despite the excellent treatment results obtained after the introduction of TKI drugs, especially Imatinib mesylate (IM), resistance to TKIs develops in approximately 35% - 40% of CML patients on TKI therapy. Since point mutations in BCR-ABL1 are a common cause of IM resistance, mutation analysis is important in IM resistant patients. Mutations are reliably detected by nested PCR amplification of the translocated ABL1 kinase domain followed by direct sequencing of the entire amplified kinase domain. The objective of this review is to highlight the importance of regular and timely CCA, FISH analysis and molecular testing in the diagnosis, prognosis, assessment of therapeutic efficacy, evaluation of MRD and in the detection of BCR-ABL1 kinase mutations which cause therapeutic resistance in adult CML patients.

Introduction : Tyrosine kinase inhibitors (TKIs), which target BCR-ABL1 kinase activity, have significantly prolonged the overall survival of patients affected by chronic myeloid leukemia (CML) and changed drastically the outcome. Evidences from several studies suggest that in patients who have achieved a sustained, stable and deep molecular response, TKI treatment can be safely discontinued with a close subsequent monitoring. Thus, a stable deep molecular response (DMR) has become a feasible treatment goal in CML. Areas covered : In this review, the main findings extrapolated from sponsored and real-life evidences regarding TKI discontinuation were discussed, through a broad research on Medline, Embase and archives from EHA and ASH congresses (including words such as discontinuation, treatment-free remission, TFR, etc). Moreover, suggestions emerged from international guidelines about treatment-free remission (TFR) are presented. Expert opinion : With the growing availability of clinical trials and real-life data on TFR, in recent years the possibility of offering to CML patients a safe, informed and shorter path to TFR, through the achievement of a stable deep molecular response (DMR), has become an increasing option. However, many controversial aspects remain regarding treatment choices and timings, predictive factors, patient communication and optimal strategies aimed at achieving a successful TFR.

Treatment-free remission (TFR) is emerging as a new therapy goal for chronic myeloid leukemia (CML) patients in the tyrosine kinase inhibitors (TKI) era. Data indicates the unfavorable success rate of TFR. This study aimed to compare and evaluate the clinical value of dd-PCR in predicting relapse in CML patients entering TFR. Using dd-PCR and RT-qPCR technology, dynamic BCR/ABL transcripts were detected in 13 CML patients who discontinued TKI treatment after sustaining undetectable BCR-ABL levels for a median time of 25 months. The results showed that in 13 patients, only 2 cases (22.2%) of 9 patients who executed planned discontinuation achieved TFR within 12 months. In the first 6 months, the detection rate of BCR/ABL transcripts by dd-PCR was higher than that by RT-qPCR and the two methods kept a positive correlation (r=0.9651, P =0.0349). Meanwhile, the time of detectable BCR/ABL by dd-PCR were significantly shorter ( P <0.05), which was an average of 2.98 months earlier than RT-qPCR. The total TKI therapy and MR4.5 duration time related with TFR were longer in patients with intermediate or high Sokal risk scores ( p <0.05). The dd-PCR could be more sensitive than RT-qPCR for monitoring BCR/ABL transcripts of CML patients with deep molecular response to TKI. The technique can be used as a preferred method to detect the transcripts in the first 6 months after TKI cessation., (© 2020 by the Association of Clinical Scientists, Inc.)

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1 T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1 T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1 T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1 T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Nanotechnology provides new tools for gene expression analysis that allow for sensitive and specific characterization of prognostic signatures related to cancer. Cancer is a complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus allows for a more accurate indication into the degree of cancerous activity than either locus alone. Metal nanoparticles have been widely used as labels for in vitro identification and quantification of target sequences.Here we describe the synthesis of nanoparticles with different noble metal compositions in an alloy format that are then functionalized with thiol-modified ssDNA (nanoprobes). We also show how such nanoprobes are used in a non-cross-linking colorimetric method for the direct detection and quantification of specific mRNA targets, without the need for enzymatic amplification or reverse-transcription steps. The different metals in the alloy provide for distinct absorption spectra due to their characteristic plasmon resonance peaks. The color multiplexing allows for simultaneous identification of different mRNA targets involved in cancer development. A comparison of the absorption spectra of the nanoprobe mixtures taken before and after induced aggregation of metal nanoparticles allows to both identify and quantify each mRNA target. We describe the use of gold and gold-silver alloy nanoprobes for the development of the non-cross-linking method to detect a specific BCR-ABL fusion gene (e.g., e1a2 and e14a2) mRNA target associated with chronic myeloid leukemia (CML) using 10 ng/μL of unamplified total human RNA. Additionally, we demonstrate the use of this approach for the direct diagnostics of CML. This simple methodology takes less than 50 min to complete after total RNA extraction with comparable specificity and sensitivity to the more commonly used methods.

Introduction: BCR-ABL fusion gene, the oncogenic driver of CML, results from a translocation between short arms of chromosome 9 and 22. Monitoring of CML patients during treatment is essential, not only for tailoring the treatment but also to detect early relapse to enable timely intervention. Commonly used methods for detection of residual disease are conventional karyotyping, FISH and molecular methods. In this study, we compared FISH with QRT-PCR for detection of residual disease in CML., Materials and Methods: CML patients on tyrosine kinase inhibitor (TKI) therapy and on regular follow up at University Kebangsaan Malaysia Medical Center (UKMMC) were selected. A comparative study was conducted between FISH and QRT-PCR for BCR-ABL transcripts at diagnosis and during follow-up., Results: There was good correlation between FISH and QRT-PCR for BCR-ABL. At 6th month of follow-up post diagnosis, FISH had a sensitivity of 83.3% and specificity of 65.2% (k >0.339, p<0.033). At 12th month, the sensitivity of FISH was 83% and the specificity was 59.1% (k >0.286, p <0.065). Similarly, at the 24th month, FISH had a sensitivity of 100% and specificity of 68.8% (k >0.642, p<0.000)., Discussion: Early achievement of major molecular response (MMR) and complete cytogenetic remission (CCyR) were reliable predictors of long-term maintenance of molecular remission.

Chronic myeloid leukemia is characterized by a t(9;22)(q34;q11.2) resulting in BCR/ABL1 fusion located on the derivative chromosome 22, also known as the Philadelphia chromosome. We present the first case, to our knowledge, of chronic myeloid leukemia with 2 cryptic insertional events resulting in BCR/ABL1 fusion on the derivative chromosome 9 and FNBP1/BCR fusion on the derivative chromosome 22. These insertional events were misinterpreted as a typical balanced BCR/ABL1 translocation by interphase fluorescence in situ hybridization studies and were cryptic by conventional chromosome analysis, resulting in a "normal" karyotype. Mate-pair sequencing, a novel next-generation sequencing technology that can detect and characterize structural variants with significantly higher resolution and precision compared with traditional cytogenetic methodologies, identified 2 insertional events and resolved the seemingly discrepant chromosome and fluorescence in situ hybridization results. This case demonstrates the complexities of genetic abnormalities unappreciable by traditional cytogenetic methodologies and highlights the clinical utility of mate-pair sequencing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To investigate the prognosis of patients with adult B-cell acute lymphoblastic leukemia (B-ALL) with Ikaros family zinc-finger 1 (IKZF1) mutation and determine the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in improving the clinical outcome, we detected the IKZF1 mutation and BCR-ABL fusion gene at diagnosis in the bone marrow of 164 adult patients with B-ALL, and analyzed the clinical data of these patients retrospectively. Our analysis showed that grade III-IV acute graft-versus-host disease and IKZF1 mutation in the transplantation group and age and IKZF1 mutation in the non-transplantation group were independent factors for poor prognosis by univariate and multivariate analyses.The 3-year overall survival (OS) and leukemia-free survival (LFS) rates were much lower in the IKZF1+/BCR-ABL+ subgroup than in the IKZF1+/BCR-ABL- and IKZF1-/BCR-ABL- subgroups in both the transplantation and non-transplantation groups. The 3-year OS and LFS rates were significantly higher in the transplantation group than in the non-transplantation group with IKZF1 mutation.The study demonstrated that IKZF1 mutation was an independent factor indicating the poor prognosis of adult B-ALL and much worse prognosis in the BCR-ABL+ subgroup in both non-transplantation and transplantation groups. However, allo-HSCT significantly improved the OS and LFS of patients and also their clinical outcomes.

Background: Patients with CML treated with TKI can have a life expectancy comparable to that of the general population. Due to the extended duration of TKI administration, treatment discontinuation has been increasingly sought., Methods: Medical records of 100 patients with CML who were in MR 4.5 and discontinued their TKI outside clinical trials were reviewed., Results: After a median follow-up of 30 months (range, 5-112 months) after discontinuation, 35% and 17% lost MR 4.5 and major molecular response (MMR), respectively. Only six patients lost MMR 12 months or more after discontinuation. Loss of MR 4.5 was observed in 29% and 7% of patients with sustained MR 4.5 duration of more than 2 and 6 years before discontinuation, respectively. By univariate analysis, there was a higher risk of loss of MR 4.5 for patients who were treated for less than 87 months, received second or subsequent line TKI, never received interferon, or those with sustained MR 4.5 for less than 6 years. By multivariate analysis, sustained MR 4.5 for 6 years or more was the only significant predictor for durable response. Overall, 30% of patients who discontinued while in MR 4.5 were retreated with 93% regaining MR 4.5 at a median of 5 months., Conclusion: These results demonstrate that under proper conditions, treatment discontinuation is feasible outside of clinical trial setting. MR 4.5 duration of 6 years or more before discontinuation is associated with very low risk of loss of MR 4.5 .

Background: Standards play an important role in detection of the BCR-ABL1 fusion gene (FG) transcript. However, the standards widely used in laboratories are mainly based on plasmids or cDNA, which cannot accurately reflect the process of RNA extraction and cDNA synthesis. Therefore, we aimed to develop armored RNA-based standards for p210 and p190 BCR-ABL1FG transcripts' quantification., Methods: Using overlapping polymerase chain reaction (PCR) technology, we first linked a segment of the p210 or p190 BCR-ABL1FG transcript with four control genes (CGs; ABL1, BCR, GUSB, and B2M) to form p210FG-CG and p190FG-CG. Subsequently, using armored RNA technology, we prepared p210FG-CG- and p190FG-CG-armored RNAs and the p210FG-CG and p190FG-CG standards, the values of which were assigned by digital PCR (dPCR)., Results: The p210FG-CG and p190FG-CG standards were stable and homogeneous, and were significantly linear with r 2  > 0.98. A field trial including 52 laboratories across China showed that the coefficient of variation (CV%) of BCR-ABL1 values among samples was in the range of 58.6%-129.6% for p210 samples and 73.2%-194.0% for p190 samples when using local standards. By contrast, when using the p210FG-CG and p190FG-CG standards, the CV% of BCR-ABL1 values was decreased to 35.6%-124.9% and 36.6%-170.6% for p210 and p190 samples, respectively. In addition, 33.3% (3/9) of the p210 and p190 samples had CV% values <50.0%, whereas 44.4% (4/9) and 77.8% (7/9) of the samples had lower CV% values when using the p210FG-CG and p190FG-CG standards., Conclusion: The overall variability of detection of BCR-ABL1 transcripts decreased significantly when using the p210FG-CG or p190FG-CG standards, especially the p190FG-CG standard., (© 2018 Wiley Periodicals, Inc.)

The unchecked tyrosine kinase activity of BCR-ABL1 contributes to the immortality of leukaemic cells. Therefore, this oncogene is a highly important therapeutic target for chronic myelogenous leukaemia (CML). Tyrosine kinase inhibitors (TKIs) are an excellent drug treatment for CML patients. However, there are still some patients who are not responsive to TKIs. We found that a novel circular RNA (circRNA), named circBA9.3, is derived from BCR-ABL1. CircBA9.3 can efficiently promote the proliferation and inhibit apoptosis of cancer cells. In addition, some patients with TKI resistance have elevated circBA9.3 expression, which is positively correlated with the level of BCR-ABL1. Furthermore, circBA9.3 is predominantly located in the cytoplasm and enhances c-ABL1 and BCR-ABL1 oncoprotein expression. Thus, circBA9.3 is a molecule associated with increased tyrosine kinase activity that promotes resistance against TKI therapy. In this study, we provided a new potential target for the treatment of TKI-resistant CML patients., (Copyright © 2018. Published by Elsevier Inc.)

For patients with chronic myeloid leukaemia (CML), treatment guidelines recommend monitoring response to treatment with tyrosine kinase inhibitors (TKIs) by testing the BCR-ABL1 fusion gene transcript level using reverse transcriptase quantitative polymerase chain reaction. Despite recent efforts to standardise protocols for BCR-ABL1 testing, some variability remains among laboratories in the UK regarding the techniques used and the approach to reporting results. This increases the risk of misinterpretation of results by both clinicians and patients. An expert panel met to discuss current issues surrounding BCR-ABL1 testing in the UK and to develop guidance for laboratories, with emphasis on the optimal approach to reporting laboratory results. Topics included the minimum required information to include in the laboratory report, units of measurement, test sensitivity and BCR-ABL1 transcript variants. To aid communication between laboratories and clinics, standard forms were generated that could be used by (i) clinics when submitting samples to laboratories, and (ii) laboratories when reporting results to clinics. Standardising the way in which BCR-ABL1 test results are reported from laboratories to clinics should help to improve communication, interpretation of results and patient care., (© 2018 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd on behalf of British Society for Haematology.)

Background: Dasatinib is a potent BCR-ABL1 and Src family tyrosine kinase inhibitor. It is approved at a dose of 100 mg orally daily for the treatment of chronic myeloid leukemia in chronic phase (CML-CP). This dose schedule is associated with myelosuppression and pleural effusions. Anecdotal data suggest that lower doses may be as effective and less toxic. The aim of this study was to assess the efficacy and safety of a lower dose of dasatinib (50 mg daily) in patients with newly diagnosed CML-CP., Methods: Seventy-five patients with newly diagnosed CML-CP received dasatinib 50 mg daily. The eligibility and response criteria were standards used in previous protocols., Results: At a median follow-up of 9 months, 60 patients were evaluable for a response at 3 months. The rates of patients achieving BCR-ABL1 transcript levels ≤ 10% and ≤ 1% at 3 months by the International Standard were 93% and 72%, respectively. The rates of complete cytogenetic response by conventional cytogenetics or fluorescence in situ hybridization at 6 and 12 months were 86% and 88%, respectively. At 12 months, 79%, 71%, and 46% of the patients had achieved a major molecular response, a molecular response with a 4.0-log reduction, and a molecular response with a 4.5-log reduction, respectively. Nine patients had a dose interruption for ≤14 days. Only 1 patient developed a pleural effusion requiring a dose reduction to 20 mg. All patients remained alive and with no transformation so far., Conclusion: Dasatinib 50 mg daily is active and well tolerated in patients with newly diagnosed CML-CP. It should be further explored as a new potential standard-of-care option for chronic myeloid leukemia. Cancer 2018;124:2740-2747. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358)., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Quantitative real-time polymerase chain reaction (qRT-PCR) is state of the art in molecular monitoring of minimal residual disease in chronic myeloid leukemia (CML). In this context, maintenance of assay fidelity and detection of technical inaccuracy are crucial. Beside multiple common negative controls for the clinical sample preparations, quality control charts (QCC) are a common validation tool to sustain high process quality by continuously recording of qRT-PCR control parameters. Here, we report on establishment and benefit of QCC in qRT-PCR-based CML diagnostics. The absolute quantification of BCR-ABL1 fusion transcripts in patient samples is based on coamplification of a serially diluted reference plasmid (pME-2). For QCC establishment the measured Ct values of each pME-2 standard dilution (4-400,000) of a test set resembling 21 sequential qRT-PCR experiments were recorded and statistically evaluated. Test set data were used for determination of warning limits (mean +/- 2-fold standard deviation) and control (intervention) limits (mean +/- 3-fold standard deviation) to allow rapid detection of defined out-of-control situations which may require intervention. We have retrospectively analyzed QCC data of 282 sequential qRT-PCR experiments (564 reactions). Data evaluation using QCCs revealed three out-of-control situations that required intervention like experiment repeats, renewal of pME-2 standards, replacement of reagents or personnel re-training. In conclusion, with minimal more effort and hands-on time QCC rank among the best tools to grant high quality and reproducibility in CML routine molecular diagnosis.

Breakpoint cluster region-Abelson (BCR-ABL1) translocation is the characteristic sign of chronic myeloid leukemia (CML). The quantitation of BCR-ABL1 messenger RNA is requisite for patients with CML, and reverse-transcription real-time quantitative polymerase chain reaction (RQ-PCR) is the method used most extensively in testing laboratories worldwide. Nevertheless, substantial variation in RQ-PCR results from different laboratories makes interlaboratory comparability inconvincible owing to the lack of standardization. To facilitate interlaboratory comparative assessment and international standardization, an international scale (IS) for BCR-ABL1 was proposed. The laboratory-specific conversion factors derived from the IS can convert local different values to the IS without changing procedures. The standardization of BCR-ABL1 also includes the whole analytical process, so it is noteworthy to pay attention to the quality control before BCR-ABL1 quantitative analysis. More importantly, the World Health Organization has validated a first genetic reference panel which is limited to the manufacturers to produce and calibrate secondary reference reagents. Also, a certified reference plasmid, ERM-AD623, was internationally accepted. This article mainly focuses on BCR-ABL1 measurement and these standardization efforts in progress.

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros ( IKZF1 ) is a hallmark of BCR-ABL1 + precursor B cell acute lymphoblastic leukemia (pre-B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1 + pre-B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1 + pre-B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1 + pre-B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage-specific expression of multiple genes through chromatin compaction at its target genes., (© 2017 Schjerven et al.)

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1 + (Ph + ) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1 , which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1 , encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease., (© 2017 Witkowski et al.)

A 53-year-old woman with a 27-year history of myeloproliferative neoplasms came to our hospital because of a marked white blood cell count increase and progressive anemia. Clinical examination demonstrated positivity for BCR-ABL1 and JAK2-V617F mutations. She was given a diagnosis of chronic myeloid leukemia. Using the international scale, a molecular response (MR) 4.5 was achieved after treatment with dasatinib, despite the persistence of marked splenomegaly. The pathological findings of myelofibrosis were demonstrated by bone marrow biopsy. After stopping dasatinib administration for 4 years and 5 months, treatment with ruxolitinib was started. Five months later, the size of her spleen was reduced. We speculated that translocation of BCR-ABL1 might have occurred in a sub-clone of the JAK2-V617F mutated tumor clone.

Molecular monitoring of BCR-ABL1 expression in chronic myeloid leukaemia (CML) is well established. As the International Scale (IS) normalised BCR-ABL1/ABL1 ratio at 3 months post-treatment is now an important milestone in patients' treatment schedule, the reliable and reproducible measurement of BCR-ABL1 levels is therefore paramount. IS conversion factors (CF) are established via sample exchange and yearly ratification with an external reference laboratory. Since any change to an established IS CF could lead to discontinuity in longitudinal results, we wished to add an internal verification step as an additional safeguard. We used the Cepheid GeneXpert qPCR and IS calibrated Xpert BCR-ABL Monitor cartridge system, parallel to our in-house pipeline on 50 CML samples, over the period of one week to verify the CF for those samples and compare it to the externally provided CF. The median non-IS in-house BCR-ABL1/ABL1 values were significantly different than that from the IS GeneXpert, but they became non-significant when adjusted to CF provided by the CXM and by the current external CF, validating it. These metrics can help decide to accept or reject an updated CF value, especially where a significant change in CF might lead to a discontinuity in ongoing patient monitoring., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To set internal quality control system of BCR-ABL (P210) transcript levels for real-time quantitative PCR (RQ-PCR). Methods: Using K562 cells and HL-60 cells, we prepared high- and low-level BCR-ABL internal quality control substance. The BCR-ABL (P210) transcript levels of internal quality control substance have been determined for 184 times together with clinical samples from August 2013 to October 2015. The slope rate, intercept and correlation coefficient of standard curve were calculated according to different reagent lots (lots number 20130303, 20131212, 20140411 and 20150327 are called R1、R2、R3 and R4 for short respectively), and the detection results of quality control substance were calculated according to different reagent lots and quality control substance lots (lots number 20130725, 20140611 are called Q1、Q2 for short respectively). Then the results were analyzed by Levey-Jennings quality control chart combined with Westgard multi-rules theory. Results: ①We analyzed the slope rate and intercept of standard curve. Fifty-three times of the R1 reagent detection, 80 times of the R3 reagent detection and 14 times of the R4 reagent detection were all under control. For 37 times detection of R2 reagent, the slope rate was out of control for 6 times. It was lower than x - s for the 2-8 tests and upper the average for the 12-37 tests. The intercept was out of control for 9 times, upper the x + s for the 1-8 tests and lower the average for the 12-37 tests. ② According to the detection results of quality control substance, for Q1 quality control substance, 49 tests by R1 reagent were under control, and 1 out of 23 tests by R2 reagent was out of control. For Q2 quality control substance, 14 tests by R2 reagent detection, 72 tests by R3 reagent detection and 14 tests by R4 reagent were all under control. Conclusion: The preparation of high- and low-level quality control substance using K562 and HL-60 cells was convenient and the detection results were reliable and stable. The application of quality control substance combined with slope rate and intercept in the internal quality control may contribute to quality assurance for quantitative detection of BCR-ABL (P210) transcript levels.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms., Competing Interests: This study was designed and funded by Novartis Pharmaceuticals Corporation. CW, DZ, SW and SSW are employed by Novartis Pharmaceuticals Corporation. None of the co-authors, participating laboratories or institutions received any payments for participating in this study. All authors have read and agreed with the contents of the manuscript. All other authors declare no conflicts of interest.

Achieving a BCR-ABL/ABL ratio <10% at 3 months has become an important goal of treatment for chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. Several evidences showed that this early molecular response (EMR) is associated with positive long-term outcome in terms of overall survival and progression-free survival, but a consensus has not been reached when this goal is not achieved. European LeukemiaNet recommendations defined patients as treatment failure only after the 6- month time point. Not all patients that lack EMR have similar outcome and it became important to identify patients before this time point of 3 months. Several groups introduced the concept of "halving time" or "velocity of ratio reduction" that could anticipate the possibility to recognize patients deserving a switch to another treatment. Aim of this review is to summarize all evidences reported on the significance of EMR and how this evaluation changed our perspectives and modified our therapeutic strategies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The current understanding of BCR-ABL1 negative myeloproliferative neoplasms pathogenesis is centred on the phenotypic driver mutations in JAK2, MPL, or CALR genes, and the constitutive activation of JAK-STAT pathway. Nonetheless, there is still a need to better characterize the cellular processes that are triggered by these genetic alterations, such as apoptosis that might play a role in the pathological expansion of the myeloid lineages and, especially, in the morphological anomalies of the bone marrow megakaryocytes. In this article we will explore the connection between the driver mutations in MPN and the abnormal apoptosis that might be translated in new therapeutic strategies.

Objective: To identify IKZF1 gene copy number abnormalities in BCR/ABL-negative B-lineage acute lymphoblastic leukemia (B-ALL) in children, and to investigate the association between such abnormalities and prognosis., Methods: Multiplex ligation-dependent probe amplification (MLPA) was applied to detect IKZF1 gene copy number abnormalities in 180 children diagnosed with BCR/ABL-negative B-ALL. These children were classified into IKZF1 deletion group and IKZF1 normal group according to the presence or absence of IKZF1 gene deletion. The association between IKZF1 copy number abnormalities and prognosis of children with BCR/ABL-negative B-ALL was analyzed retrospectively., Results: Among 180 children, 27 (15.0%) had IKZF1 deletion; among the 27 children, 4 had complete deletions of 8 exons of IKZF1 gene, 17 had deletion of exon 1, 3 had deletions of exons 4-7, and 3 children had deletions of exons 2-7. Compared with those in the IKZF1 normal group, children in the IKZF1 deletion group had higher white blood cell (WBC) count and percentage of individuals with high risk of minimal residual disease at the first visit. IKZF1 deletions often occurred in BCR/ABL-negative children with no special fusion gene abnormalities. They were frequently accompanied by abnormalities in chromosomes 11, 8, 5, 7, and 21. The analysis with Kaplan-Meier method showed that disease-free survival (DFS) in the IKZF1 deletion group was significantly lower than that in the IKZF1 normal group (0.740 ± 0.096 vs 0.905 ± 0.034; P=0.002). Cox analysis showed that after exclusion of sex, age, initial WBC count, cerebrospinal fluid state at the first visit, prednisone response, and chromosome karyotype, IKZF1 deletion still affected the children's DFS (P<0.05)., Conclusions: Some children with BCR/ABL-negative B-ALL have IKZF1 deletion, and IKZF1 deletion is an independent risk factor for DFS in children with BCR/ABL-negative B-ALL.

This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chronic myeloid leukemia (CML) is diagnostically defined by the reciprocal translocation t(9;22)(q34;q11). This aberration can be detected by the BCR-ABL fluorescence in situ hybridization (FISH) technique. This article presents a comparative analysis of different commercially available FISH probes and different FISH protocols in order to optimize this technique on formalin-fixed and paraffin-embedded bone marrow trephine biopsies.

Few patients in remission of Ph-positive chronic myelogenous leukemia (CML) develop Ph-negative MDS/AML, usually with clonal cytogenetic abnormalities. Isolated Ph-negative myeloid sarcoma (MS) is presented here as a form of such disorder, different from Ph-positive MS establishing CML relapse in blastic phase. We describe 11-year-old male who developed Ph-negative isolated MS with NPM1 mutation, remaining in complete molecular remission of Ph-positive chronic myeloid leukemia treated with allo-HSCT in first chronic phase and with imatinib and donor lymphocyte infusion in molecular relapse. The possible mechanisms of the tumor formation are reviewed with stress on importance of comprehensive molecular/cytogenetic evaluations., (© 2015 Wiley Periodicals, Inc.)

Background: Chronic myelogenous leukemia (CML) has been reportable to the Pennsylvania Cancer Registry (PCR) since the 1980s, but the completeness of reporting is unknown. This study assessed CML reporting in eastern Pennsylvania where a cluster of another myeloproliferative neoplasm was previously identified., Methods: Cases were identified from 2 sources: 1) PCR case reports for residents of Carbon, Luzerne, or Schuylkill County with International Classification of Diseases for Oncology, Third Edition (ICD-O-3) codes 9875 (CML, BCR-ABL+), 9863 (CML, NOS), and 9860 (myeloid leukemia) and date of diagnosis 2001-2009, and 2) review of billing records at hematology practices. Participants were interviewed and their medical records were reviewed by board-certified hematologists., Results: PCR reports included 99 cases coded 9875 or 9863 and 9 cases coded 9860; 2 additional cases were identified by review of billing records. Of the 110 identified cases, 93 were mailed consent forms, 23 consented, and 12 medical records were reviewed. Hematologists confirmed 11 of 12 reviewed cases as CML cases; all 11 confirmed cases were BCR/ABL positive, but only 1 was coded as positive (code 9875)., Conclusions: Very few unreported CML cases were identified, suggesting relatively complete reporting to the PCR. Cases reviewed were accurately diagnosed, but ICD-0-3 coding often did not reflect BCR-ABL-positive tests. Cancer registry abstracters should look for these test results and code accordingly.

The Therapeutic Intensification in De Novo Leukaemia (TIDEL)-II study enrolled 210 patients with chronic phase chronic myeloid leukemia (CML) in two equal, sequential cohorts. All started treatment with imatinib 600 mg/day. Imatinib plasma trough level was performed at day 22 and if <1000 ng/mL, imatinib 800 mg/day was given. Patients were then assessed against molecular targets: BCR-ABL1 ≤10%, ≤1%, and ≤0.1% at 3, 6, and 12 months, respectively. Cohort 1 patients failing any target escalated to imatinib 800 mg/day, and subsequently switched to nilotinib 400 mg twice daily for failing the same target 3 months later. Cohort 2 patients failing any target switched to nilotinib directly, as did patients with intolerance or loss of response in either cohort. At 2 years, 55% of patients remained on imatinib, and 30% on nilotinib. Only 12% were >10% BCR-ABL1 at 3 months. Confirmed major molecular response was achieved in 64% at 12 months and 73% at 24 months. MR4.5 (BCR-ABL1 ≤0.0032%) at 24 months was 34%. Overall survival was 96% and transformation-free survival was 95% at 3 years. This trial supports the feasibility and efficacy of an imatinib-based approach with selective, early switching to nilotinib. This trial was registered at www.anzctr.org.au as #12607000325404., (© 2015 by The American Society of Hematology.)

We report here the case of a sixty-eight-year old woman with chronic myeloid leukemia. Molecular techniques identified the presence of the rare e19a2 BCR-ABL1 transcript. The patient was treated by 1(st) generation tyrosine-kinase inhibitor (TKI) (imatinib). Disease monitoring was performed by cytogenetic analyses and quantification of the BCR-ABL1 transcript. After 3 months, the treatment was modified due to an absence of biological response and poor tolerance. After 21 months with 2nd generation TKIs (nilotinib), the patient was responding optimally to treatment, with a complete cytogenetic response and a major molecular response. This observation emphasizes the importance of determining the chromosomal breakpoints at diagnosis to enable adequate molecular monitoring of residual disease. Careful monitoring of minimal residual disease is important to thoroughly assess the response to treatment, detect resistance and adapt the therapeutic strategy. The kinetics and the depth of the response to TKI also represent major prognostic factors. Molecular monitoring is performed using real-time quantitative PCR, which has to be adapted to each type of transcript. For rare BCR-ABL1 transcripts, an international standardization, as it is developed for conventional transcripts, is lacking. Yet, such a harmonization would be useful to assess in an optimal and large scale way the response to TKI in these patients, and to determine what the best management is.

Imatinib is the frontline therapy for chronic myeloid leukemia (CML) management. Most of the CML patients achieve major responses, but a proportion (nearly 25-35%) of them develop drug resistance. Molecular monitoring using quantitative real-time PCR at regular intervals according to European LeukemiaNet (ELN) helps in the assessment of long-term outcomes in imatinib-treated CML patients. Eighty-four CML patient samples (42 at diagnosis and 42 at 3-month intervals from the same patients) were analyzed for Bcr-Abl transcript levels. Quantification results revealed that the patients with <10% Bcr-Abl levels at 3 months had higher rates of complete cytogenetic response (CCyR) and optimal responses compared to patients with >10% Bcr-Abl levels (P < 0.0001). Patients with >10% Bcr-Abl levels were found to have 25.0% of suboptimal response and 3.57% of failure to imatinib at standard dose. Hence, the present study confirms that early molecular monitoring at 3 months after imatinib initiation helps in predicting the concurrent cytogenetic response and treatment optimization in CML patients.

We retrospectively evaluated the outcome of 156 adults age 17-60 with BCR-ABL negative acute lymphoblastic leukemia treated with a pediatric-inspired protocol. The CR rate was 93%; 5-year overall survival (OS) and disease-free survival were 66% and 70%, respectively. Age <34 years and low presenting WBC were independent predictors of favorable OS (p<0.0001). The 5 year OS of patients age <34 and 34-60 presenting with a low WBC were 85% and 57%, respectively; the 5 year OS for those presenting with a high WBC were 57% and 30%, respectively. Cytogenetics and phenotype were not independent predictors of OS., (Copyright © 2014. Published by Elsevier Ltd.)

Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the in situ detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence in situ hybridization (sm-FISH), in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing's sarcoma cells. This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts.

An emerging problem in patients with Philadelphia (Ph)-positive leukaemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukaemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukaemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. Polymerase chain reaction (PCR) amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)