Monitoring Of Chronic Myeloid Leukemia Research Articles

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Establishment of a BCR-ABL p190 Copy Number Estimator for Xpert ® BCR-ABL Ultra p190 Test

Statement of the Problem: BCR-ABL p190 (e1a2) is expressed in 20-30% of Acute Lymphoblastic Leukemia (ALL) and 1-2% of Chronic Myeloid Leukemia (CML). The co-expression of p190 and p210 is rare in both CML and ALL. Xpert ® BCR-ABL Ultra p190, an automated cartridge-based test for measuring BCR-ABL p190 transcript level, is standardized to quantify the amount of BCR-ABL p190 relative to ABL1 control gene based on delta Ct in peripheral blood of patients. Since BCR-ABL p190 level is crucial for risk stratification and treatment decisions in ALL as well as diagnosis and continuous therapeutic monitoring in CML, it can be useful to obtain the copy number (CN) of BCR-ABL p190. The purpose of this study is to establish a BCR-ABL p190 CN estimator with known CN of e1a2-ABL IVT-RNA as well as to compare %BCR-ABL p190/ABL1 reporting between delta Ct-based and CN-based methods. Methodology & Theoretical Orientation: Nine levels of e1a2-ABL IVT-RNA as well as two lots of Xpert ® BCR-ABL Ultra p190 tests were used to generate standard curves for CN and %CN reporting. BCR-ABL p190 contrived samples and ALL clinical samples containing the BCR-ABL p190 transcript were examined to evaluate the CN and %CN between two lots of the Xpert ® BCR-ABL Ultra p190 tests and to compare the delta Ct-based and CN-based methods for reporting %BCR-ABL p190/ABL1. Findings: Linearity was demonstrated in Ct vs CN input for BCR-ABL p190 (R 2>0.99) and ABL1 (R 2>0.98). Less than 1.4-fold difference was exhibited for CN and %CN across two different lots. Less than 2-fold difference was observed in %BCR-ABL p190/ABL1 reporting between delta Ct-based and CN-based approaches. Conclusion & Significance: A BCR-ABL p190 copy number estimator for Xpert ® BCR-ABL Ultra p190 test was established, which will provide helpful information for diagnosis and prognosis of diseases relating to BCR-ABL p190.

Comparable b2a2/e13a2 and b3a2/e14a2 Reporting in BCR-ABL Assay When Calibrated By Who Is and By IVT-RNA Copy Number

Introduction: Xpert® BCR-ABL Ultra, automated cartridge-based assay for monitoring BCR-ABL transcript levels, is calibrated by WHO IS to standardize the %reporting for both b2a2/e13a2 BCR-ABL(e13a2) and b3a2/e14a2 BCR-ABL(e14a2) relative to control ABL gene in peripheral blood of patients as a standard management of Chronic Myeloid Leukemia (CML) [1]. Studies have shown differences in patients carrying e13a2 or e14a2 in molecular response to Tyrosine Kinase Inhibitor (TKI) treatment [2-4]. Therefore, it is necessary to understand if there is %reporting differences between two transcripts calibrated by WHO IS[5] and explore the calibration method using breakpoint specific RNA input copy number (CN) for accurate TKI treatment monitoring. Objectives: To establish % reporting method calculated with known CN of e13a2, e14a2 and ABL IVT-RNA within a wide CN range and compare % e13a2 and % e14a2 to WHO IS quantification system. Methods: Three IVT-RNAs, containing e13a2-ABL-BCR, e14a2-ABL-BCR and ABL-BCR, were synthesized as shown in Figure 1. Serial diluted e13a2 RNA, e14a2 RNA and ABL RNA panels were tested on Xpert BCR-ABL Ultra assay to generate individual standard curve of e13a2 Ct, e14a2 Ct and ABL Ct as a function of CN for % reporting calculation. Four levels of IVT-RNA panels with same CN of e13a2 and e14a2 were tested for breakpoint specific % reporting comparison. K562(e14a2), BV173 (e13a2) cell lysates and CML clinical samples carrying e13a2 or e14a2 transcripts were tested to evaluate the %CN reporting compared to WHO IS. Result: Good linearity demonstrated in Ct vs CN input for e13a2, e14a2 and ABL IVT-RNA (Figure2) with comparable efficiency (E) between e13a2 (E=0.992) and e14a2 (E=0.986). %e13a2 reporting was ~1.50-fold (by WHO IS) and ~1.46-fold (by %CN) higher than %e14a2, by testing IVT-RNA panel (Table1). Minor differences in %reporting observed between % CN and WHO IS for e13a2 (84.5%~110.8%) vs e14a2 (82.5%~89.5%) from cell lysates (Table2) and e13a2 (92.6%~105.5%) vs e14a2 (88.1%~92.2%) from clinical samples (Table3). Conclusions: A %CN e13a2/ABL and e14a2/ABL reporting method with known CN of IVT-RNAs has been successfully established in Xpert BCR-ABL Ultra. Both WHO IS and %CN showed very minor differences between e13a2 and e14a2 for % reporting. %CN method demonstrated comparable %reporting to WHO IS in Xpert BCR-ABL Ultra. Recent Publications 1. Dominy KM, Simon IM, Khorashad JS (2021) Evaluation of Xpert® BCR-ABL Ultra for the confirmation of BCR-ABL1 international scale conversion factors for the molecular monitoring of chronic myeloid leukaemia, Int J Lab Hematol, Feb;43(1):e31-e34. 2. Hanfstein B, Lauseker M, Hehlmann R, Saussele S, Erben P, Dietz C (2014) Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib. Haematologica.99:1441-7. 3. Jain P, Kantarjian H, Patel KP, Gonzalez GN, Luthra R, Shamanna RK (2016) Impact of BCR-ABL transcript type on outcome in patients with chronic-phase CML treated with tyrosine kinase inhibitors. Blood.127:1269-75. 4. Castagnetti F, Gugliotta G, Breccia M, Iurlo A, Levato L, Albano F (2017) The BCR-ABL1 transcript type influences response and outcome in Philadelphia chromosome-positive chronic myeloid leukemia patients treated frontline with imatinib. Am J Hematol. 92:797-805. 5. Matthew S, White HE, Hana Z, Nicholas C.P C (2022) Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy. Leukemia 36(7):1-8

Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

Imatinib dose optimization based on therapeutic drug monitoring in Chinese patients with chronic-phase chronic myeloid leukemia.

Imatinib treatment often produces various adverse reactions in patients with chronic myeloid leukemia (CML), and increasing patients are pursuing dose optimization. In this study, the authors aimed to explore imatinib dose optimization based on therapeutic drug monitoring (TDM) in CML patients. The relationship between imatinib concentration and clinical response and adverse reactions was evaluated, then the dose-reduction data in 110 Chinese CML patients was also explored. Patients with a major molecular response (MMR) had higher imatinib plasma concentration compared with those not achieving MMR (1473.70±419.13 vs. 985.8±213.32ng/ml) when receiving 400mg daily. Imatinib concentration >1000ng/ml predicted improved event-free survival and failure-free survival. In addition, imatinib concentration was significantly correlated with leukopenia or neutropenia, diarrhea, edema, and rash. Patients receiving imatinib concentration >1685ng/ml were more susceptible to diarrhea and those with levels >1575ng/ml were more susceptible to periorbital and limb edema. Thirty-nine (35.5%) patients underwent low-dose therapy and seven (6.4%) patients received discontinuation therapy. Patients with a higher imatinib concentration were more likely to maintain MMR or deep molecular response after dose reduction. No significant difference in molecular relapse-free survival rate was observed between the low-dose and standard-dose groups over 1year and 2years. Furthermore, most adverse reactions significantly improved after dose reduction. Imatinib concentration was closely associated with clinical response and adverse reactions, suggesting that dose optimization based on TDM might achieve beneficial clinical outcomes. Dose reduction based on TDM is feasible and safe for patients exhibiting optimal response, which could improve adverse reactions.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

Health economic evidence for the use of molecular biomarker tests in hematological malignancies: A systematic review.

ObjectivesMolecular biomarker tests can inform the clinical management of genomic heterogeneous hematological malignancies, yet their availability in routine care largely depends on the supporting health economic evidence. This study aims to systematically review the economic evidence for recent molecular biomarker tests in hematological malignancies.MethodsWe conducted a systematic search in five electronic databases for studies published between January 2010 and October 2020. Publications were independently screened by two reviewers. Clinical study characteristics, economic methodology, and results were extracted, and reporting quality was assessed.ResultsFourteen studies were identified, of which half (n = 7; 50%) were full economic evaluations examining both health and economic outcomes. Studies were predominantly conducted in a first‐line treatment setting (n = 7; 50%) and adopted a non‐lifetime time horizon to measure health outcomes and costs (n = 7; 50%). Five studies reported that companion diagnostics for associated therapies were likely cost‐effective for acute myeloid leukemia, chronic myeloid leukemia, diffuse large B‐cell lymphoma, and multiple myeloma. Four studies suggested molecular biomarker tests for treatment monitoring in chronic myeloid leukemia were likely cost‐saving.ConclusionsAlthough there is initial confirmation of the promising health economic results, the present research for molecular biomarker tests in hematological malignancies is sparse with many applications of technological advances yet to be evaluated.

NATURAL KILLER CELLS PHENOTYPE CORRELATES WITH TYROSINE KINASE INHIBITORS TREATMENT RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS

Despite well-known Natural Killer (NK) cells antitumor activity, its potential in regulating Chronic Myeloid Leukemia (CML) is not fully stablished. Tyrosine kinase inhibitors (TKI) treatment, besides reducing BCR-ABL1 transcripts, seems to result in immunomodulation that partly restore the immune dysregulation of the disease. Numerical, functional and maturation defects might be associated with CML risk stratification, molecular response profile or even with resistance to treatment with TKI. Therefore, we aimed to immunophenotypically characterize frequency, subtypes and maturation profile of NK cells in CML patients, by Multiparametric Flow Cytometry (MFC), during monitoring of TKI treatment. 11 Peripheral Blood samples from patients with CML (Clinical Hospital of Ribeirão Preto - HCRP-USP) and 10 healthy donors (controls) were analyzed. Clinical, laboratory and ELN2017 risk stratification were assessed. NK cells were defined as CD19-CD3-CD56+ and: CD56brightCD16- (secretory), CD56dimCD16+ (cytotoxic), CD11b-CD27- (tolerant); CD27+CD11b- (immature secretor), CD27+CD11b+ (secretory) and CD11b+CD27- (cytotoxic). Based on molecular response levels assessed by BCR-ABL1 RT-qPCR, patients were classified in G1 (without loss of molecular response to the 1 st line of TKI; n = 3), G2 (loss of molecular response to 1 st line of TKI; n = 3) and G3 (loss of molecular response to 2 nd generation TKI; n = 5). Mann Whitney or Kruskal-Wallis tests (p < 0.05) were applied using SPSS (v.20). A higher frequency of lymphocytes in CML patients was observed when compared to controls (p = 0.0242), but no difference was found in the frequency of NK cells. G1 and G2 showed a higher frequency of NK cells than G3 (p = 0,7774). Even though no statistical significance was found, CD27-CD11b- and CD27+CD11b- NK cells showed a higher frequency, while CD27+CD11b+ NK cells showed a lower frequency in CML when compared to controls. Frequency of CD27+CD11b- NK cells was higher in G2 and G3 when compared to G1. G3 showed an immature profile with higher percentages of CD27-CD11b- and CD27+CD11b- NK cells when compared to controls, while the more mature CD27+CD11b+ NK cells showed a lower frequency in CML. Furthermore, a lower frequency of CD56brightCD16- (p = 0.0002) and CD56dimCD16+ (p = 0.0448) NK cells was observed in CML, as wells as the frequency of CD27-CD11b+ cytotoxic NK cells, consistent to what we found in G3 and supporting our hypothesis. We observed that patients with loss of molecular response in treatment with 1 st and 2 nd TKI generation presented an immature non-effective NK cell profile, with deficient cytotoxicity. This scenario might result in impaired tumor surveillance of CML, meaning that phenotypic changes in NK cells may directly affect risk stratification and treatment response in CML. However due to the small number of patients and the lack of a functional assay, it is not possible to confirm that the cytotoxic cell may actually be deficient. Our findings and further functional studies can contribute to a better understanding of the physiopathology, prognosis and monitoring of CML, response to TKI therapy, as well as help to define therapeutic strategies based on immunotherapy mediated by NK cells.

Economic Modeling of the Potential Impact of Chronic Myeloid Leukemia (CML) Monitoring on Healthcare Costs

Introduction: Molecular monitoring (qPCR testing) of patients with chronic myeloid leukemia (CML) is necessary, as it allows physicians to optimize treatment and make timely changes to therapy. National Comprehensive Cancer Network (NCCN) guidelines recommend molecular testing as often as every 3 months, but previous studies indicate that only approximately 30% of patients receive testing in accordance with these guidelines and up to 50-79% of patients do not receive testing within 3-6 months of starting treatment (Latremouille-Viau D, et al. J Manag Care Spec Pharm 2017, Guérin A, et al. J Med Econ 2014, Di Bella NJ, et al. Leuk 2015, Saleh MN, et al. Curr Med Res Opin 2014). Appropriate molecular testing has been shown to be associated with a lower risk of disease progression (Haque R, et al. J Am Pharm Assoc 2017).Studies have also shown that testing according to guidelines is expected to lead to cost savings (Latremouille-Viau D, et al. J Manag Care Spec Pharm 2017).Finally, previous research has concluded that the total cost of care for patients with CML progression is significantly greater than for patients with no evidence of CML progression (Jabbour EJ, et al. J Med Econ 2017).Currently data that can directly link these elements are lacking, thus an economic model was developed to estimate the potential impact of poor monitoring on patient healthcare costs.Methods: An Excel based decision analytic economic model was developed to evaluate the potential impact of CML monitoring on healthcare costs from a U.S. payer perspective. This model calculates the expected cost savings from molecular monitoring of CML patients as compared to not monitoring. The following model inputs were estimated or obtained based on published literature (Haque R, et al. J Am Pharm Assoc 2017, Jabbour EJ, et al. J Med Econ 2017): CML progression rates among patients with vs. without monitoring, the annual cost of CML progression, the average number of monitoring tests per year, and the average cost per CML monitoring test. Such information was used to assess the total healthcare cost and the cost differences per patient among patients with vs. without monitoring. The total healthcare costs were defined as the combined costs of CML monitoring tests and CML progression. Additionally, univariate and multivariate sensitivity analyses were conducted to test the robustness of the results by varying the model inputs.Results: Estimated average CML progression rates of 0.35% per patient year for patients with monitoring and 5.12% per patient year among patients without monitoring were used in the calculations (Table). The annual incremental cost of CML progression per patient of $136,308 was obtained from the published literature. The average number of monitoring tests per year was estimated at 3 and 0 among patients with and without molecular monitoring, respectively, with an average cost of $223 per monitoring test. The total healthcare costs including costs associated with CML progression and molecular monitoring tests were estimated to be $1,142 for patients with monitoring and $6,982 for patients without monitoring (difference = $5,840). In a hypothetical cohort of 100 CML patients, achieving a 90% monitoring rate (i.e., 90 out of 100 patients were monitored) was associated with a total cost reduction of $525,604 as compared to a 0% monitoring rate. This cost reduction remained consistent under both univariate and multivariate sensitivity analyses, demonstrating the validity of the model calculations.Conclusion: Improving CML monitoring rates is associated with improved patient outcomes in CML and therefore reduced healthcare costs. [Display omitted] DisclosuresJabbour:Bristol-Myers Squibb: Consultancy. Siegartel:Bristol-Myers Squibb: Employment. Lin:Novosys Health: Employment; Bristol-Myers Squibb: Consultancy. Lingohr-Smith:Novosys Health: Employment. Menges:Novosys Health: Employment. Makenbaeva:Bristol-Myers Squibb: Employment.

Impact of Earlier Vs. Later Monitoring on Disease Progression and Economic Outcomes Among Patients with Chronic Myeloid Leukemia in the Real-World Setting

Introduction: The National Comprehensive Cancer Network recommends monitoring of patients with chronic myeloid leukemia (CML) beginning at diagnosis. Such testing is necessary for optimal treatment selection, however, patients are often not monitored until later in the course of disease or when clinical symptoms present. In this study, we evaluated the impact of molecular monitoring earlier in the course of disease as compared to later monitoring with the aim to determine a potential association between timing of monitoring and CML disease progression and healthcare resource utilization in a real-world setting in the US.Methods: Patients (≥ 18 years of age) with ≥1 inpatient or ≥2 distinct outpatient (≥30 days apart) diagnoses for CML, January 01, 2006 through June 30, 2016, were identified from the MarketScan® databases. Patients were defined as having disease progression based on the presence of treatments for advanced phase and blast crisis CML. A case control study design was used; cases were defined as patients with disease progression and patients not captured as having progression were identified as controls. Among cases, the index date was defined as the first date of treatment for CML progression and among controls, the index date was a randomly selected date (using simple random sampling) after the first CML diagnosis. Patients were required to have 24 months of continuous medical and prescription insurance coverage prior to the index date (baseline period). Patients were also required to have ≥1 CML monitoring test in the baseline period. Patients with earlier monitoring were identified as those with ≥1 monitoring test during the 7-24 months prior to the index date (Figure); patients with later monitoring were identified as those with ≥1 monitoring test only during the last 6 months prior to the index date and not during the 7-24 months prior to the index date (Figure). Patient demographics and clinical characteristics were evaluated during the baseline period. All-cause healthcare resource utilization and associated costs were evaluated per patient per month (PPPM) during the baseline period and a variable length follow-up period lasting ≥1 day and up to 1 year. Multivariate generalized linear modeling (GLM) was used to evaluate differences in all-cause healthcare costs between the earlier vs. later monitoring cohorts. Multivariate logistic regression was used to evaluate the association between monitoring status and progression.Results: A total of 2,730 CML patients were identified, among which 60% (n=1,633) received earlier monitoring and 40% (n=1,097) received later monitoring only. The two groups were similar in terms of mean age and gender: 56 years vs. 57 years and 56% vs 57% females among patients with earlier vs. later monitoring, respectively. The mean Charlson Comorbidity Index score was lower among patients with earlier vs. later monitoring (3.15 vs. 3.41, p=0.0001), which indicates that patients with earlier monitoring were healthier. The three most frequently observed comorbid conditions of cardiovascular disease, gastrointestinal disease and lipid abnormalities were present in 67%, 62% and 44%, respectively, of patients with earlier monitoring vs. 71%, 66% and 52% of patients with later monitoring. Evidence of progression was present in 8.9% of patients with earlier monitoring, vs. 9.3% of patients with later monitoring. After the multivariable regression model was used to adjust for patient characteristics, patients with earlier vs. later monitoring were shown to be associated with a lower likelihood of CML progression (odds ratio: 0.715, confidence interval (CI) [0.530 - 0.964]; p=0.0280), during the follow-up period. After adjusting for covariates, the mean all-cause healthcare costs (PPPM) for inpatient (hospitalization), outpatient medical service, total (inpatient + outpatient) medical service, outpatient pharmacy and total healthcare were lower for patients with earlier monitoring as compared to those with later monitoring (Table).Conclusion: Patients who are monitored earlier in the course of their disease may have better outcomes and lower total cost of care, as evidenced by the lower likelihood of CML progression and lower healthcare resource utilization in this group as compared to patients with later monitoring only. [Display omitted] DisclosuresJabbour:Bristol-Myers Squibb: Consultancy. Siegartel:Bristol-Myers Squibb: Employment. Lin:Novosys Health: Employment; Bristol-Myers Squibb: Consultancy. Lingohr-Smith:Novosys Health: Employment. Menges:Novosys Health: Employment. Makenbaeva:Bristol-Myers Squibb: Employment.

Philadelphia chromosome positive chronic myeloid leukemia with 5q deletion at diagnosis

BackgroundAlthough, molecular genetic analyses became more and more important to guide therapy decisions in leukemia, banding cytogenetic analysis has retained its vital role in diagnosis and monitoring of chronic myeloid leukemia (CML), by quick and easy enabling identification of pathognomonic Philadelphia chromosome (Ph).Case presentationA 45 year old female presented with characteristic hematological features of CML in chronic phase; cytogenetic studies revealed the presence of the typical Ph and a deletion of almost entire long arm of a chromosome 5.Conclusion5q deletions have rarely been reported in CML. Those seen yet were either associated with tyrosine kinase inhibitor therapy or detected post allogeneic stem cell transplantation. To our knowledge, this is the first case of Ph positive CML accompanied by a 5q deletion.

Chronic myeloid leukemia-from the Philadelphia chromosome to specific target drugs: A literature review.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm and was the first neoplastic disease associated with a well-defined genotypic anomaly ― the presence of the Philadelphia chromosome. The advances in cytogenetic and molecular assays are of great importance to the diagnosis, prognosis, treatment, and monitoring of CML. The discovery of the breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) 1 fusion oncogene has revolutionized the treatment of CML patients by allowing the development of targeted drugs that inhibit the tyrosine kinase activity of the BCR-ABL oncoprotein. Tyrosine kinase inhibitors (known as TKIs) are the standard therapy for CML and greatly increase the survival rates, despite adverse effects and the odds of residual disease after discontinuation of treatment. As therapeutic alternatives, the subsequent TKIs lead to faster and deeper molecular remissions; however, with the emergence of resistance to these drugs, immunotherapy appears as an alternative, which may have a cure potential in these patients. Against this background, this article aims at providing an overview on CML clinical management and a summary on the main targeted drugs available in that context.

Digital PCR for BCR‐ABL1 Quantification in CML: Current Applications in Clinical Practice

Molecular monitoring of the BCR-ABL1 transcript for patients with chronic phase chronic myeloid leukemia (CML) has become increasingly demanding. Real-time quantitative PCR (qPCR) is the routinely used method, but has limitations in quantification accuracy due to its inherent technical variation. Treatment recommendations rely on specific BCR-ABL1 values set at timed response milestones, making precise measurement of BCR-ABL1 a requisite. Furthermore, the sensitivity of qPCR may be insufficient to reliably quantify low levels of residual BCR-ABL1 in patients in deep molecular response (DMR) who could qualify for an attempt to discontinue Tyrosine Kinase Inhibitor (TKI) therapy. We reviewed the current use of digital PCR (dPCR) as a promising alternative for response monitoring in CML. dPCR offers an absolute BCR-ABL1 quantification at various disease levels with remarkable precision and a clinical sensitivity reaching down to at least MR5.0. Moreover, dPCR has been validated in multiple studies as prognostic marker for successful TKI treatment discontinuation, while this could not be achieved using classical qPCR. dPCR may thus prospectively be the preferred method to reliably identify patients achieving treatment milestones after initiation of TKI therapy as well as for the selection and timing for TKI discontinuation.

Inclusion of molecular monitoring (BCR-ABL1) in the treatment of chronic myeloid leukemia in the Brazilian Public Health System (SUS): an urgent need for treatment management

IntroductionChronic Myeloid Leukemia (CML) is a myeloproliferative disease that affects mainly adults between 50 and 55 years. In Brazil, information from the Sistema Único de Saúde (SUS) Outpatient Information System indicates that 12,531 patients had the Autorização de Procedimento Ambulatorial (APAC) approved for the CML treatment in 2017. Disease monitoring through molecular response evaluation is critical to the care of CML patients. The quantitative PCR test (real-time polymerase chain reaction) provides adequate evaluation parameters that allow the health professional to intervene at the right moments in order to reduce the chance of progression of the disease, providing the best outcome to the patient, including the possibility of treatment discontinuation for eligible patients. Although the test is included in the Clinical Protocol and Therapeutic Guidelines (PCDT) of CML, it is not possible to monitor the molecular response within SUS since there is no reimbursement for this test.ObjectiveObtain expert recommendations on the importance, financing, and reimbursement of molecular monitoring in SUS.MethodsSix CML experts with different perspectives participated in the panel. The discussion was based in the main publications about the quantitative PCR test in CML monitoring.ResultsExperts’ recommendations:1)Molecular monitoring should be part of the integral treatment of patients with CML to reduce the chances of disease progression and costs to the health system;2)The government should put into practice what is provided in the PCDT of Chronic Myeloid Leukemia in Brazil: performing the monitoring of the molecular response via quantitative PCR;3)The government should create a code with adequate nomenclature and reimbursement value in SIGTAP, so that the test is carried out and covered by the public health network, as it is contained in the PCDT of the disease and the existing APAC does not cover the operational costs for its performance;4)Patients with chronic phase CML should perform a quantitative PCR every 3 months and, after reaching the MMR, should perform the examination every 6 months, as recommended by international guidelines;5)Patients should be monitored in reference laboratories that are standardized according to the international scale;6)The laboratories that are within the reference public centers could absorb all the test demand in Brazil, and other centers could be qualified through an ABHH accreditation;7)Adequate molecular monitoring may allow some patients to stop taking drugs and selffinancing the molecular test for all SUS patientsConclusionA solution for the molecular test (BCR-ABL1) funding is urgent to ensure the monitoring of CML patients in SUS. The savings that might be generated with patients that stop taking the medication when adequately monitored may finance the test.

A Comparison of Droplet Digital PCR and RT-qPCR for BCR-ABL1 Monitoring in Chronic Myeloid Leukemia

Background: Monitoring of BCR-ABL1 molecular levels is essential for the management of Chronic Myeloid Leukemia (CML) patients treated with Tyrosine Kinases Inhibitors (TKIs). Real Time Quantitative PCR (RT-qPCR) is currently the standard method for assessing molecular remission (MR) in patients with CML. Recently, droplet digital PCR (ddPCR) has emerged to provide a more accurate detection of minimal residual disease (MRD). In order to hypothesize the use of this new technology in the clinical practice, in the era of TKI discontinuation, we designed a multi-centric study to evaluate the potential value of ddPCR in diagnostic routine. Aims of the study were:1) the evaluation of the agreement between the measures obtained by ddPCR and RT-qPCR and 2) the assessment of the repeatability of the two methods. Methods: Total RNA was extracted from 37 CML patients using Maxwell 16 LEV simplyRNA Blood kit (Promega), following the manufacturer's instructions. Samples were divided in 5 groups based on molecular response (MR) as follow: group 1, MR&lt;3, n=5; group 2, MR 3, n=5; group 3, MR 4, n=9; group 4, MR 4.5, n=9 and group 5, MR 5, n=9. BCR-ABL1 p210 was quantified by RT-qPCR and ddPCR in 3 different Italian laboratories namely Lab A, Lab B and Lab C. Lab B and C performed 1 amplification session each, while Lab A performed 3 independent sessions. All ddPCR experiments were performed using Kit QXDxTM BCR-ABL%IS (Bio-Rad) on the QX200 system (Bio-Rad), according to the manufacturer's instructions. For RT-qPCR experiments, BCR-ABL1 p210 was quantified with three different methods: by using the One-Step BCR-ABL P210 ELITe MGB Kit (ELITech Group), according to manufacturer's protocol (Lab A); by using the One-Step Philadelphia SensiQuant Kit (Bioclarma), according to the manufacturer's instructions (Lab B) and as described in Gabert et al (Lab C). The results obtained were corrected for the laboratory-specific conversion factor, as recommended by Standard Operating Procedures of Labnet CML network (GIMEMA group). The target quantification for each sample was expressed as BCR-ABL1/ABL1 %IS for both RT-qPCR and ddPCR. Statistical analysis: Bland-Altman analysis was performed. For the measurement of the agreement we reported the bias, which is the mean of the difference between the methods, the 95% limits of agreement and the coefficient of variation. Residual variance and coefficients of repeatability (i.e. the upper limits of a prediction interval for the absolute difference between two measurements by the same method on the same item) were computed to achieve the second endpoint. An analysis of sensitivity on the labs was also carried out. All analyses were stratified by the level of disease. Results: A total of 370 measures were included in the analysis, 185 for ddPCR and 185 for RT-qPCR divided as follow: 50 for group 1 and for group 2, 90 for group 3, 4 and 5. In Table 1 we reported the median and interquartile range (IQR) for all levels of disease. Results of the Bland-Altman analysis are shown in Table 2. The coefficients of variation, which expresses the standard deviation as a percentage of the mean, were 2.35, 2.31, 1.10, 1.34, 39.12 in group 1, 2, 3, 4 and 5 respectively. The repeatability coefficients of ddPCR were smaller than qRT-PCR across all the levels of disease, showing a slightly better precision of ddPCR (Table 2). Conclusions: Higher coefficients of variation in group 1 and 2 were probably due to a greater heterogeneity of the patients. In fact, BCR-ABL1/ABL1 levels by RT-qPCR ranged between 1.43 and 6.94 and between 0.10 and 0.25 in group 1 and in group 2 respectively (Table 1). Sensitivity analysis showed that the high coefficient of variation in group 5 can be explained almost all by the variability observed in Lab B. Coefficient of repeatability of ddPCR was always smaller than RT-qPCR for all level of disease showing a slightly better precision. Our results showed that ddPCR has a good agreement with RT-qPCR and it is more precise to quantify BCR-ABL1 transcript levels, particularly for MR 4 and MR 4.5. Thus, ddPCR may be valuable in diagnostic routine. Disclosures Fava: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Martino:Bio-Rad: Employment. Saglio:Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy. Martinelli:Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Molecular Monitoring of Chronic Myeloid Leukemia.

Molecular diagnosis and measurement of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) is essential for clinical management. In the era of tyrosine kinase inhibitor therapy molecular tests including BCR-ABL1 transcript monitoring and kinase domain mutation analysis are the main tools used to inform choice of treatment, appropriate dosage and even whether therapy can be safely withdrawn. Quantitation of BCR-ABL1 oncogene transcript by real-time quantitative PCR (qPCR) is currently the gold-standard method for monitoring as it provides superior sensitivity over karyotyping and fluorescent in situ hybridization (FISH). Here we describe step-by-step methods of RNA conversion to cDNA along with the qPCR protocol which is used in one of the main reference laboratories for this test.

Differences in treatment and monitoring of chronic myeloid leukemia with regard to age, but not sex: Results from a population-based study.

There are established guidelines for treatment and monitoring of chronic myeloid leukemia (CML) but little is known about routine care. Data on ICD-10 codes as well as prescribed medications were available for 10.5million patients in the statutory health insurance system in Bavaria for the years 2010 to 2016. Also, data on the molecular and cytogenetic monitoring were integrated. A total of 1714 adult patients with CML were observed. Only 50.8% received more than 67.5 daily doses per quarter year (target: 91.5) while 18.2% did not receive any tyrosine kinase inhibitor (TKI). The median number of daily doses was at least 80 doses per quarter year for all age groups in men, but decreased to 62 doses in elderly women. With this exception, no differences between men and women were observed. The percentage of patients without any TKI increased with age. The median number of molecular examinations was 3.54 independent of age and sex. Even in a highly developed country, still a considerable number of patients with CML seem to not receive adequate treatment, whereas molecular monitoring can be considered satisfactory.

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA

This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration–cleared assay designed to monitor breakpoint cluster region–Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients.

Molecular Monitoring of Chronic Myeloid Leukemia in Chronic Phase (CML-CP)

Objective: Quantification of the BCR-ABL transcript is recommended to follow-up CML patients that treated by Imatinib mesylate (IM) as a tyrosine kinase inhibitor. BCR-ABL transcripts have been recognized as a molecular marker for response to therapy in CML patients (pts). Monitoring of this marker to be more effective for identifying optimal responses and can help to inform the decision to switch to alternative therapies. Quantitative reverse transcriptase PCR (Q-PCR) of BCR-ABL1 RNA is a critical laboratory technique for accurate and sensitive monitoring of the efficiency of tyrosine kinase inhibitor therapy. The aim of our study was to analyze the molecular response (MR) in Kurdish CML patients who are treated with Imatinib.Materials and Methods: We studied 60 blood samples from CML patients in chronic phase (CP), 36 females and 24 males, under IM treatment and monitored by Q-PCR on 12 months. The median duration of CML was 5 years (range: 1-15). The median duration of IM treatment was 4 years (range: 1-10). Results: 40% (24 pts), 28.33% (17 pts) and 15% (9 pts) and respectively had reached early molecular response (EMR) at 1.0-2.0 log, major molecular response (MMR) at 3.0 log and deep molecular response (DMR) at 4.0-5.0 log and also undetectable BCR‑ABL1 levels (CMR) were achieved in 16.67% (10 pts) at 12 months.Conclusion: We highlighted the possibility to use of Q-PCR as a warning at diagnosis, and may use to identify patients who could benefit from a more scrupulous follow-up.

Molecular monitoring in CML: how deep? How often? How should it influence therapy?

With the advent of tyrosine kinase inhibitors (TKIs), the goals of therapy in chronic myeloid leukemia (CML) are steadily shifting. Long-term disease control on TKI therapy has been the goal and expectation for most patients. More recently, treatment-free remission (TFR) has entered mainstream practice and is increasingly being adopted as the main goal of therapy. This therapeutic shift not only influences TKI selection but also, has necessitated the refinement and dissemination of highly sensitive and accurate molecular monitoring techniques. Measurement of BCR-ABL1 messenger RNA expression through reverse transcription quantitative polymerase chain reaction, reported according to the International Scale, has become the primary tool for response assessment in CML. Achieving specific time-dependent molecular milestones, as defined by global therapeutic guidelines, has been established as critical in maximizing optimal outcomes while identifying patients at risk of therapy failure. Depth and duration of a deep molecular response have become the new therapeutic targets in patients considered for TFR. Consequently, molecular monitoring in CML has become even more critical to ongoing response assessment, identifying patients with TKI resistance and poor drug adherence, and enabling TFR to be attempted safely and effectively.