Molecular BCR::ABL1 Quantification and ABL1 Mutation Detection as Essential Tools for the Clinical Management of Chronic Myeloid Leukemia Patients: Results from a Brazilian Single-Center Study.

Chronic myeloid leukemia (CML) is a well-characterized oncological disease in which virtually all patients possess a translocation (9;22) that generates the tyrosine kinase BCR::ABL1 protein. This translocation represents one of the milestones in molecular oncology in terms of both diagnostic and prognostic evaluations. The molecular detection of the BCR::ABL1 transcription is a required factor for CML diagnosis, and its molecular quantification is essential for assessing treatment options and clinical approaches. In the CML molecular context, point mutations on the ABL1 gene are also a challenge for clinical guidelines because several mutations are responsible for tyrosine kinase inhibitor resistance, indicating that a change may be necessary in the treatment protocol. So far, the European LeukemiaNet and the National Comprehensive Cancer Network (NCCN) have presented international guidelines on CML molecular approaches, especially those related to BCR::ABL1 expression. In this study, we show almost three years' worth of data regarding the clinical treatment of CML patients at the Erasto Gaertner Hospital, Curitiba, Brazil. These data primarily comprise 155 patients and 532 clinical samples. BCR::ABL1 quantification by a duplex-one-step RT-qPCR and ABL1 mutations detection were conducted. Furthermore, digital PCR for both BCR::ABL1 expression and ABL1 mutations were conducted in a sub-cohort. This manuscript describes and discusses the clinical importance and relevance of molecular biology testing in Brazilian CML patients, demonstrating its cost-effectiveness.

A Ten-Year Experience of Treating Chronic Myeloid Leukemia in Rural Rwanda: Outcomes and Insights for a Changing Landscape.

PURPOSE: In describing our ten-year experience with treating chronic myeloid leukemia (CML) as part of the Glivec Patient Assistance Program (GIPAP) in rural Rwanda, we evaluate (1) patient characteristics and treatment outcomes, (2) resource-adapted management strategies, and (3) the impact of diagnostic capacity development. METHODS: We retrospectively reviewed all patients with BCR-ABL-positive CML enrolled in this GIPAP program between 2009 and 2018. Clinical data were analyzed using descriptive statistics, Kaplan-Meier methods, proportional hazards regression, and the Kruskal-Wallis test. RESULTS: One hundred twenty-four patients were included. The median age at diagnosis was 34 (range 8-81) years. On imatinib, 91% achieved complete hematologic response (CHR) after a median of 49 days. Seven (6%) and 12 (11%) patients had primary and secondary imatinib resistance, respectively. The 3-year overall survival was 80% (95% CI, 72 to 87) for the cohort, with superior survival in imatinib responders compared with those with primary and secondary resistance. The median time from imatinib initiation to CHR was 59 versus 38 days (P = .040) before and after in-country diagnostic testing, whereas the median time to diagnosis (P = .056) and imatinib initiation (P = .170) was not significantly different. CONCLUSION: Coupling molecular diagnostics with affordable access to imatinib within a comprehensive cancer care delivery program is a successful long-term strategy to treat CML in resource-constrained settings. Our patients are younger and have higher rates of imatinib resistance compared with historic cohorts in high-income countries. High imatinib resistance rates highlight the need for access to molecular monitoring, resistance testing, and second-generation tyrosine kinase inhibitors, as well as systems to support drug adherence. Hematologic response is an accurate resource-adapted predictor of survival in this setting. Local diagnostic capacity development has allowed for continuous, timely CML care delivery in Rwanda.

NOVEL-1st: an observational study to assess the safety and efficacy of nilotinib in newly diagnosed patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase in Taiwan.

Nilotinib has been approved for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase (Ph+ CML-CP). However, the real-world evidence of nilotinib in newly diagnosed untreated Ph+ CML-CP is limited in Taiwan. The NOVEL-1st study was a non-interventional, multi-center study collecting long-term safety and effectiveness data in patients with newly diagnosed and untreated Ph+ CML-CP receiving nilotinib. We enrolled 129 patients from 11 hospitals. Overall, 1,466 adverse events (AEs) were reported; among these, 151 were serious and 524 were nilotinib-related. Common hematological AEs were thrombocytopenia (31.0%), anemia (20.9%), and leukopenia (14.0%); common nilotinib-related AEs were thrombocytopenia (29.5%), anemia (14.7%), and leukopenia (12.4%). Early molecular response, defined as BCR-ABL ≤ 10% at Month 3, was seen in 87.6% of patients. By 36 months, the cumulative rates of complete hematologic response, complete cytogenetic response, major molecular response, molecular response 4.0-log reduction, and molecular response 4.5-log reduction were 98.5, 92.5, 85.8, 65.0, and 45.0%, respectively. Nilotinib is effective and well-tolerated in patients with newly diagnosed Ph+ CML-CP in the real-world setting. Long-term holistic care and a highly tolerable AE profile may contribute to good treatment outcomes in Ph+ CML-CP under first-line treatment with nilotinib.

A Machine Learning Model to Successfully Predict Future Diagnosis of Chronic Myelogenous Leukemia With Retrospective Electronic Health Records Data.

BACKGROUND: Chronic myelogenous leukemia (CML) is a clonal stem cell disorder accounting for 15% of adult leukemias. We aimed to determine if machine learning models could predict CML using blood cell counts prior to diagnosis. METHODS: We identified patients with a diagnostic test for CML (BCR-ABL1) and at least 6 consecutive prior years of differential blood cell counts between 1999 and 2020 in the largest integrated health care system in the United States. Blood cell counts from different time periods prior to CML diagnostic testing were used to train, validate, and test machine learning models. RESULTS: The sample included 1,623 patients with BCR-ABL1 positivity rate 6.2%. The predictive ability of machine learning models improved when trained with blood cell counts closer to time of diagnosis: 2 to 5 years area under the curve (AUC), 0.59 to 0.67, 0.5 to 1 years AUC, 0.75 to 0.80, at diagnosis AUC, 0.87 to 0.92. CONCLUSIONS: Blood cell counts collected up to 5 years prior to diagnostic workup of CML successfully predicted the BCR-ABL1 test result. These findings suggest a machine learning model trained with blood cell counts could lead to diagnosis of CML earlier in the disease course compared to usual medical care.

TKI dose reduction can effectively maintain major molecular remission in patients with chronic myeloid leukaemia.

Targeted therapy for chronic myeloid leukaemia (CML) has allowed for a near-normal patient life-expectancy; however, quality of life and aggravation of existing co-morbidities have posed new treatment challenges. In clinical practice, TKI dose reduction occurs frequently, often on multiple occasions, because of intolerance. We conducted a retrospective 'real-world practice' review of 246 patients receiving lower than standard dose (LD) TKI after the achievement of major molecular response (MR3), because of intolerable adverse events. In 274 of 298 cases of dose reduction (91·9%), MR3 was maintained at median follow-up of 27·3 months. One patient progressed to blast crisis while on LD TKI. Two patients developed two new ABL kinase domain mutations (T315I and V299L), of whom one had achieved deep molecular response on an alternative LD TKI at last follow-up. Seventy-six patients eventually discontinued LD TKI and the two-year treatment-free remission (TFR) rate in these patients was 74·1%. The majority of patients with CML in at least MR3 appear to be safely managed with LD TKI, although three of 246 patients had new events (progression and new mutation), indicating that this approach requires vigilance. TKI LD does not prevent the achievement of TFR in this patient population.

Illuminating novel biological aspects and potential new therapeutic approaches for chronic myeloproliferative malignancies.

This review reflects the presentations and discussion at the 14th post-American Society of Hematology (ASH) International Workshop on Chronic Myeloproliferative Malignancies, which took place on the December 10 and 11, 2019, immediately after the 61st ASH Annual Meeting in Orlando, Florida. Rather than present a resume of the proceedings, we address some of the topical translational science research and clinically relevant topics in detail. We consider how recent studies using single-cell genomics and other molecular methods reveal novel aspects of hematopoiesis which in turn raise the possibility of new therapeutic approaches for patients with myeloproliferative neoplasms (MPNs). We discuss how alternative therapies could benefit patients with chronic myeloid leukemia who develop BCR-ABL1 mutant subclones following ABL1-tyrosine kinase inhibitor therapy. In MPNs, we focus on efforts beyond JAK-STAT and the merits of integrating activin receptor ligand traps, interferon-α, and allografting in the current treatment algorithm for patients with myelofibrosis.

Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib.

Measurement of BCR-ABL1 transcripts on the International Scale in the United States: current status and best practices.

Chronic myeloid leukemia (CML) results from the Philadelphia chromosome (Ph) translocation and expression of its fusion oncoprotein BCR-ABL1. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the standard therapy for Ph-positive CML. Achievement of deep molecular responses (typically defined as ≥4-log reduction in BCR-ABL1 RNA levels) is an emerging treatment goal becoming attainable for more patients due to the availability of second-generation TKIs. Deep molecular responses are associated with improved long-term outcomes and are required prior to attempting cessation of treatment in treatment-free remission clinical trials. The National Comprehensive Cancer Network and European LeukemiaNet recommend regular monitoring of BCR-ABL1 RNA levels using real-time quantitative polymerase chain reaction (RQ-PCR). However, BCR-ABL1 RQ-PCR is a complex laboratory-developed test; routine quantitative results from clinical diagnostic laboratories may differ from those used to establish the recommendations. Although an International Scale (IS) was developed for standardized reporting of BCR-ABL1 RNA levels, IS adoption has been slow in the United States, but is now used by the vast majority of laboratories. Here, we discuss the importance of molecular monitoring in CML, gaps between current and best molecular monitoring practices in the United States, and challenges and potential solutions for universal IS adoption in the United States.

Controlled deposition of functionalized silica coated zinc oxide nano-assemblies at the air/water interface for blood cancer detection.

We report results of the studies relating to controlled deposition of the amino-functionalized silica-coated zinc oxide (Am-Si@ZnO) nano-assemblies onto an indium tin oxide (ITO) coated glass substrate using Langmuir-Blodgett (LB) technique. The monolayers have been deposited by transferring the spread solution of Am-Si@ZnO stearic acid prepared in chloroform at the air-water interface, at optimized pressure (16 mN/m), concentration (10 mg/ml) and temperature (23 °C). The high-resolution transmission electron microscopic studies of the Am-Si@ZnO nanocomposite reveal that the nanoparticles have a microscopic structure comprising of hexagonal assemblies of ZnO with typical dimensions of 30 nm. The surface morphology of the LB multilayer observed by scanning electron microscopy shows uniform surface of the Am-Si@ZnO film in the nanometer range (<80 nm). These electrodes have been utilized for chronic myelogenous leukemia (CML) detection by covalently immobilizing the amino-terminated oligonucleotide probe sequence via glutaraldehyde as a crosslinker. The response studies of these fabricated electrodes carried out using electrochemical impedance spectroscopy show that this Am-Si@ZnO LB film based nucleic acid sensor exhibits a linear response to complementary DNA (10(-6)-10(-16) M) with a detection limit of 1 × 10(-16) M. This fabricated platform is validated with clinical samples of CML positive patients and the results demonstrate its immense potential for clinical diagnosis.

Rationale and motivating factors for treatment-free remission in chronic myeloid leukemia.

With BCR-ABL1 tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib, many patients with chronic myeloid leukemia in chronic phase (CML-CP) can expect to live near-normal life spans. Current treatment recommendations of the National Comprehensive Cancer Network and the European LeukemiaNet state that patients with CML-CP should remain on TKI therapy indefinitely. However, there is increasing evidence from clinical trials that some patients with sustained deep molecular responses may be able to achieve treatment-free remission (TFR), whereby they can suspend TKI therapy without losing previously achieved responses. With many patients achieving deep molecular responses to TKI therapy, there is growing interest in whether such patients can achieve TFR. In addition, adverse events (AEs) with long-term TKI therapy, including both the potential for later-emerging AEs and chronic, low-grade AEs, represent a major motivator for oncologists and their patients to investigate the feasibility of TFR. In this review, we provide an overview of data from TFR clinical trials, discuss the importance of achieving a deep molecular response to TKI treatment, and consider potential reasons for investigating TFR following TKI therapy.

Molecular monitoring of chronic myeloid leukemia: present and future.

INTRODUCTION: Fusion of BCR-ABL1 genes causes chronic myeloid leukemia (CML). As a reliable marker of disease burden, it also serves as the target of tyrosine kinase inhibitors (TKIs). New more sensitive molecular diagnostic tools for BCR-ABL1 can contribute to therapeutic decision-making, especially in considering drug discontinuation for patients enjoying prolonged deep molecular response. Areas covered: Several novel platforms are transforming CML molecular diagnostics to enable faster point-of-care devices, better understanding of clonal diversity and resistance mutations. Here, we review these molecular platforms, knowing implementation in other hematological malignancies will ensue. Expert commentary: Treatment with TKI in CML is the first example of a highly effective targeted therapy. Monitoring of BCR-ABL1 mRNA is standard in assessing disease burden being highly predictive of outcomes recommended by both European LeukemiaNet (ELN) and National Comprehensive Cancer Network (NCCN); however, studies has demonstrated poor adherence to these recommendations. In both clinical practice and assay performance, further optimizing of BCR-ABL1 monitoring can be envisioned including point-of-care methods for increased availability of rapid, standardized testing and increasingly sensitive molecular assays that allow for quantification of MRD and detecting resistance mutations.

Monitoring Chronic Myeloid Leukemia in the Real World: Gaps and Opportunities.

In clinical trials, in which treatment algorithms and monitoring schedules are tightly prescribed by research protocols, outcomes for patients with chronic myeloid leukemia (CML) have been excellent, with > 90% 5-year survival rates. However, outside of clinical trials in the so-called real world, monitoring schedules are more variable, with < 40% of patients undergoing quantitative polymerase chain reaction (qPCR) molecular testing 3 to 4 times during the first year after diagnosis as recommended by National Comprehensive Cancer Network/European Leukemia Net (NCCN/ELN) evidence-based guidelines. Results from chart reviews, claims-based databases, and observational databases suggest that carefully monitored patients with CML are more likely to be adherent to medications, incur fewer hospitalizations, experience lower overall treatment costs, and have better progression-free survival and overall survival compared with patients who are not monitored. Regular monitoring provides valuable early information on treatment responses that physicians can use to modify treatment. Unfortunately, physician-perceived resource barriers, lack of familiarity, and lack of agreement have restricted monitoring guideline adoptions. Multifaceted approaches to encourage appropriate monitoring are needed to improve clinical outcomes and reduce costs in the real world.

Milestones and monitoring.

In chronic myeloid leukemia (CML), the presence of a specific chromosome marker (Ph-chromosome) as well as of the corresponding molecular marker (BCR-ABL fusion transcripts) provides suitable and precise tools to monitor the burden of the disease present at diagnosis and that of the residual disease present at specific time points during treatment. A huge number of studies have clearly demonstrated that in CML cytogenetic and molecular responses are strictly correlated to the final outcome of the patients and the correct use of standardized methods to assess the achievement of specific degrees of disease reduction at specific time points during treatment has become an essential part of proper clinical management of CML. The target to be achieved and the corresponding "optimal response" definition are however evolving, and at least for some patients, they may be represented not only by best possible overall survival (OS) but also by the possibility to discontinue the tyrosine-kinase inhibitor (TKI) treatment and therefore to live in a treatment-free remission (TFR) status. Therefore, at least for some patients, deep degrees of molecular response, as MR(4) and MR(4.5), whose precise definition has been recently introduced and that are prerequisites to try to discontinuation, are becoming the target to be achieved even in common clinical practice. As a fast initial decline of the disease burden after therapy start may be highly predictive for the final outcome of patients not only in terms of progression-free survival (PFS) and of PS but also in terms of possibility of achieving deep molecular responses, a more intense and punctual monitoring of the response of CML patients during the first 6 months of TKI therapy is now recommended by the more recent versions of the European Leukemia Net (ELN) and National Comprehensive Cancer Network (NCCN) guidelines, as this represents the major driver to decide therapy.

Molecular monitoring of chronic myeloid leukemia: international standardization of BCR-ABL1 quantitation.

The BCR-ABL1 translocation is a hallmark of chronic myeloid leukemia. Because patients treated with imatinib and other tyrosine kinase inhibitors achieve lower levels of detectable disease, quantitation of BCR-ABL1 transcripts with quantitative RT-PCR has become an essential tool in chronic myeloid leukemia monitoring. The prognostic significance of molecular responses was recently established by large-scale clinical trials. Achieving defined levels of BCR-ABL1 on the International Scale within specific time frames is an important measure for assessing patient response and probability for relapse and progression. However, extensive variation in quantitative RT-PCR procedures and reporting makes it difficult to interpret these results. More important, lack of standardization, particularly in the United States, prevents the comparison of individual patient results to the data from the clinical trials, which thereby prohibits the meaningful use of such results in the direction of patient care. In this article, we will present an overview of the clinical trial discoveries that drive the need for standardization, review the most updated monitoring guidelines by the National Comprehensive Cancer Network, and highlight recommendations for laboratory practice regarding internal controls and reference materials. Finally, we will provide an update on the recent efforts in the standardization of quantitative RT-PCR reporting using the International Scale.

BCR-ABL1 RT-qPCR for monitoring the molecular response to tyrosine kinase inhibitors in chronic myeloid leukemia.

The pathognomonic genetic alteration in chronic myeloid leukemia is the formation of the BCR-ABL1 fusion gene, which produces a constitutively active tyrosine kinase that drives leukemic transformation. Targeted tyrosine kinase inhibitor treatment with imatinib, nilotinib, dasatinib, bosutinib, and ponatinib is the cornerstone of modern therapy for this hematologic malignancy. Real-time quantitative RT-PCR (RT-qPCR, also RQ-PCR) of BCR-ABL1 RNA is a necessary laboratory technique for monitoring the efficacy of tyrosine kinase inhibitor therapy and quantitatively assessing minimal residual disease. The molecular response measured by BCR-ABL1 RT-qPCR assists in identifying suboptimal responses and can help inform the decision to switch to alternative therapies that may be more efficacious (or to pursue more stringent monitoring). Furthermore, the tyrosine kinase inhibitor-mediated molecular response provides valuable risk stratification and prognostic information on long-term outcomes. Despite these attributes, informed, universal, practical utilization of this well-established monitoring test will require heightened efforts by the molecular diagnostics laboratory community to adopt the standardized reporting units of the International Scale. Without widespread adoption of the International Scale, the consensus major molecular response and early molecular response treatment thresholds will not be definable, and optimal clinical outcomes for patients with chronic myeloid leukemia may not be achieved.

Molecular monitoring 101: helping your patients with chronic myeloid leukemia to understand the meaning of molecular response.

For patients with chronic myeloid leukemia (CML), measurement of molecular response (i.e. the level of BCR-ABL1 transcripts) is firmly established as a key element of disease monitoring. Assessment of BCR-ABL1 levels may help to identify early signs of resistance to treatment and enable a timely switch to alternative therapies. Hence, regular and accurate monitoring of BCR-ABL1 transcripts helps to maximize the chance of successful outcomes in CML. Because the incidence of CML is relatively low, many community oncologists encounter only a limited number of cases; measuring and interpreting BCR-ABL1 measurements in a clinically relevant fashion may be challenging. The team at our institution often encounters questions regarding real-time quantitative polymerase chain reaction assessments of BCR-ABL1 levels, International Scale standardization, the implications of achieving or losing molecular responses and mutation monitoring. The aim of this article is to provide practical advice for effective long-term monitoring of patients with CML by addressing frequently asked questions and common case scenarios using guideline- and evidence-based approaches.

Translating trial-based molecular monitoring into clinical practice: importance of international standards and practical considerations for community practitioners.

The success of tyrosine kinase inhibition of the BCR-ABL fusion gene with imatinib in the treatment of chronic myeloid leukemia (CML) has resulted in the use of molecular detection techniques for routine clinical management. Current clinical guidelines recommend the use of molecular testing of BCR-ABL transcript levels by quantitative real-time transcriptase polymerase chain reaction (qRT-PCR) every 3 to 6 months. However, qRT-PCR methods have not yet been standardized, particularly in the United States, where most patients are initially treated outside of academic practices. The lack of standard methods for molecular monitoring has resulted in the failure to follow National Comprehensive Cancer Network and European LeukemiaNet guideline recommendations and in the misinterpretation of test results. Standardization of molecular monitoring methods and adherence to guideline recommendations are important for optimal patient management. In this article, we provide an update on the current clinical trial results by using the molecular technique to monitor patient response. Current problems and efforts in standardizing the qRT-PCR technique and reporting are reviewed. We provide examples of potential problems of various reference laboratory reports and present recommendations for assessing molecular test results. These recommendations seem particularly important because nilotinib and dasatinib appear to have improved the molecular response in the initial treatment of CML.

Determining the rise in BCR-ABL RNA that optimally predicts a kinase domain mutation in patients with chronic myeloid leukemia on imatinib.

In imatinib-treated chronic myeloid leukemia (CML), secondary drug resistance is often caused by mutations in the BCR-ABL kinase domain (KD). As alternative therapies are available for imatinib resistance, early identification of mutations may prevent disease progression. Because most patients are routinely monitored by BCR-ABL quantitative polymerase chain reaction (PCR), it is important to define the optimal increase in BCR-ABL that should trigger mutation testing. Expert panels have provisionally recommended a 10-fold BCR-ABL increase as the trigger for mutation screening, acknowledging the lack of consensus. To address this question, we monitored 150 CML patients by quantitative PCR and DNA sequencing. Thirty-five different mutations were identified in 53 patients, and, during 22.5 months (median) of follow-up after sequencing, mutations were significantly predictive of shorter progression-free survival. An unbiased receiver operating characteristic analysis identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). The 2.6-fold threshold approximated the analytic precision limit of our PCR assay. In contrast, transcript rise cutoffs of 5-fold or greater had poor diagnostic sensitivity and no significant association with mutations. We conclude that the currently recommended 10-fold threshold to trigger mutation screening is insensitive and not universally applicable.