Chronic Myeloid Leukemia BCR-ABL1 Research Articles

The TKI Era in Chronic Leukemias.

Tyrosine kinases are proteins involved in physiological cell functions including proliferation, differentiation, and survival. However, the dysregulation of tyrosine kinase pathways occurs in malignancy, including hematological leukemias such as chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Particularly, the fusion oncoprotein BCR-ABL1 in CML and the B-cell receptor (BCR) signaling pathway in CLL are critical for leukemogenesis. Therapeutic management of these two hematological conditions was fundamentally changed in recent years, making the role of conventional chemotherapy nearly obsolete. The first, second, and third generation inhibitors (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) of BCR-ABL1 and the allosteric inhibitor asciminib showed deep genetic and molecular remission rates in CML, leading to the evaluation of treatment discontinuation in prospective trials. The irreversible BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and spebrutinib) covalently bind to the C481 amino acid of BTK. The reversible BTK inhibitor pirtobrutinib has a different binding site, overcoming resistance associated with mutations at C481. The PI3K inhibitors (idelalisib and duvelisib) are also effective in CLL but are currently less used because of their toxicity profiles. These tyrosine kinase inhibitors are well-tolerated, do have some associated in-class side effects that are manageable, and have remarkably improved outcomes for patients with hematologic malignancies.

Acute Myeloid Leukemia with Platelet-Derived Growth Factor Receptor β (PDGFRβ) Rearrangement Successfully Treated with Intensive Chemotherapy in Combination Withimatinib- A Case Report

Acute myeloid leukemia (AML) is a heterogeneous clonal stem cell malignancy. Several subgroups of AML have been identified based on multiple genomic and proteomic aberrations; these demonstrate different clinical features, response to treatment and prognosis. Rearrangements in the platelet-derived growth factor receptor β (PDGFRβ) gene result in greater constitutive enzymatic activity (tyrosine kinase) and deregulation of haematopoiesis, similarly to BCR-ABL1 in chronic myeloidleukemia (CML). PDGFRβ gene rearrangements are infrequent entities, which are mostly diagnosed in patients presenting with atypical CML, chronic myelomonocytic leukemia (CMML), myelodysplastic/myeloproliferative disorders (MDS/MPN) or juvenile myelomonocytic leukemia (JMML). PDGFRβ-positive AML is extremely rare, accounting for well below 1% of all cases. In this article, we present the case of a 31-year-old woman with AML and abnormal karyotype with PDGFRβ rearrangement, who was treated with intensive chemotherapy combined with low-dose imatinib. Complete remission (CR) was achieved and imatinib was used as maintenance treatment. The patient remains in CR with disease-free survival (DFS) of 41 months.

Abstract PR04: Clonal landscapes of hematologic malignancies redefined by ultrasensitive duplex sequencing

Abstract Hematologic malignancies (HM) are driven by clonal evolution—from preneoplastic clones through acquired drug resistance. Clonal hematopoiesis of indeterminate potential (CHIP) is a process whereby healthy individuals accumulate low-frequency clones with driver gene mutations. CHIP is associated with increased risk of HM but is challenging to detect. Measurable residual disease (MRD) after therapy in acute myeloid leukemia (AML) and other HM is a strong predictor of relapse; however, current MRD assays either have limited sensitivity or are narrowly applicable. Tyrosine kinase inhibitors (TKI) target BCR-ABL1 in chronic myeloid leukemia (CML) and Ph+ acute lymphocytic leukemia (ALL), yet TKI-resistance (TKI-R) mutations can drive relapse. Early detection of MRD and TKI-R mutations can inform important clinical interventions. Next-generation sequencing (NGS) can detect HM subclones, but not below a frequency of approximately 1%. Common error correction methods have difficulty detecting mutations below 0.1%. Duplex sequencing (DS) is a sophisticated NGS error correction assay capable of detecting individual mutations down to ultralow frequencies of at least 0.001% with a background error frequency below 1 in 10 million bases. We used DS for three clinically relevant HM applications: (1) CHIP: We performed DS on DNA from 10 hematopoietic cell transplant (HCT) donor/recipient pairs 7-46 years post-HCT. We targeted 29 genes recurrently mutated in AML, many of which have been described in CHIP. DS error-corrected depth up to 45,402x was generated. We identified dozens of clonal exonic variants (CEV) in every donor and recipient, most nonsynonymous, down to variant allele frequency (VAF) of 5.7E-5. The majority of the 154 shared donor/recipient CEVs were present at VAF <0.1%, with many increasing as well as decreasing in the recipient. (2) AML MRD: Dozens of genes are recurrently mutated in AML, with multiple mutations per patient. To model MRD, we mixed mutant cell line DNA into normal DNA from a healthy young control. 9 unique mutations were titrated to predicted VAF of 1%-0.001%. Replicate DS libraries generated over 1,000,000x total molecular depth with a targeted capture panel. All mutations were identified close to expected values with r2= 0.97. (3) HM drug resistance: We performed DS on Ph+ ALL samples in TKI-induced remission. The relapse TKI-R mutation was detected in samples banked 1-5 months prior to relapse at VAFs from 0.1%-1%. DS analysis of genomic DNA prior to TKI therapy demonstrated extremely low prevalence of resistance mutations in TKI-naïve patients, and cDNA analysis revealed extensive false positives. DS is a flexible, ultrasensitive technology for detection of low-frequency mutations in HM. DS redefines the landscape and prevalence of CHIP and reveals complex HCT donor/recipient dynamics. DS can detect MRD mutations at levels below 1/100,000 and identify TKI-R mutations months prior to relapse, which will better predict relapse and inform personalized therapy options. This abstract is also being presented as Poster A48. Citation Format: Jake Higgins, Lindsey N. Williams, Charles C. Valentine, Gabriel Pratt, Rashmi Kanagal-Shamanna, Nicholas J. Short, Masumi Ueda, Rainer Storb, Jerald P. Radich, Jesse J. Salk. Clonal landscapes of hematologic malignancies redefined by ultrasensitive duplex sequencing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR04.

Cdx Report Program: Heterogeneity Revealed in Current Reporting Practices for Hemato-Oncology Companion Diagnostic (CDx) Markers in Multiple Countries

Introduction: In the current era of precision medicine, CDx tests have become the basis for optimal patient care and targeted treatment. While technical aspects of a CDx assay's performance are audited regularly by participation in external quality assessment (EQA) programs, the reporting of these CDx markers is rarely scrutinized. The aim of this first of its kind CDx report program is to get a detailed picture of hemato-oncology CDx marker reporting across multiple countries. Therefore, we evaluated multiple laboratory parameters and reviewed actual content of clinical CDx reports. Methods: Thirty-four clinical labs from Germany, Italy, Spain, and the United Kingdom provided information for 7 biomarkers used for treatment decisions in hemato-oncology. An identical program is already running in Australia and the United States and will be initiated shortly in India and China. The CDx markers covered in this CDx report program were: BCR-ABL1 in chronic myeloid leukemia (CML), at diagnosis; BCR-ABL1 in CML, minimal residual disease (MRD); IDH1/2 in acute myeloid leukemia (AML); FLT3-ITD in AML; FLT3-TKD in AML; IGHV in chronic lymphocytic leukemia (CLL); and TP53 in CLL. The information requested from participating laboratories included two anonymized or blank reports: one with a positive/mutant result and one with a negative/wild-type result. The received anonymized reports were reviewed by experts within each country according to pre-agreed criteria. In addition, labs participated in a short online survey evaluating test volumes, turnaround times (TATs), positivity rates, participation in an EQA program, and status regarding accreditation and reimbursement. The results of the questionnaires were forwarded as anonymized and aggregated data to reviewing experts. Results: Overall, we received 184 survey datasets and 179 sets of anonymized reports. The review of the anonymized reports according to pre-defined criteria revealed differences in the way CDx results are represented and interpreted in clinical reports. Since not all markers covered in this program were tested by all participating labs, the number of survey datasets per CDx marker ranged from 16 to 34 (IGHV: 16; TP53: 19; IDH1/2: 24; FLT3-TKD: 28; FLT3-ITD: 30; BCR-ABL1 [MRD]: 33; and BCR-ABL1 [diagnosis]: 34). The 184 survey datasets represented more than 7000 tests per month (Figure). The stated average TAT across all covered markers was 6.9 days, ranging from 5.3 days to 8.6 days in the covered countries. The TATs for the individual markers ranged from 4.4 days to 9.4 days (FLT3-ITD: 4.4 days; FLT3-TKD: 4.6 days; BCR-ABL1 [diagnosis]: 5.3 days; BCR-ABL1 [MRD]: 6.9 days; IDH1/2: 8.4 days; IGHV: 9.3 days; and TP53: 9.4 days). In 103/179 (58%) datasets labs participated in EQA programs, and in 76/179 (42%) datasets labs did not participate in EQA programs. For 84/180 (47%) datasets, labs were ISO15189-accredited; for 12/180 (7%) datasets, labs were College of American Pathologists (CAP)-accredited; and for 84/180 (47%) datasets, labs were not accredited by either ISO15189 or CAP. Conclusion: CDx report program results reveal a broad range globally in CDx reporting practices. The identified differences in laboratory parameters, such as TAT and the actual content of clinical CDx reports, suggest a need for international harmonization of CDx reporting. The anonymized and aggregated data generated provide the basis for other initiatives and may support guideline updates and the international harmonization of CDx reporting. Figure. Estimated number of companion diagnostic tests per month covered in the CDx report program. Figure Disclosures Delic: Diaceutics: Employment, Equity Ownership. Blombery:Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Calasanz:Janssen: Honoraria; Diaceutics: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria. Colomer:Novartis: Honoraria; Incyte: Honoraria. Evans:Diaceutics: Honoraria; Novartis: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mason:Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria; AbbVie: Honoraria. Thiede:Daiichi Sankyo: Honoraria; Diaceutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AgenDix GmbH: Employment, Equity Ownership. Clark:Diaceutics: Employment, Equity Ownership.

Targeting BCR-ABL1 in Chronic Myeloid Leukemia by PROTAC-Mediated Targeted Protein Degradation.

Although the use of ATP-competitive tyrosine kinase inhibitors of oncoprotein BCR-ABL1 has enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. To test whether the degradation of BCR-ABL1 kinase could offer improved response, we developed a series of proteolysis-targeting chimera (PROTAC) that allosterically target BCR-ABL1 protein and recruit the E3 ligase Von Hippel-Lindau, resulting in ubiquitination and subsequent degradation of the oncogenic fusion protein. In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared with diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34+ cells, with no effect on healthy CD34+ cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells. Together, these findings suggest that combined BCR-ABL1 kinase inhibition and protein degradation may represent a strategy to address BCR-ABL1-dependent drug resistance, and warrant further investigation into the eradication of persistent leukemic stem cells, which rely on neither the presence nor the activity of the BCR-ABL1 protein for survival. SIGNIFICANCE: Small-molecule-induced degradation of BCR-ABL1 in CML provides an advantage over inhibition and provides insights into CML stem cell biology. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/18/4744/F1.large.jpg.

Validation of a Drosophila model of wild-type and T315I mutated BCR-ABL1 in chronic myeloid leukemia: an effective platform for treatment screening.

Chronic myeloid leukemia (CML) is caused by a balanced chromosomal translocation resulting in the formation of BCR-ABL1 fusion gene encoding a constitutively active BCR-ABL1 tyrosine kinase, which activates multiple signal transduction pathways leading to malignant transformation. Standard treatment of CML is based on tyrosine kinase inhibitors (TKI); however, some mutations have proven elusive particularly the T315I mutation. Drosophila melanogaster is an established in vivo model for human diseases including cancer. The targeted expression of chimeric human/fly and full human BCR-ABL1 in Drosophila eyes has been shown to result in detrimental effects. In this study, we expressed human BCR-ABL1p210 and the resistant BCR-ABL1p210/T315I fusion oncogenes in Drosophila eyes. Expression of BCR-ABL1p210/T315I resulted in a severe distortion of the ommatidial architecture of adult eyes with a more prominent rough eye phenotype compared to milder phenotypes in BCR-ABL1p210 reflecting a stronger oncogenic potential of the mutant. We then assessed the efficacy of the currently used TKI in BCR-ABL1p210 and BCR-ABL1p210/T315I expressing flies. Treatment of BCR-ABL1p210 expressing flies with potent kinase inhibitors (dasatinib and ponatinib) resulted in the rescue of ommatidial loss and the restoration of normal development. Taken together, we provide a CML tailored BCR-ABL1p210 and BCR-ABL1p210/T315I fly model which can be used to test new compounds with improved therapeutic indices.

Studying Methylation Status of Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) in Chronic Myeloid Leukemia Patients in the State of Qatar

Background:Chronic myeloid leukemia (CML) is a clonal myeloid stem cell disorder associated with increased myeloid cells spectrum in peripheral blood. The disease is due to the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL1 fusion oncogene which is the hallmark of CML. The resultant chimeric protein P210 kD of BCR-ABL1 causes a constitutive activation of tyrosine kinase domain that leads to continuous activation of several cell signaling pathways.The CML is currently treated with several generations of tyrosine kinase inhibitors (TKIs). However, treatment failure, resistance and disease progression are still reported and the molecular mechanisms still remain poorly understood.On the other hand, protein tyrosine phosphatase receptor gamma (PTPRG) is the natural regulatory mechanism to tyrosine kinase family, acts as tumor suppressor gene, well documented to be downregulated in CML patients while its re-expression led to reduced tyrosine phosphorylation and induced apoptosis in CML cells.PTPRG is also an important protein in the JAK/STAT pathway, has a vital role in the downstream of BCR-ABL protein kinase. Several studies suggested that the aberrant DNA methylation is involved in TKIs resistance in CML leading to leukemic clone escape and disease propagation.In different types of tumors, several mechanisms have been suggested for the loss of function of PTPRG activities such as mutations, deletions, or loss of heterozygosity LOH and gene silencing by methylation.Aim:In this study, we aimed to evaluate and characterize the methylation status of PTPRG gene function in 15 CML Patients 1- Group A: CML patients at diagnosis and 2- Group B) CML patients failed treatments according to European Leukemia Net (ELN) 2013 criteria and 10 healthy individuals. (Labeled as Normal in the figure 1)Materials and Methods:Thirty subsequent peripheral blood samples have been collected from the 15 CML patients at diagnosis and at time of failure. Genomic DNA was isolated from 400 ml of blood on EDTA tubes using Maxwell DNA Purification Kits, the quality and quantity of DNA were measured on Nanodrop Spectrophotometer and gel electrophoresis. The methylation-specific PCR and bisulfite-sequencing were adapted, EZ DNA Methylation Kit was utilized for bisulfite-conversion. Methylation Primers were designed to target the promotor and intronic CpG islands of the PTPRG gene. Gradient PCR was performed to detect the two bands of 321bp and 218bp for CpG islands of promoter and intron of PTPRG respectively. Bisulfite-specific PCR technique was carried out to amplify a fragment of bisulfite-converted DNA that has been pre-defined by the designed bisulfite-specific primers.The Methylation PCR amplified product was sequenced via Sanger Sequencing (Applied Biosystems 3130 Genetic Analyzer). The methylation analysis was performed to study the methylation status of the CpG islands using the ESME (Epigenetic Sequencing Methylation) Analysis Software.Results:Results of the methylation in 25 CpG sites of Promoter region of PTPRG:There are significant differences in the mean of methylation status amongst CML patients at diagnosis and healthy individuals of CpG promoter of PTPRG (p value is ˂0.04). However, there are no significant differences in the mean of methylation status between patients failed treatments and other groups (Figure1)Results of the methylation in 26 CpG sites of Intronic region of PTPRG:There are also significant differences in the mean of methylation status amongst all groups (p value is ˂0.003). (Figure1)Discussion and Conclusions:This is the first prospective study to evaluate epigenetic changes of PTPRG gene from CML patients in Qatar. We previously documented that the expression level of PTPRG is inversely correlated with the expression of BCR-ABL1 in CML patients.In this study, the mechanism behind the downregulation of PTPRG in our cohorts of CML patients was explored and the aberrant methylation of PTPRG could be a mechanism associated with CML disease progression and resistance to TKIs.However, further studies are still needed including larger patient's cohort and CML patients responding to the TKIs.Finally, the methylation of PTPRG in the CML patients at diagnosis and failed treatment may allow the identification of new therapeutic targets and using methylation inhibitors could be also suggested to prevent or reverse TKIs resistance and restore TKIs sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Ultra-Deep Sequencing Leads to Earlier and More Sensitive Detection of Known or Novel Mutations of BCR-ABL1 in Chronic Myeloid Leukemia

Detection of BCR-ABL1 mutations that inhibit tyrosine kinase inhibitors is important for the treatment of CML patients. Sanger sequencing (SS) technique is considered the gold standard for mutation detection in a clinical laboratory. We analyzed 40 clinical samples from 22 CML patients with TKI resistance by ultra-deep sequencing (UDS) and compared the results with classical SS-based tests. UDS facilitates the detection of low levels of mutations (<20% allele frequencies) that SS does not detect. In subgroups of cases with multiple mutations, UDS was also able to determine whether two mutations were on same BCR-ABL1 allele (compound) or not (polyclonal). We found a pair of compound mutations unreported: F359C/E450 which confer high-level resistance to first-generation TKI imatinib, second-generation TKIs dasatinib and nilotinib, third-generation TKI ponatinib and a new TKI GZD824. UDS provides a reliable method for identifying low levels of BCR-ABL1 mutations. DisclosuresNo relevant conflicts of interest to declare.

Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia.

Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.

Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy

Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.

Molecular monitoring of chronic myeloid leukemia: a personalized approach to optimizing treatment response.

Personalized medicine is rapidly developing a purposeful niche in the field of oncology. Monitoring the activity of the oncogenic fusion gene BCR-ABL1 in chronic myeloid leukemia (CML) is a good example of individualizing CML treatment for patients using patient-specific genetic information. However, the frequency at which molecular monitoring for BCR-ABL1 transcripts occurs during treatment with tyrosine kinase inhibitors (TKIs) for CML in clinical practice is much lower than that recommended by either the National Cancer Center Network or the European LeukemiaNet guidelines. Adherence, one of the most critical factors affecting response to TKIs, is often less than desirable and rarely communicated to physicians by patients or managed by care providers. Less than optimal molecular monitoring and low adherence to TKI treatment can lead to rising transcripts levels, that when not detected, have been shown to contribute to poor outcomes. This review reports the basis for and describes the design of a state-of-the-art program intended to improve communication with physicians through real-time messaging about sequential test results for BCR-ABL1 and patients' adherence to TKI therapy.