Response In Chronic Myeloid Leukemia Patients Research Articles

Correlation of T Regulatory Cells, Cytotoxic T-lymphocyte-associated Antigen 4, and Transforming Growth Factor-β1 with Treatment Response in Patients with Chronic Myeloid Leukemia Receiving Dasatinib Therapy.

Tyrosine kinase inhibitors improve chronic myeloid leukemia (CML) outcomes. Dasatinib inhibits breakpoint cluster region-Abelson 1 proto-oncogene tyrosine kinase better than imatinib in CML. T-regulatory cells prevent autoimmune diseases and aberrant immune responses by reducing oncoprotein antigen reactivity. They also reduce self-antigen-induced immune responses to maintain peripheral tolerance. In this study, T-regulatory cells in peripheral blood of chronic myeloid leukemia-chronic phase patients were measured, together with serum levels of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and transforming growth factor (TGF)-β1 at diagnosis and 3 months postdasatinib therapy. The Pathology and Clinical Haematology Departments at King George's Medical University, Lucknow, India, conducted this prospective analytical study. Forty CML-chronic patients and 10 healthy controls were analyzed. Flow cytometry was used to determine T-regulatory cell percentage in peripheral blood mononuclear cells of newly diagnosed CML patients before and after 3 months of dasatinib treatment; ELISA was used to measure serum levels of CTLA-4 and TGF-β1. T-regulatory cells, CTLA-4, and TGF-β1 significantly decreased in CML-chronic phase patients after 3 months of dasatinib therapy compared to the initial diagnosis. No significant change in T-regulatory cell, CTLA-4, or TGF-β1 percentages were seen between responders and poor responders. However, responders had a lower percentage of T-regulatory cells than suboptimal responders. The study concluded that dasatinib treatment improved response in CML patients with decreased Treg cells. Dasatinib reduces Treg-mediated immunological suppression, reducing CTLA-4 and TGF-β1 levels.

Successful maintenance of a sustained molecular response in CML patients receiving low-dose tyrosine kinase inhibitors.

The development of tyrosine kinase inhibitor (TKI) therapy has positively impacted the survival rates of patients with chronic myeloid leukemia (CML). It is common in medical practice to adjust the dosage of TKI downward because of TKI-associated adverse events, financial burden, comorbidity, or an attempt at treatment-free remission. This investigation sought to explore the feasibility of employing a reduced dosage of TKI for treating CML. This was a retrospective study. Patients with CML in its chronic phase who had been on a reduced dose of TKI for a minimum of 3 months for various reasons in a practical clinical environment, irrespective of molecular response, were included. Regular molecular monitoring was performed, and changes in adverse events were recorded after dose reduction. This research included a total of 144 participants. Upon reducing the dosage, 136 of 144 patients achieved major molecular response or deeper, and 132 of 144 achieved molecular response 4 (MR4). Following a median observation period of 16 months, the calculated 1- and 2-year survival rates free from MR4 failure were estimated to be 96.5% (95% CI: 90.8-98.7) and 90.5% (95% CI: 81.3-95.3), respectively. MR4 failure-free survival was better in patients with longer MR4 durations (⩾34 months) before dose reduction (p = 0.02). The median interval from dose reduction to MR4 loss was 15 months. Improved TKI-associated adverse events after dose reduction were observed in 61.3% of patients. Lowering the TKI dose can effectively preserve a deep molecular response over time while relieving adverse events caused by TKIs.

Mechanism of Late Recurrences of Chronic Myeloid Leukemia after Discontinuation of TKI Therapy: BCR::ABL1 Ins35bp Loss-of-Function Splicing Mutation and Regulatory T Cells

Introduction: Treatment-free remission (TFR) as one of the goals of treatment of BCR::ABL-positive chronic phase chronic myeloid leukemia (CML) has been proposed. Actually, approximately 40-60% of CML patients maintain TFR without recurrence after discontinuation of tyrosine kinase inhibitors (TKIs). In some of such patients, there are fluctuated cases in which BCR::ABL positive clones continue to be detected for long periods at levels of international scale (IS) BCR::ABL < 0.1%. Such fluctuated cases show no apparent molecular recurrence and maintain TFR for a long time, but they have also been shown to be more likely to eventually develop late recurrence (Haematologica 2022, Blood adv. 2020). We have reported that a higher percentage of PD-1 positive CD8 + T cells before and after TKI discontinuation is associated with molecular recurrence, and together with the possibility of assessing host immune response capacity after TKI discontinuation by using regulatory T (Treg) cells (Fujioka, et al. Cancers 2021). On the other hands, mutations in BCR::ABL caused by the alternative splicing, such as BCR::ABL Ins35bp, are known to be one of the TKI resistance in CML. Loss of BCR::ABL kinase activity is thought to result in loss of TKI sensitivity and thus failure to achieve deep molecular remission (Yuda, et al. Cancer Science 2017). In this study, we analyzed the mutations that cause splicing variants such as BCR::ABL Ins35bp and host immune response in CML patients to elucidate molecular mechanisms in the long-term maintenance of TFR and late recurrence. Methods: Twenty-four CML patients who discontinued TKI treatment for any reason at Akita University Hospital were included in the study. The trends of IS level after TKI discontinuation were monitored, and immune profile analysis by multi-color flow cytometry (FCM) was performed in all patients. Mutation analysis of the BCR::ABL region including splicing variants was performed in selected patients. Mutation analysis was performed on samples with sufficient cell count and IS > 0.0032% (MR 4.5), and the BCR::ABL region of these samples was amplified by long nested PCR and then analyzed by the next-generation sequencer (NGS). Results: First, BCR::ABL Ins35bp was evaluated by mutation analysis. Among CML patients who underwent mutation analysis, BCR::ABL Ins35bp was detected in all cases in which the samples could perform analysis at newly diagnosis (ND). Even after TKI discontinuation, a certain percentage of BCR::ABL Ins35bp was detected in all patients, and the percentage of BCR::ABL Ins35bp increased at the time of recurrence regardless of whether early or late. These results suggest that function-dead BCR::ABL Ins35bp clones may remain in CML patients with detectable BCR::ABL under and after TKI treatment, and eventually leading to recurrence with transcriptional promotion of native BCR::ABL. On the other hand, there were cases in which the BCR::ABL Ins35bp was lost at the time of recurrence. There is a possibility of immunological involvement in such cases. CML patients with detectable BCR::ABL in the TFR for a long time had a decreased immunosuppressive Treg cells, while analysis using late recurrence increased Treg cells prior to recurrence (figure). These suggest the presence of cases in which early recurrence is suppressed by host immune responses. Conclusion: Although the loss of function mutations such as BCR::ABL Ins35bp was previously thought to occur after TKI administration as a cause of TKI resistance, this analysis reveals that the clone is included from the ND before TKI treatment. Examining the BCR::ABL Ins35bp clone at ND may be possible to predict the possibility of recurrence after TFR. Host immune capacity, as assessed by the kinetics of Treg after TKI discontinuation, may contribute to prolonged TFR. Thus Treg monitoring may be important in predicting molecular recurrence, especially in BCR::ABL Ins35bp cases.

KF1601, a Novel Orally Bioavailable Inhibitor of BCR-ABL1 T315I without Inducing Thrombotic Microangiopathy

Targeting Bcr-Abl with tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML) by significantly improving patients' survival. However, the gatekeeper mutation T315I confers resistance to TKIs targeting Bcr-Abl, presenting a formidable task in CML's clinical management. Ponatinib significantly reduces the mortality of CML patients with Bcr-Abl T315I, but often accompanies severe, life-threatening side effects. Herein, we report KF1601, a novel orally bioavailable TKI: 1) KF1601 inhibited kinase functions of both Bcr-Abl WT and Bcr-Abl T315I with nanomolar IC 50 values; 2) KF1601 and ponatinib had similar potency in terms of inhibiting CRKL phosphorylation, which correlates to major molecular responses in CML patients. 3) In murine models using Ba/F3 Bcr-Abl T315I cell line, it showed promising in vivo antitumor efficacy, comparable to that of ponatinib; 4) In animal models, it did not cause thrombotic microangiopathy, a major mechanism of ponatinib's toxicity. In summary, KF1601 is a promising drug candidate for safely treating CML patients with drug-resistant Bcr-Abl T315I mutation.

How we monitor the therapy effectiveness in patients with chronic myeloid leukemia - A Single center experience

Chronic myeloid leukemia (CML) is a hematological disorder characterized by increased proliferation of the granulocytic cell lineage. We diagnose CML by presence of the breakpoint cluster region-abelson (BCR-ABL1) oncogene using the quantitative real-time polymerase chain reaction (QRT-PCR). This method provides an accurate and unambiguous follow-up of the treatment response to tyrosine kinase inhibitors (TKIs). This study aimed to determine the molecular response to the first and second generation of TKIs in first and second line of treatment using the QRT-PCR method. We conducted a retrospective study on 48 CML patients treated with the first and second generation of TKIs in first and second-line treatment. Treatment responses have been followed-up every 3 months using the QRT-PCR method. Patients were divided into three groups according to molecular responses to the first line of TKIs. Results obtained in this study showed that the first group of patients did not achieve major molecular response (MMR) in the first 18 months of TKI treatment. The second and third group of patients achieved MMR and deep molecular response (DMR) in the first 18 months of TKI treatment. These results indicate that patients with MMR and DMR in the first 18 months of TKIs treatment had a favourable clinical course of the disease. Inadequate molecular responses to the first line of TKIs can be improved with in increase of the dose of TKIs or by switching to other TKIs. Continuous and timely molecular monitoring of TKI’s response in CML patients provides a careful observation of the disease's course and a proper treatment approach.

Role of miRNA -150 between different BCR-ABL p210 transcript levels and between different levels of imatinib optimal response in CML patients

The dysregulation of miRNA expression patterns is one of the many effects developments of cancer, miRNA has been found to express abnormally in hematological neoplasia such as chronic myeloid leukemia and solid malignancies. Resistance and the degree of response following tyrosine kinase inhibitor treatment are correlated with miRNA expression. Hence, in this study we tried to study the relationship of miRNA-150 between different breakpoint cluster region–Abelson (BCR- ABL) P210 transcript levels and the role miRNA- 150 between different levels of imatinib optimal response in chronic myeloid leukemia (CML). Our study included sixty chronic myeloid leukemia (CML) patients they were divided into two groups based on response to imatinib therapy, thirty samples of the optimal molecular response of chronic myeloid leukemia (CML) patients, and thirty samples of failure molecular response chronic myeloid leukemia (CML) patients. Thirty samples of apparently healthy volunteers were included and evaluated as control. According to the P210 BCR-ABL%, the results showed a significant difference (P= < 0.0001) between the responder and the failure response CML patients. Assessed the result of miRNA-150 showed a significant difference between both CML patients (P = < 0.0001), assessed miRNA-150 level among different response groups, and failure response of CML patients (P = 0.0002). A cutoff value of response vs. failure response (1.784) with high sensitivity can be a diagnostic value to differentiate between response and failure response. Changing gene expression with different amounts of miRNAs had an impact on drug-gene interactions, with consequences for cell growth and death. Gene expression of different levels miRNA-150 among of CML patients of imatinib therapy showed high expression in response patients than failure response patients. The gene expression level of miRNA-150 differs through different responses in CML patients.

Evaluation of HOXA5 methylation–gene expression in response to Imatinib mesylate treatment in chronic myeloid leukemia

Introduction and Aim: Genetic and epigenetic factors affect chronic myeloid leukemia (CML) treatment response. This study aimed to investigate HOXA5 methylation and gene expression with Imatinib mesylate (IM) response in CML patients and assess their predictive value as response markers. Materials and Methods: Blood samples were collected from 50 CML patients (25 responders and 25 non-responders to Imatinib mesylate) and 50 healthy controls of the same age and sex. Methylation-specific quantitative PCR (MS-qPCR) was used to analyze HOXA5 methylation, and quantitative PCR was used to study gene expression (qPCR). Results: This study revealed a significant higher level of HOXA5 gene demethylation and expression in CML patients compared to control (P <0.001). Non-responder CML patients had significantly higher levels of HOXA5 gene demethylation and expression than responder CML patients (P<0.001 and P<0.01 respectively). According to the optimal cut-off point determined by receiver operating characteristics (ROC) analysis, a significant associated risk was observed between HOXA5 demethylation and expression levels (P<0.01 and P<0.01, respectively) and the development of IM resistance in CML patients. Conclusion: HOXA5 gene demethylation can be used as a biomarker to predict IM resistance in CML patients.

A Systematic Review on Second Treatment-Free Remission (TFR) Attempt in Chronic Myeloid Leukemia (CML): Can it be Applied in Clinical Practice?

The prognosis and life expectancy of chronic myeloid leukemia (CML) patients have improved significantly with the launch of first tyrosine kinase inhibitor (TKI), imatinib. Maintaining at least one major molecular response in CML patients without the use of TKI is known as treatment-free remission (TFR). The safety of the first TFR (TFR1) effort has been reported by numerous studies. However, some patients relapse during TFR1. A second TFR (TFR2) can be tried again in those patients. This systematic review aims to evaluate individual patient characteristics for a TFR2, factors predicting successful TFR2, monitoring, consequences of the cessation of TKI, and studies about TFR2. We identified 5 studies related TFR2. The results showed that the first failed TKI discontinuation attempt is not an indicator of a second TKI discontinuation failure. TKIs could safely and successfully be discontinued for a second time in chronic phase CML patients despite a TFR1 failure. The most important factors for estimating TFR2 success are the speed of molecular relapse and the TKI-free duration after the first TKI discontinuation attempt. New trends in the management of CML patients are reducing the side effects of treatment, lessening the financial burden, and improving the quality of life of patients as CML has developed into a manageable chronic disease rather than an aggressive cancer. Although there are many studies and guidelines on TFR1, there are few studies on TFR2 and predictive factors. More data is still needed regarding TFR2 attempt in patients with CML.

Targeting BCR-ABL1-positive leukaemias: a review article.

Treatment and understanding of BCR::ABL1-positive leukaemias is a precision medicine success story. Our appreciation of the BCR::ABL1 gene and resulting BCR::ABL1 oncoprotein in chronic myeloid leukaemia (CML) and Philadelphia chromosome-positive (Ph+) acute leukaemias, has led to treatment advances associated with exceptional improvements in patient outcomes with normal life expectancy for many patients with chronic phase (CP-)CML. However, despite these major therapeutic advances, the management of Ph+ leukaemias remains complex, with development of specific resistance mutations on treatment, as well as the need for lifelong therapy in most patients due to the persistence of CML stem cells despite prolonged tyrosine kinase inhibitors (TKIs) treatment. BCR::ABL1-specific TKIs are associated with chronic toxicities affecting quality-of-life in many patients but can also result in more serious pulmonary and cardiovascular complications. Dose optimisation is increasingly being used to manage side effects and maintain molecular response in CML patients. Here, we review the development of BCR::ABL1-specific TKIs from the discovery of imatinib in 1996 to the more recent second- and third-generation TKIs and emerging specifically targeting the ABL myristoyl pocket (STAMP) inhibitors. We will also evaluate the current evidence for treatment of BCR::ABL1-positive leukaemias, including TKI discontinuation in optimally responding CP-CML patients.

Description of PTPRG genetic variants identified in a cohort of Chronic Myeloid Leukemia patients and their ability to influence response to Tyrosine kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) have remarkably transformed Ph+ chronic myeloid leukemia (CML) management; however, TKI resistance remains a major clinical challenge. Mutations in BCR-ABL1 are well studied but fail to explain 20-40% of resistant cases, suggesting the activation of alternative, BCR-ABL1-independent pathways. Protein Tyrosine Phosphatase Receptor Gamma (PTPRG), a tumor suppressor, was found to be well expressed in CML patients responsive to TKIs and remained at low level in resistant patients. In this study, we aimed to identify genetic variants in PTPRG that could potentially modulate TKIs response in CML patients. DNA was extracted from peripheral blood samples collected from two CML cohorts (Qatar and Italy) and targeted exome sequencing was performed. Among 31 CML patients, six were TKI-responders and 25 were TKI-non-responsive. Sequencing identified ten variants, seven were annotated and three were novel SNPs (c.1602_1603insC, c.85+14412delC, and c.2289-129delA). Among them, five variants were identified in 15 resistant cases. Of these, one novel exon variant (c.1602_1603insC), c.841-29C>T (rs199917960) and c.1378-224A>G (rs2063204) were found to be significantly different between the resistant cases compared to responders. Our findings suggest that PTPRG variants may act as an indirect resistance mechanism of BCR-ABL1 to affect TKI treatment.

Activated na\xefve \u03b3\u03b4 T cells accelerate deep molecular response to BCR-ABL inhibitors in patients with chronic myeloid leukemia

Tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the frontline treatments in chronic myeloid leukemia (CML). Growing evidence has shown that TKIs also enhance immunity. Since gamma-delta T (γδT) cells possess the potent anticancer capability, here we investigated the potential involvement of γδT cells in TKI treatments for CML. We characterized γδT cells isolated from chronic-phase CML patients before and during TKI treatments. γδT expression increased significantly in CML patients who achieved major molecular response (MMR) and deep molecular response (DMR). Their Vδ2 subset of γδT also expanded, and increased expression of activating molecules, namely IFN-γ, perforin, and CD107a, as well as γδT cytotoxicity. Mechanistically, TKIs augmented the efflux of isopentenyl pyrophosphate (IPP) from CML cells, which stimulated IFN-γ production and γδT expansion. Notably, the size of the IFN-γ+ naïve γδT population in TKI-treated CML patients was strongly correlated with their rates to reach DMR and with the duration on DMR. Statistical analysis suggests that a cutoff of 7.5% IFN-γ+ naïve subpopulation of γδT in CML patients could serve as a determinant for MR4.0 sustainability. Our results highlight γδT cells as a positive regulator for TKI responses in CML patients.

Aberrantly reduced expression of miR-342-5p contributes to CCND1-associated chronic myeloid leukemia progression and imatinib resistance

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3’-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation.

Regulation of miR-126 and miR-122 Expression and Response of Imatinib Treatment on Its Expression in Chronic Myeloid Leukemia Patients

Background: MicroRNAs (miRNAs) have been observed to exhibit altered expression patterns in chronic myeloid leukemia (CML). Therefore, this study was aimed to evaluate the clinical importance of miR-126 and miR-122 expression in concert to imatinib response in CML patients. Methods: The present study included 100 CML and 100 healthy subjects. The expression of the 2 miRNAs was performed using TaqMan probe chemistry, and snU6 was used as internal control. Results: The expression of miR-126 and miR-122 was downregulated in CML patients, with a mean fold change ± SD 0.20 ± 0.33 and 0.22 ± 0.37, respectively. While the expression of both miRNAs was analysed before and after imatinib treatment, it was observed that the expression levels of both were increased after imatinib treatment by 26.25-fold (5.33 against 0.20) and 13.95-fold (3.07 against 0.22) and the increase was statistically significant (p < 0.0001 and p < 0.0001, respectively). The expression of miR-126 was not conclusive when compared in different clinical stages of the CML disease as it showed a decreased expression in patients with accelerated phase compared to chronic phase (mean fold change = 0.03 and 0.27, respectively), but patients with chronic phase and blastic phase had comparable expression (mean fold change = 0.27 and 0.24, respectively). We also observed an increased expression of both miRNAs after imatinib therapy in each clinical phase. Conclusion: The study concludes that expression of miR-126 and miR-122 increases after imatinib treatment in CML patients and that miR-126 defines the good responders of imatinib therapy.

Concomitant Medications and Comorbidities Affect Early Response in CML Patients Treated with Imatinib: A Retrospective Analysis of 340 Patients from the Polish Adult Leukemia Group (PALG) Registry

Background : It has been consistently shown that early molecular response is associated with positive long-term outcome in terms of overall survival and progression-free survival in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Achieving a BCR-ABL(IS) <10% at three months has thereby become an important goal of treatment. According to current European LeukemiaNet recommendations, the optimal treatment response is defined as BCR-ABL(IS) ≤ 10% at three months and BCR-ABL(IS)<1% and/or complete cytogenetic response (CCyR) at six months. Factors influencing the early response are still largely unknown.Our previous study demonstrated that more than half of newly diagnosed CML patients in Poland had comorbid conditions and received concomitant medications. TKIs are metabolized by the CYP3A4 isoenzyme and have been reported to be substrates of ABCB1 (MDR1) and ABCG2 (BCRP) transporters. Importantly, numerous commonly used drugs are transported and metabolized through the same pathways as TKIs. Drug interactions should therefore be considered when administering inhibitors or inducers of CYP3A4, ABCB1 and ABCG2 in combination with TKIs. Furthermore, it has been shown that the presence of comorbidities and the necessity of additional therapies may significantly affect treatment compliance.Objectives: The primary objective of the study was to investigate whether the presence of comorbidities and additional therapies affected early response in CML patients treated with imatinib.Patients and methods : We retrospectively analyzed patients with newly diagnosed CML who were receiving imatinib as the initial therapy for CML at nine Polish hematological tertiary care centers. Inclusion criteria were as follows: 1) age ≥18 years and 2) diagnosis of chronic phase CML between January 1st 2005 and December 31st 2014. Data on presence of comorbid conditions, concomitant drugs, cytogenetic and molecular response were anonymously collected through electronic case reports forms. Patients in whom therapy was changed because of imatinib intolerance were excluded. Influence of potential prognostic factors on probability of response at three and six months was tested by univariate and multivariate logistic regression.Results : We enrolled 340 patients with CML in the chronic phase, with median age 53 years (range, 41-62) where 50 % were males. According to the Sokal score, 190 patients (55.9%) were classified as low risk, 107 (31.5%) as intermediate risk and 43 (12.5%) as high risk. Overall, 142 (41.8%) patients reported no comorbidities, 121 (35.6%) reported one or two comorbidities and 77 (22.7%) had more than two comorbid conditions. The most common comorbidities were: hypertension (30%), hypercholesterolemia (16%), ischemic heart disease (10%) and diabetes (9%). One hundred thirty (38.2%) patients received no additional therapies, 110 (32.4%) patients received one or two therapies and 100 patients (29.4%) more than two. The most frequently used drugs were: beta blockers (22% of patients), ACE inhibitors (19%) and statins (15%). At three months, a molecular analysis was done in 194 patients and 144 (74%) achieved BCR ABL(IS) of less than 10%. At six months, 217 patients underwent molecular analysis and 305 had cytogenetic assessment. One hundred thirty five patients (62%) achieved BCR-ABL(IS) < 1% and 198 (65%) achieved CCyR. Multivariate analysis revealed that at three months patients who had a low Sokal score, more than two comorbidities and received more than two concomitant therapies, significantly more often achieved BCR-ABL <1% (p=0.013, p=0.054 and p=0.005 respectively). At six months, patients who received more than two concomitant medications still tended to have a higher probability of optimal response: BCR-ABL(IS) <1% (p=0.062) and CCyR (p=0.035). Other potential prognostic factors analyzed including patients' age, sex and BMI had no impact on the probability of optimal response.Conclusions: We found that comorbidities and concomitant therapies did not adversely affect early response to imatinib in CML patients. In contrast, patients with more than two additional therapies and comorbidities seem to more often achieve treatment goals. The potential explanations for these findings are drug interactions that increase imatinib exposure and better adherence to therapy of patients accustomed to systematic use of medications. DisclosuresGora Tybor:BMS: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Sacha:Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Lewandowski:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Warzocha:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Jamroziak:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria.

Comparation of Digital PCR and Quantitative Pcr for Molecular Monitorization of Chronic Myeloid Leukemia Patients

Introduction: Chronic myeloid leukemia (CML) is a neoplasm produced by the formation of the BCRABL fusion gene. Tyrosine kinase inhibitors (TKI) have changed dramatically the clinical course of the disease. Quantitative PCR (qPCR) is the reference technique for measuring response in CML patients. Droplet digital PCR (ddPCR) is, in brief, a technology based on partitioning of the sample in a high number of small fractions which, in a further step, are read as positive or negative by its fluorescence. ddPCR performs an absolute quantification of the number of transcripts and their results do not depend on PCR efficiency. In spite of these theoretical advantages over qPCR, ddPCR is not routinely applied for BCRABL monitoring. In this study, we sought to compare qPCR and ddPCR techniques in similar amplification conditions.Methods: We included samples from 43 CML patients with different levels of response determined by qPCR: 23 patients with BCRABL/ABL1 levels <15%, 7 patients with MR3.0, 10 patients with MR4.0 and 3 patients who showed MR4.5 (according to European LeukemiaNet guidelines). Additionally, 3 non-CML patients were included. High-Capacity cDNA Reverse Transcription Kit was employed, following manufacturer instructions, in a unique reaction for both subsequent technologies utilized. qPCR was carried out with primers and probes for BCRABL and ABL1 from Europe Against Cancer report with 5 microL of non-measured cDNA in duplicates. The standards were BCR-ABL P210 ELITe Standard (ELITech Group) and the conversion factor was obtained with the Philadelphia P210 RNA Reference (Elitech Group). qPCR assys were run in an ABI 7500 platform in standard quantitation mode. EUTOS recommendations for BCRABL quantification were followed.ddPCR reaction was performed with the same primers and probes. Median cDNA concentration was 4,485ng/µL (1,771-7,787ng/µL). In order to avoid saturation of the technology, 2microL of cDNA at a concentration of 600ng/microL (i.e. 1,200ng) were added and run in duplicates in a C1000 touch Thermal Cycler (Bio-RAD) with the same amplification conditions used in qPCR. The same RNA reference material was used to obtain the calibration factor for the ddPCR technique. Bio-Rad QX200 Droplet Digital PCR System was employed for analysis. We used the mode of analysis for rare event detection and thresholds were set individually in each reaction. Qualitative comparations were carried out using Kappa coefficient with quadratic weight. For quantitative comparations, we applied Pearson correlation, intraclass correlation coefficient for absolute agreement and Passing-Bablok tests. A subgroup analysis, including samples with MR3.0 or deeper, was also performed.Results: All the samples showed ≥10000 ABL copies per well by qPCR. More than 10000 events were analyzed in every ddPCR reaction well. Good replicates were observed in qPCR and ddPCR. Adequate separation of positive and negative events could be performed and no saturation was observed in the ddPCR reations. No signal from BCRABL was detected in non-CML patients in ddPCR. The Kappa coefficient between qPCR and ddPCR measurements was 0.76 (P< 0.001, 95% confidence interval (CI): 0.63-0.88). In the 20 samples with MR 3.0 or deeper, kappa=0.13 (P >0.05 ) .For quantitative comparison, 32 samples with detectable BCRABL were included. Pearson test yielded an R2=0.91 (P< 0.001). The intraclass correlation coefficient for absolute agreement showed a value of 0.96 (P <0.001). Passing-Bablok test provided a non significant constant difference (0.041, CI 95%: -0.04-0.05) and a significant proportional difference (0.8, CI 95%: 0.7-0.96) (i.e. ddPCR results= 0.041 + (0.8*qPCR results). There were only 9 samples with a BCRABL/ABL1 ratio and quantitative test were not performed.Conclusion: Although a global excelent qualitative correlation is observed, in patients with MR3.0 or deeper which are the more important to classify correctly, qualitative correlation is absent. The R2 and intraclass correlation coefficient for absolute agreement showed a high concordance but Passing-Bablok test yielded a significant proportional difference among qPCR and ddPCR. We sought to test both technologies in similar conditions but different ddPCR strategies could be implemented in order to improve reproducibility of both techniques: higher amount of replicates or, given the lack of saturation, a higher cDNA concentration in the reactions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Relevance of ABCB1, ABCG2, and ABCC2 Gene Polymorphisms in Chronic Myeloid Leukemia Patients Treated With Nilotinib.

Tyrosine kinase inhibitors (TKIs) have radically changed the outcome of chronic myeloid leukemia (CML) patients in the last 20 years. Moreover, the advent of second generation TKIs, namely nilotinib and dasatinib, have largely increased the number of CML patients achieving deep and sustained molecular responses. However, the possible mechanisms capable of influencing the maintenance of the long-term molecular response are not yet fully known and understood. In this light, polymorphisms in MDR-ABC transporters may influence the efficacy and safety of TKIs. In this study, we examined seven single nucleotide polymorphisms (SNPs) in four ABC transporter genes: ABCC1 rs212090 (5463T>A), ABCC2 rs3740066 (3972C>T), ABCC2 rs4148386 G>A, ABCC2 rs1885301 (1549G>A), ABCG2 rs2231137 (34G>A), ABCG2 rs2231142 G>C, ABCB1 rs1045642 (3435C>T), to determine their effect on the achievement and/or loss of molecular response in 90 CML patients treated with nilotinib. We found that ABCC2 rs3740066 CC and CT as well as the ABCB1 rs1045642 TT genotypes correlated with a higher probability to achieve MR3 in a shorter time (p=0.02, p=0.004, and p=0.01), whereas ABCG2 rs2231137 GG was associated with lower probability of MR3 achievement (p=0.005). Moreover, ABCC2 rs3740066 CC genotype, the ABCB1 rs1045642 CC and TT genotypes were positively correlated with MR4 achievement (p=0.02, p=0.007, and p=0.003). We then generated a predictive model incorporating the information of four genotypes, to evaluate the combined effect of the SNPs. The combination of SNPs present in the model affected the probability and the time to molecular response. This model had a high prognostic significance for both MR3 and MR4 (p=0.005 and p=0.008, respectively). Finally, we found ABCG2 rs2231142 GG genotype to be associated with a decrease risk of MR3 loss. In conclusion, MDR-transporters SNPs may significantly affect the achievement and loss of molecular response in CML patients treated with nilotinib.

Imatinib Mesylate Adherence in Chronic Myeloid Leukemia Patients: Data from Middle Euphrates Region of Iraq

Background: Chronic myeloid leukemia (CML) represents about one quarter of leukemia cases in our region making it a great challenge to the health system. Aims: Since adherence to imatinib contributes to optimal cytogenetic and molecular responses in CML patients, ours. study tries to evaluate imatinib adherence in Middle Euphrates region of Iraq. Materials & Methods: This study was conducted in Al- Hussein cancer center in Karbala province of Iraq between November 2011 and August 2019. There were 67 CML patients treated with imatinib, their adherence was assessed by using the 9-items Morisky Medication Adherence Scale, while response was evaluated by measuring BCR-ABL1 transcription level in peripheral blood. Results: Only 26.87% of our patients were adherent. Unavailability of drug was the most common cause of poor adherence in 73.13% of patients. Patients with good adherence were more likely to achieve an optimum response (P value =0.007). Conclusion: Our results showed that adherence to imatinib was very low in our region. Economic factor (Unaffordable drug prices) was the main cause leading to suboptimal response. Therefore, efforts should be made to provide drugs to our patients. That will help in improve adherence and treatment outcomes.

Impact of Fas/Fasl Gene Polymorphisms on Susceptibility Risk and Imatinib Mesylate Treatment Response in Chronic Myeloid Leukaemia Patients

Background:The FAS mediated apoptosis pathway involving the FAS and FASL genes plays a crucial role in the regulation of apoptotic cell death and imatinib mesylate (IM) mechanism of action. Promoter polymorphisms FAS-670 A>G and FAS-844 T>C which alter the transcriptional activity of these genes may grant a risk to develop cancer and revamp the drug activities towards the cancer cell. We investigated the association of these two polymorphisms with the susceptibility risk and IM treatment response in Malaysian chronic myeloid leukaemia (CML) patients. Methods:This is a retrospective study, which included 93 CML patients and 98 controls. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method was used to genotype the FAS and FASL polymorphisms. Data nanlysis was done using SPSS Version 22. The associations of the genotypes with susceptibility risk and IM response in CML patients were assessed by means of logistic regression analysis and deriving odds ratio with 95% CI. Results:We observed a significant association between FASL-844T>C polymorphism and CML susceptibility risk and IM response. Variant C allele and FASL-844 CC variant genotype carriers had significantly higher risk for CML susceptibility (OR 1.756, CI 1.163-2.652, p=0.007 and OR 2.261, CI 1.013-5.047, p=0.047 respectively). Conversely, the heterozygous genotype FASL-844 TC conferred lower risk for CML susceptibility (OR 0.379, CI 0.176-0.816, p=0.013). The heterozygous and homozygous variant genotypes and variant C alleles were found to confer a lower risk for the development of IM resistance with OR 0.129 (95% CI: 0.034-0.489 p=0.003), OR 0.257 (95% CI: 0.081-0.818, p=0.021), and OR 0.486 (95% CI: 0.262-0.899, p=0.021) respectively. We also found that FAS-670 A>G polymorphism was not associated with CML susceptibility risk or IM response. Conclusion:The genetic polymorphism FASL-844 T>C may contribute to the CML susceptibility risk and also IM treatment response in CML patients. Accodringly, it may be useful as a biomarker for predicting CML susceptibility risk and IM resistance.

Several different cytogenetic clones arising during treatment of Philadelphia positive chronic myeloid leukemia with tyrosine kinase inhibitors lead to the progression into Philadelphia negative acute myeloid leukemia

Introduction. Additional karyotype abnormalities in the Philadelphia-positive (Ph+) clone can emerge during the progression of chronic myeloid leukemia (CML) and are of-ten associated with the resistance to treatment with tyrosine kinase inhibitors (TKI). Sometimes, during the TKI treatment, karyotype abnormalities can appear in the Philadelphia-negative (Ph-) cells as well but do not seem to adversely affect the outcome except for chromosome 7 abnormalities. Case report. The patient presented was in the chronic phase of Ph+ CML with highly diverse karyotype abnormalities. The abnormalities appeared in three unrelated clones during the TKIs treatment, followed by the evolution of the disease into acute myeloid leukemia (AML). The primary Ph+ clone was revealed during the chronic phase of CML, and therapy with imatinib mesylate was commenced. After a three-year hematologic and cytogenetic remission period, the evolution of the primary clone was noticed. Nilotinib was introduced, leading to a good molecular response and the disappearance/loss of the Ph+ clone with additional abnormalities but with the appearance of the Ph-clone with trisomy 8. Finally, after 5.5 years of nilotinib therapy, the Ph-clone with monosomy 7 occurred during the deep molecular response for BCR-ABL. At that time, the FISH analysis for trisomy 8 was negative, but the rise in blast count was noticed in the bone marrow, and the diagnosis of the secondary AML was established soon after. Conclusion. The achievement of the deep molecular response in CML patients does not rule out regular cytogenetic testing of their bone marrow. This is of crucial importance for detecting adverse karyotype abnormalities leading to the development of the myelodysplastic syndrome and AML.

Association Between Glutathione-S-Transferase Gene Polymorphisms and Responses to Tyrosine Kinase Inhibitor Treatment in Patients with Chronic Myeloid Leukemia: A Meta-analysis.

Although many earlier studies revealed an effect of glutathione-S-transferase (GST) gene polymorphisms on tyrosine kinase inhibitor (TKI) treatment responses in chronic myeloid leukemia (CML) patients, the significance of this relationship remains controversial. This study aimed to review and meta-analyze treatment responses to TKIs in patients with CML and GST gene polymorphisms, including GSTT1, GSTM1, and GSTP1. We searched four medical databases, PubMed, Web of Science, the Cochrane Library, and Embase, by using keywords related to GST gene polymorphisms and clinical responses in CML patients receiving TKI treatment. The meta-analysis was performed using RevMan version 5.3 and Comprehensive Meta-Analysis software version 3.0. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to examine the association between GSTT1, GSTM1, and GSTP1 polymorphisms and TKI treatment responses in patients with CML. The null polymorphisms of GSTT1 and GSTM1 did not affect TKI treatment responses, while the GSTP1 Ile105Val polymorphism had a significant impact on responses to TKI. Patients who were GSTP1 variant allele carriers (AG + GG) had poor responses to TKI treatment compared to patients who were wild-type homozygote carriers (AA) (OR 1.85, 95% CI 1.31-2.62; p < 0.001). This meta-analysis of patients with CML showed that G allele carriers with GSTP1 Ile105Val polymorphism had significantly worse responses to TKI treatment than wild-type homozygote carriers.