Advanced Phase Chronic Myeloid Leukemia Research Articles

Clinical Validation of the Somatic FANCD2 Mutation (c.2022-5C>T) as a Novel Molecular Biomarker for Early Disease Progression in Chronic Myeloid Leukemia: A Case–Control Study

Background: Chronic myeloid leukemia (CML) results from chromosomal translocation t(9;22) leading to the formation of the BCR-ABL fusion oncogene. CML has three stages: the chronic phase (CP), the accelerated phase (AP), and the blast crisis (BC). Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML. TKIs work well in CP-CML, and these patients have a survival rate similar to the normal population, but TKIs are less effective in advanced-phase CML. Even with current advances in treatment, BC-CML patients have an average overall survival of less than a year. Early recognition of CML patients at risk of disease progression can help in timely interventions with appropriate TKIs or other therapeutic modalities. Although some markers of disease progression like BCR-ABL kinase domain, ASXL1, and GATA2 mutations are available, no universal and exclusively specific molecular biomarkers exist to early diagnose CML patients at risk of CML progression for timely therapeutic interventions to delay or minimize blast crisis transformation in CML. A recent study found that all BC-CML patients harbored the FANCD2 (c.2022-5C>T) mutation. Therefore, the current study was designed to detect this FANCD2 mutant in AP-CML (early progression phase) and to clinically validate its potential as a novel molecular biomarker of early CML progression from CP to AP. Methods: Our study comprised 123 CP-CML (control group) and 60 AP-CML patients (experimental group) from 2 oncology centers, from January 2020 to July 2023. Mean hemoglobin level, WBC count, platelet count, treatment type, hepatomegaly, splenomegaly, and survival status of AP-CML patients were significantly different from those of CP-CML patients. However, as these clinical parameters cannot help in the early detection of patients at risk of CML progression, there was a need for a clinically validated biomarker of AP-CML. DNA was extracted from the patients’ blood samples, and the FANCD2 gene was sequenced using an Illumina NextSeq500 next-generation sequencer (NGS). Results: The NGS analysis revealed a unique splice-site mutation in the FANCD2 gene (c.2022-5C>T). This mutation was detected in the majority (98.3%) of AP-CML patients but in none of the CP-CML patients or healthy control sequences from genomic databases. The mutation was confirmed by Sanger sequencing. FANCD2 is a member of the Fanconi anemia pathway genes involved in DNA repair and genomic stability, and aberrations of this gene are associated with many cancers. Conclusions: In conclusion, our study shows that the somatic FANCD2 (c.2022-5C>T) mutation is a new molecular biomarker for early CML progression. We recommend further clinical validation of this biomarker in prospective clinical trials.

Ponatinib-review of historical development, current status, and future research.

Ponatinib is a third-generation BCR::ABL1 tyrosine kinase inhibitor (TKI) with high potency against Philadelphia chromosome (Ph)-positive leukemias, including T315I-mutated disease, which is resistant to first- and second-generation TKIs. Ponatinib was approved for T315I-mutated chronic myeloid leukemia (CML), CML resistant/intolerant to ≥2 prior TKIs, advanced phase CML and Ph-positive acute lymphoblastic leukemia (ALL) where no other TKIs are indicated, and T315I-mutated CML and Ph-positive ALL. The response-based dosing of ponatinib in chronic phase CML (CP-CML) improved treatment tolerance and reduced the risk of toxicities, including cardiovascular risks. Ponatinib-based therapy also resulted in significantly better outcomes in frontline Ph-positive ALL compared with prior TKIs and is becoming a new standard of care in this setting. As the clinical development of third-generation TKIs and their rational combinations progresses, we envision further transformative changes in the treatment of CML and Ph-positive ALL.

Beyond BCR::ABL1-The Role of Genomic Analyses in the Management of CML.

Chronic myeloid leukemia (CML) is a model of genomically based diagnosis and management where BCR::ABL1 is successfully targeted by tyrosine kinase inhibitor (TKI) therapy in most patients. The dynamics of BCR::ABL1 transcript decline during therapy is a dependable biomarker of response, relapse, and drug resistance. Missense mutations acquired within the BCR::ABL1 kinase domain that disrupt TKI binding can evolve during therapy and are frequently detected in patients for whom TKI treatment fails. Importantly, specific BCR::ABL1 missense mutations are targetable alterations and direct therapeutic decisions based on the individual mutant TKI sensitivity profile. Nevertheless, BCR::ABL1 mutations are only implicated in approximately half of the cases of acquired resistance. Furthermore, not all patients with a single BCR::ABL1 mutation that is predicted to be sensitive to a specific TKI will experience a response when switched to that TKI. Progression to blast phase heralds independence from BCR::ABL1, and this phase of the disease is notoriously difficult to treat. The independent drivers of resistance and disease progression have long been investigated to both predict progression and to find targets for therapeutic intervention. Recent data reaffirm that drug resistance and disease progression is a mutation-driven process in CML, and somatic variants in genes that are known to drive acute myeloid and lymphoid leukemia have been detected in patients in the advanced phases of CML. Genomic testing over the last few decades for patients with blood cancer has revealed of variety of genomic aberrations that drive disease. Consequently, incorporation of genomic factors into patient management for a range of blood cancers has led to the implementation of high-throughput gene testing to detect clinically actionable variants. Is it time to integrate broader genomic screening into clinical management strategies for patients with CML?

Molecular Pathogenesis and Treatment Strategies of Chronic Myeloid Leukemia (CML)

Chronic Myeloid Leukemia (CML) is a myeloproliferative disease diagnosed in bone marrow, arising from a chromosomal translocation between chromosomes 9 and 22, resulting in the formation of fusion oncogene BCR–ABL. The product of this fusion oncogene is a new oncoprotein bcr–abl which possesses abnormal tyrosine kinase activity. In response to this, abnormal signaling pathway activation occurs, leading to cell transformation. BCR–ABL oncogene could be targeted by tyrosine kinase inhibitors (TKIs) to delay or inhibit the disease progression. Imatinib is the first drug designed against CML but resistance to this has led to the development of the second- and third generations of inhibitors that are active against many types of BCR–ABL gene mutations. However, somehow, due to disease progression, TKIs do not remain as effective. There are three well-characterized phases of CML: The chronic phase (CP), the accelerated phase, and the terminal stage which is the blast crisis (BC) stage. In the CP of CML, mature granulocytes and myeloid precursors become aggregated majorly in the bone marrow and peripheral blood. The accelerated phase is marked by increased disease severity and an increase in progenitor/precursor cell number. In the BC stage, undifferentiated blast cells grow in number. Many patients with CML are diagnosed during the CP of the disease, so the survival rate of CML is high. However, 20% of CML patients proceed to advanced stages that result in drug resistance, intolerance, and mortality. So, for proper CML treatment, drugs are needed to target multiple BCR– ABL mutations, delay or stop disease progression, and overcome resistance caused by BCR–ABL independent mechanisms, especially during advanced phases of CML. Moreover, drugs could be developed to eradicate the stem cells of CML. These targets could be achieved by understanding mechanisms of disease progression, disease relapse, and drug resistance by utilizing high throughput molecular genetics, cell biology and immunology techniques.

Combinatorial Treatment Options for Highly Resistant Compound Mutations in the Kinase Domain of the BCR::ABL1 Fusion Gene

The introduction of tyrosine kinase inhibitor (TKI)-based therapies has dramatically changed the outcome in patients with chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ALL). However, the occurrence of point mutations in the kinase domain (KD) of the BCR::ABL1 fusion gene can impair or inhibit the efficacy of individual TKIs. In advanced phases of CML and in Ph +ALL, patients frequently develop compound mutations (CMs), defined as the presence of more than one mutation on the same BCR::ABL1 molecule in a leukemic cell, which are often highly or even completely resistant to any individual TKI. Some CMs respond to high doses of the currently most potent available TKI, ponatinib, but the dose-related toxicity is an important impediment. The most frequently occurring CMs, which include the gatekeeper mutation T315I, are often resistant to any currently available TKI. We performed in vitro analyses using a common cell line model based on Ba/F3 cells expressing a panel of 12 different CMs (V299L/G250E, V299L/E255V, V299L/F359V, F317L/Y253H, F317L/E255V, F317L/F359V, F359V/Y253H, T315I/M244V, T315I/G250E, T315I/E255K, T315I/E255V, T315I/F359V) in BCR::ABL1 constructs introduced into the cells by a transposon-mediated approach. The presence of single insertion copies of the mutant constructs was verified by FACS analysis to preclude skewed test results potentially caused by multiple copies of the mutant construct. The cells pre-tested in this way were exposed in vitro to different concentrations of various drugs including ponatinib combined with a number of other agents such as asciminib, hydroxyurea, palbociclib, venetoclax, ibrutinib, vodobatinib, and crizotinib. The drug concentrations tested corresponded to the spectrum of the respective dosing regimens used in the clinical setting. The cell viability assays performed indicated that CMs responding only to elevated concentrations of ponatinib could be suppressed by combinatorial treatment with lower concentrations of ponatinib and different other agents tested. Moreover, CMs displaying high levels of resistance to TKIs applied as single agents mostly revealed adequate in vitro responses to individual drug combinations with ponatinib, most commonly including asciminib, hydroxyurea or crizotinib. Only the most resistant CM constellation tested, T315I/F359V, failed to respond to the compounds investigated and showed a limited response to the combination of high concentrations of ponatinib with hydroxyurea. A good response of this CM in vitro was only achieved by a combination of ponatinib with a very high concentration of crizotinib indicating the need for further testing of additional candidate drugs for this constellation. Overall, combinatorial treatment of BCR::ABL1 CMs indicated that relatively low concentrations of the drugs used are capable of effectively inhibiting in vitro survival of mutant cells in most instances, which is important with regard to the expected toxicity of combinatorial treatment approaches. Since our earlier data indicated a good correlation between in vitro test results and clinical responses, our observations may serve as a basis for novel treatment options in patients with Ph +leukemias displaying challenging BCR::ABL1 KD-mutations. [This project was supported by an educational grant from Incyte Biosciences International Sàrl]

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

An Update on the Management of Advanced Phase Chronic Myeloid Leukemia.

While most patients with chronic myeloid leukemia (CML) present in a chronic phase and are expected to have a normal life expectancy, some patients present with or progress to a more aggressive accelerated phase (AP) or blast phase (BP) of CML. Herein, we discuss the diagnostic considerations of advanced phase CML and review its contemporary management. Later-generation, more potent BCR::ABL1 tyrosine kinase inhibitors (TKIs) such as ponatinib may result in superior outcomes in patients with advanced phase CML. For CML-BP, combination approaches directed against the blast immunophenotype appear superior to TKI monotherapy. The role of allogeneic stem cell transplantation is controversial in CML-AP but has consistently been shown to improve outcomes for patients with CML-BP. Advanced phase CML, particularly CML-BP, remains a poor risk subtype of CML. However, novel combination approaches using later-generation TKIs are being explored in clinical trials and may lead to improved outcomes.

The Role of Allogeneic Transplantation in Chronic Myeloid Leukemia in 2023: A Case-Based Concise Review

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML), granting patients a life expectancy close to that of the normal population and, in a subset of patients, the possibility to discontinue therapy. Nonetheless, for a not negligible minority of patients, TKIs are not able to control CML. Allogeneic hematopoietic cell transplantation (HCT) has long been a pivotal therapy for CML. At present, allogeneic HCT is considered an option in CML patients diagnosed or progressing to blast phase (BP), for those in chronic phase (CP) resistant to multiple lines of TKI therapy or for those experiencing severe toxicity, mostly hematologic, under TKIs. Moving from real-world cases, we reviewed the results of allogeneic HCT in the setting of advanced-phase CML or failure of TKIs, with a focus on the progresses in transplant technology that has extended transplant options in elderly CML patients and in those lacking a sibling donor, and on the post-HCT strategies for prevention and treatment of disease relapse.

Next-generation DNA sequencing identifies mutated FANCD2 Gene as a novel Biomarker for monitoring early disease progression and timely therapeutic interventions in advanced phase Chronic Myeloid Leukemia patients

Next-generation DNA sequencing identifies mutated FANCD2 Gene as a novel Biomarker for monitoring early disease progression and timely therapeutic interventions in advanced phase Chronic Myeloid Leukemia patients

Transplantation in CML in the TKI era: who, when, and how?

Molecular therapy with tyrosine kinase inhibitors (TKIs) has significantly reduced the indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in chronic myeloid leukemia (CML). Treatment-free remission can be obtained in about 50% of patients with an optimal response. However, cure rates up to 90% are restricted to patients receiving HSCT. Timing is essential since HSCT in the early stages of the disease has the best outcome. Patients in a more advanced phase (AdP) than chronic-phase (chP) CML undergo HSCT with suboptimal outcomes, and the gap between chP and AdP disease is widening. First-line therapy should start with first- or second-generation (G) TKIs. Patients failing treatment (BCR-ABL1 transcripts of greater than 10% at 3 and 6 months and greater than 1% at 12 months) should be switched to second-line TKIs, and HSCT should be considered. Patients not responding to 2G-TKI therapy as well as patients in an accelerated phase (AP) or blast crisis (BC) are candidates for HSCT. Therapy resistant BCR-ABL1 mutations, high-risk additional cytogenetic abnormalities, and molecular signs of leukemia progression should trigger the indication for HSCT. Patients who, despite dose adjustments, do not tolerate or develop severe adverse events, including vascular events, to multiple TKIs are also candidates for HSCT. In AdP CML, TKIs do not show long-lasting results, and the outcome of HSCT is less optimal without pretransplant therapy. In these patients the induction of chP2 with TKIs, either alone (AP) or in combination with intensive chemotherapy (BC), followed by HSCT should be pursued.

A Phase II Study of the Combination of Decitabine, Venetoclax and Ponatinib in Patients with Chronic Myeloid Leukemia (CML) in Myeloid Blast Phase (MBP) or Philadelphia-Chromosome Positive (Ph+) Acute Myeloid Leukemia (AML)

Background: Advanced phases of CML and Ph+ AML are therapeutic challenges. Preclinical and early clinical data have shown that combining venetoclax (VEN) with a tyrosine kinase inhibitor (TKI) works synergistically in Ph+ myeloid leukemias (Leonard et. al., Sci Transl Med 2016 and Maiti et. al., Acta Haematologica 2020). We hypothesized that combining a potent TKI (ponatinib) with a hypomethylating agent plus VEN backbone might lead to high rates of response that could facilitate potentially curative allogeneic stem cell transplantation (SCT). Methods: This is a single-arm phase II study (NCT04188405) that enrolled patients (pts) ≥ 18 years (yrs) of age with newly diagnosed or relapsed/refractory advanced phase CML [CML in accelerated phase (CML-AP) or CML-MBP] or Ph+ AML. Pts were required to have an ECOG PS ≤ 3 and adequate liver and renal function. Pts with clinically significant cardiovascular comorbidities, including recent or active myocardial infarction, heart failure, clinically significant arrhythmias, cerebrovascular accident, venous thromboembolism or uncontrolled hypertension were excluded. Pts were treated with a combination of decitabine (DAC) (20 mg/m2/day) x 5 days, VEN (400 mg equivalent dose/day) x 21 days, and ponatinib (45mg/day) x 21 days in cycle 1 and then continuously in cycle 2+ (Fig. 1A). Ponatinib was reduced to 30mg/day for pts in CR/CRi and to 15 mg/day for pts in CMR. For C1 only, pts who had not received ponatinib prior to trial initiation had a lead-in phase of ponatinib monotherapy for 7 days before the initiation of DAC and VEN. Cycles were repeated every 28 days, as permitted by peripheral blood count recovery, and pts could receive a maximum of 24 cycles. Intrathecal prophylaxis with 4 doses of cytarabine was recommended. The primary objective was to determine the overall response rate of the regimen. Results: From July 2020 to May 2022, 12 pts were treated, 1 pt with de novo Ph+ AML and 11 pts with advanced phase CML (2 pts with CML-AP and 9 pts with CML-MBP). The median age of the pts was 45 yrs (range, 28-71 yrs). Three pts (25%) had received no prior therapy (1 each with Ph+ AML, CML-AP and CML-MBP). Nine pts (75%) had prior TKI exposure (including prior ponatinib exposure in 4 pts) and 3 pts (25%) had received prior chemotherapy. Eleven pts (92%) had the p210 transcript of BCR::ABL1. Eight pts (67%) had additional cytogenetic abnormalities, including 5 pts with complex karyotype and 2 pts with a MECOM rearrangement. 4 of 11 tested pts (36%) had an ABL1 kinase domain mutation detected at enrollment (T315I, Q252H, L384M and E255K in 1 pt each). The median number of cycles received was 2 (range, 1-7). Nine pts (75%) responded, including 1 with CR, 3 with CRi and 5 with MLFS as best response. The CR/CRi rate was 33%. Best response was achieved after cycle 1 in 7 of 9 responders. Both pts with CML-AP responded, and 2 of 3 pts without prior TKI exposure responded. One of the responders achieved an MMR. After a median follow-up of 9.1 months, the median overall survival (OS) is 9.5 months, and the estimated 9-month OS is 64%. The median relapse-free survival 5.1 months. Among the 9 responders, 3 pts are in continuous response without SCT, 1 pt proceeded to SCT and is still alive, 3 pts did not undergo SCT and relapsed (2 of whom had a complex karyotype), 1 pt died in MLFS, and 1 pt was lost to follow-up (Fig. 1B). The one pt who proceeded to SCT achieved MLFS as best response pre-SCT and remains in MRD-negative CR 22 months post-SCT. The toxicity profile of the regimen was similar to historical expectations with DAC plus VEN. One pt developed grade 3 mucositis, which was considered related to study therapy. No grade 4-5 related non-hematological adverse events were observed. Related grade 1-2 non-hematological adverse events included rash in 2 pts, and transaminitis, creatinine elevation and mucositis in 1 pt each. Of the 8 pts who received > 1 cycle of study therapy, 4 pts had delay (>1 week) in initiation of a subsequent treatment cycle due to cytopenias; for 2 of these pts the duration of VEN was reduced. The 60-day mortality rate was 0%. Two pts died on the study, one from leukemia and one from infection while in MLFS. Conclusion: The triplet combination of DAC, VEN and ponatinib was well-tolerated in pts with advanced Ph+ myeloid leukemias. In this poor-risk population, a marrow remission rate of 75% was achieved, although durations of remission were modest in the absence of consolidative SCT. This study continues to accrue pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery of Mutated ZNF208 Gene As a Novel Transcription Factor Exclusively Associated with Accelerated Phase and Blast Crisis Chronic Myeloid Leukemia: Hunt for Common Biomarker of Fatal CML Progression

Other Authors and their Affiliations: Muhammad Absar8, Tanveer Akhtar8, Masood Shammas9, Salman Basit10, Aamer Aleem11, Amer Mahmood11 1College of Applied Medical Sciences (CoAMS-A), King Saud Bin Abdulaziz University for Health Sciences / KAIMRC/ SSBMT, Al-Ahsa, Saudi Arabia; 8Hematology, Oncology and Pharmaco-genetic Engineering Sciences (HOPES) Group, Health Sciences Laboratories (HaSiL), Department of Zoology, Faculty of Biological Sciences, University of the Punajb, Lahore, Pakistan; 9Dana Farbar Cancer Institute, University of Harvard , Boston , United States of America; 10Centre for Genetics and Inherited Diseases, Taibah University, Madinah; 11College of Medicine, King Saud University, Riyadh, Saudi Arabia. #Corresponding author: Dr. Zafar Iqbal, drzafar.medgen1@gmail.com; iqbalz@ksau-hs.edu.sa Introduction: Chronic Myeloid Leukemia (CML) primarily develops as a result of t(22;9) that creates Philadelphia chromosome and oncogenic BCR-ABL fusion transcript (1). Discovery of BCR-ABL paved the way for designing signal transduction inhibitor drugs called tyrosine kinase inhibitors (TKIs) that have revolutionized CML treatment in 21st century, by making survival of CML equal to general population (2). Nevertheless, TKIs are minimally effective CML patients progressed to blast crisis (BC-CML). Unfortunately mechanism of CML progression is poorly understood (2). Various transcription factors (TFs) are associated with different diseases and progression of many cancers (3). Although a large number of TFs are involved in pathogenesis of acute leukemias, very little is known about role of TFs in CML progression. Therefore, objective of this study was to find about novel TFs associated with CML progression. Materials and Methods: Patient selection: The study subjects included accelerated phase (AP-CML) and blast crisis phase CML (BC-CML) patients (Experimental groups 1 & 2, respectively) while chronic phase treatment-naïve CML patients (CP-CML) were used as control 1, CP-CML CML long-term TKI responders (at least 3 continuous years of MMR) as control group 2 and TK-resistant CML patients as control group 3, along with healthy controls. Sample collection: DNA extraction and Clinical follow-up: 10 ml peripheral blood was collected from all study subjects. DNA was extracted and patient follow-up was carried out during course of this study. All treatment criteria per ENL guidelines were adopted. Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool (4). Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio (4). R package was employed to align specific TF-gene mutants to disease phenotypes (5). TF-genes mutated in all AP/BC-CML patients but not mutated in any of control groups were selected. Variants were confirmed using Sanger sequencing. Results: We found ZNF208 as a novel TF-gene mutated in all AP/BC-CML but in none of the controls (missense mutations G64A). It shows that mutated ZNF208 is a novel biomarker exclusively for advanced phases of CML. Discussion: ZNF208, a member of zing-finger binding transcription factor family, has been reported to be play role as tumor suppressor gene in various cancers through distant signaling pathways (3,6,7). Further studies are required to find exact role of ZNF208 in CML progression and its evaluation as potential biomarker and novel drug target in AP- and BC-CML. Conflict of interest: The authors declare that they have no conflict of interests.

High-Throughput DNA Sequencing Identifies a Novel Splice-Site Mutation in Fanconi Anemia Pathway Gene FANCD2 As a Potential Biomarker of Early and Late Phases of Disease Progression in Chronic Myeloid Leukemia

OtherAuthors: Muhammad Absar4, Tanveer Akhtar4, Masood Shammas8, Salman Basit9, Aamer Aleem10, Amer Mahmood10 8Dana Farbar Cancer Institute, University of Harvard , Boston USA, 9Centre for Genetics and Inherited Diseases, Taibah University, Madinah 10College of Medicine, King Saud University, Riyadh, Saudi Arabia. Introduction: Chronic Myeloid Leukemia (CML) results due to t(22;9) that leads to formation of Philadelphia chromosome and subsequently BCR-ABL fusion transcript, the later leading to leukemogenesis(1). This led to identification of tyrosine kinase inhibitors (TKIs) as novel drugs that revolutionised CML treatment in 21st century by improving the survival of CML equal to general population at least in developed countries (2). Nevertheless TKIs lose their effectiveness if CML patients progress to blast crisis (BC-CML). Despite extensive research in CML field, mechanism of its progression is still poorly understood and universal molecular biomarkers of CML progression are unknown (2). There are different mechanisms associated with initiation and progression of various cancers. DNA repair defects mediated through biologically very important Fanconi anemia (FA) pathway genes are associated with many cancers (3). Nevertheless, FA pathway genes have not been reported in myeloid malignancies. In this study, in searching for mutated genes associated with many mechanism, we hereby report mutations in a very important FA-pathway gene associated exclusively with CML progression for the first time in literature. Materials and Methods: Patient selection: Whole-exome sequencing (WES) was employed to find out mutations in novel TF-genes associated with accelerated phase (AP-CML) and blast crisis phase CML (BC-CML) patients (Experimental groups 1 & 2, respectively). Chronic phase treatment-naïve CML patients (CP-CML) were used as control 1, CP-CML CML long-term TKI responders (at least 3 continuous years of MMR) as control group 2 and TK-resistant CML patients as control group 3, along with healthy controls. Sample collection: DNA extraction and Clinical follow-up: 10 ml peripheral blood was collected from all study subjects. DNA was extracted and patient follow-up was carried out during course of this study. All treatment criteria per ENL guidelines were adopted. Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool (4). Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio (4). R package was employed to align gene mutants to disease phenotypes (5). DNA repair genes mutated in all AP/BC-CML patients but not mutated in any of control groups were selected. Variants were confirmed using Sanger sequencing. Results: A novel splice-site substitution (C2022T) in FANCD2 gene (that is a very important member of FA-pathway genes) was detected in all AP/BC-CML but in none of the controls. It shows that mutated FANCD2 can serve as a novel biomarker exclusively for advanced phases of CML. Discussion: It is the first report that a member of FA-pathway genes (FANCD2) has been reported mutated in progression of myeloid malignancies (6). It shows that DNA repair defects and genomic instability mediated through FA-pathway could be one of the important mechanisms in CML progression. Mutations in FANCD2 and such other genes could be reason why CML blast crisis patients have poor outcome of stem cell transplantation. Moreover, anti-cancer drugs targeting DNA repair defects (PARP inhibitors) like Olaparib in combination with TKIs could be effective in patients with mutated FANCD-2 and other such genes. Conflict of interest: The authors declare that they have no conflict of interests.

Pathogenesis and management of accelerated and blast phases of chronic myeloid leukemia.

The treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) has been a model for cancer therapy development. Though most patients with CML have a normal quality and duration of life with TKI therapy, some patients progress to accelerated phase (AP) and blast phase (BP), both of which have a relatively poor prognosis. The rates of progression have reduced significantly from over >20% in the pre-TKI era to <5% now, largely due to refinements in CML therapy and response monitoring. Significant insights have been gained into the mechanisms of disease transformation including the role of additional cytogenetic abnormalities, somatic mutations, and other genomic alterations present at diagnosis or evolving on therapy. This knowledge is helping to optimize TKI therapy, improve prognostication and inform the development of novel combination regimens in these patients. While patients with de novo CML-AP have outcomes almost similar to CML in chronic phase (CP), those transformed from previously treated CML-CP should receive second- or third- generation TKIs and be strongly considered for allogeneic stem cell transplantation (allo-SCT). Similarly, patients with transformed CML-BP have particularly dismal outcomes with a median survival usually less than one year. Combination regimens with a potent TKI such as ponatinib followed by allo-SCT can achieve long-term survival in some transformed BP patients. Regimens including venetoclax in myeloid BP or inotuzumab ozogamicin or blinatumomab in lymphoid BP might lead to deeper and longer responses, facilitating potentially curative allo-SCT for patients with CML-BP once CP is achieved. Newer agents and novel combination therapies are further expanding the therapeutic arsenal in advanced phase CML.

Asciminib for chronic myeloid leukaemia: Next questions.

Recent approval of asciminib, a novel "specifically targeting the ABL myristoyl pocket" (STAMP) BCR-ABL1 inhibitor, for the treatment of chronic myeloid leukaemia (CML) patients who have either failed ≥2 lines of therapy or have the T315I mutation, has provided clinicians with a wider selection of potentially effective treatment options. Asciminib has the attractive twin attributes of high potency directed against BCR-ABL1 and good tolerability based on its limited off-target effects. However, it is unclear exactly where asciminib will be positioned amongst the other available tyrosine kinase inhibitors (TKIs), especially ponatinib which is also available for the same indications. There are many questions yet to be answered with regard to the optimal use of asciminib which include its role in the first- and second-line settings, combination therapy with other TKIs, and effectiveness in advanced phase CML. In this review, we discuss the available data on asciminib while exploring a number of clinical trials in progress. Finally, we provide our opinion based on the current data about where asciminib is most likely to be the optimal choice, which will hopefully assist clinicians with therapy selection.

CLINICAL VALIDATION OF ANKRD36 MUTATIONS AS A NOVEL BIOMARKER FOR MONITORING EARLY PROGRESSION AND TIMELY CLINICAL INTERVENTIONS IN BLAST CRISIS CML.

Chronic Myeloid Leukemia (CML) is initiated in the bone marrow due to the chromosomal translocation t(9;22), resulting in the fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have transformed fatal CML into an almost curable disease. However, TKIs lose efficacy during disease progression, and the mechanism of CML progression remains to be fully understood. Additionally, common molecular biomarkers for CML progression are lacking. Our studies previously detected ANKRD36 (c.1183_1184 delGC and c.1187_1188 dupTT) associated exclusively with advanced phase CML. However, clinical validation of this finding was pending. Therefore, this study aimed to clinically validate mutated ANKRD36 as a novel biomarker of CML progression. The study enrolled 124 patients in all phases of CML, recruited from Mayo Hospital and Hameed Latif Hospital in Lahore, Punjab, between January 2019 and August 2021. All response criteria were adopted from the European LeukemiaNet guideline 2020. Informed consent was obtained from all study subjects. The study was approved by scientific and ethical review committees of all participating centers.Sanger sequencing was employed to detect ANKRD36 mutations in CML patients in accelerated phase (AP) (n=11) and blast crisis (BC) (n=10), with chronic-phase CML (CP-CML) patients as controls (n=103). Samples were processed using Big Dye Terminator Cycle Sequencing Ready Reaction kits and sequenced using ABI Prism 3730 Genetic Analyzer, and sequencing using forward and reverse primers for ANKRD36. During our study, 17% of CML patients progressed to advanced phases AP-CML n=11 (8.9%) and BC-CML n=10 (8.1%). The chronic- and advanced-phase patients showed significant difference with respect to male-to-female ratio, hemoglobin level, WBC count, and platelet count. Sanger sequencing detected ANKRD36 mutations c. 1183 1184 delGC and c. 1187 1185 dupTT exclusively in all AP- and BC-CML patients but in none of the CP-CML patients. Nevertheless, mutations status was not associated with male-to-female ratio, hemoglobin level, WBC count, and platelet count, which makes ANKRD32 as an independent predictor of early and terminal disease progression in CML. The study confirms ANKRD36 as a novel genomic biomarker for early and late CML progression. Further prospective studies should be carried out in this regard. ANKRD36, although fully uncharacterized in humans, shows the highest expression in bone marrow, particularly myeloid cells. Functional integrated genomic studies are recommended to further explore the role of ANKRD36 in the biology and pathogenesis of CML.

Risk factors for outcome after allogeneic stem cell transplantation in patients with advanced phase CML

Allogeneic hematopoietic stem-cell transplantation (HSCT) remains the only curative option for patients with advanced chronic myeloid leukemia (CML). However, outcome is dismal and of short follow-up. The objective of the study was to determine long-term outcome and risk factors in patients with a history of CML Blast Crisis (BC; n = 96) or accelerated phase (n = 51) transplanted between 1990 and 2018. At transplant, patients had a median age of 39 (range 7–76) years and were in ≥CP2 (n = 70), in AP (n = 40) or in BC (n = 37) with a diagnosis-HSCT interval of median 1.9 (range 0.3–24.4) years. Overall survival (OS) amounted 34% (95% CI 22–46) and progression-free survival (PFS) 26% (95% CI 16-36) at 15 years. Adverse risk factors for OS and PFS were low CD34+ count in the graft, donor age (>36 years) and BC. Cumulative incidence of Non-Relapse Mortality (NRM) was 28% (95% CI 18–38) and of relapse (RI) 43% (95% CI 33–53) at 15 years. PB-HSCT and HSCT after 2008 were favorable prognostic factors for NRM, while family donor and patient age >39 years were independently associated with higher RI. HSCT resulted in long-term OS in patients with advanced CML. OS was improved in non-BC patients, with donors ≤36 years and with higher CD34+ dose in the graft.

SETD2 Loss of Function Is a Recurrent Event in Advanced-Phase Chronic Myeloid Leukemia

Abstract In recent years, deregulation of epigenetic modifiers has turned out to be a recurrent event in cancer evolution. The SETD2 tumor suppressor gene encodes a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone H3 (H3K36Me3), the major chromatin mark associated with active transcription and alternative splicing. Recent studies have also implicated SETD2 in homologous recombination repair, mismatch repair, mitosis and cytokinesis. SETD2 loss of function was initially uncovered in clear cell renal cell carcinomas and was subsequently found in many other solid tumors. More recently, it has also been reported in a variety of hematologic myeloid and lymphoid malignancies, including acute lymphoblastic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphoma, systemic mastocytosis and myeloproliferative neoplasms. We thus wondered whether advanced-phase chronic myeloid leukemia (CML), that shareS similarities with acute leukemias including a high level of genetic instability, may also exhibit SETD2 loss of function. To this purpose, we studied a relatively large cohort of 69 advanced phase CML patients (pts), including pts with accelerated phase (AP; n=15), myeloid blast crisis (my-BC; n=30), and lymphoid blast crisis (ly-BC; n=15) and pts with tyrosine kinase inhibitor (TKI)-resistant chronic phase harboring 2 or more BCR-ABL kinase domain mutations (mut-CP; n=9). Samples collected at diagnosis from CP pts (n=8) who achieved optimal response to therapy were also studied for comparison. A recent study in advanced systemic mastocytosis has demonstrated that SETD2 loss of function may result not only from genomic disruption but also from post-translational events, suggesting that the frequency of SETD2 involvement in solid tumors or hematologic malignancies might be higher than sequencing and copy number data alone would suggest. We thus decided to use a Western Blotting (WB) approach to assess H3K36Me3 levels as a surrogate marker of SETD2 loss of function and to screen for the expression of SETD2 full-length vs truncated protein isoforms, that are the most frequent result of inactivating nonsense or frameshift mutations. Reduced or null H3K36Me3 (as compared to a pool of healthy donors) was detected in the great majority of pts with advanced-phase CML (58/69, 84.1%; 12/15 AP, 24/30 my-BC, 12/15 ly-BC, 8/9 mut-CP). In contrast, CP pts at diagnosis showed H3K36Me3 levels superimposable to those of healthy donors. In some of them, however, the CD34+ cell fraction was available and reduced H3K36Me3 levels were detected in all cases. No evidence of abnormal SETD2 protein isoforms was detected, but pts with no H3K36Me3 also had no detectable SETD2 protein and pts with reduced H3K36Me3 had similarly reduced levels of full-length SETD2 protein as assessed by densitometric analysis of WB (Spearman R=0.95, p To rule out the possibility that SETD2 loss of function might be another age-associated alteration in an epigenetic regulator, we collected a series of 80 healthy individuals with an age ranging from 65 to 95 years. In this cohort, we checked for SETD2 mutations by sequencing and for SETD2 and H3K36 trimethylation levels by WB. No mutations were detected and protein levels were not significantly lower in older adults than in a pool of younger adults. Advanced-phase CML is characterized by a profound genetic heterogeneity. We here show the first evidence for a novel, nearly unifying alteration. However, SETD2 downmodulation and, consequently, H3K36Me3 deficiency seem to be most frequently accomplished at the post-translational level rather than being the results of genetic/genomic hits or transcriptional repression. Further studies aimed to assess whether H3K36Me3 loss might contribute to the genetic instability that is the hallmark of advanced-phase CML are warranted. Restoring physiological H3K36Me3 levels may help improve the outcome of this critical subset of pts. Supported by AIL and AIRC (project code 16996) Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Gugliotta: Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti: Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Iurlo: Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Abruzzese: Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; BMS: Consultancy. Crugnola: BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Bonifacio: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Tiribelli: Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Galimberti: Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau. Baccarani: Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Martinelli: Celgene: Consultancy; Amgen: Consultancy; JohnsonJ Pfizer: Consultancy; Ariad/Incyte: Consultancy; Roche: Consultancy. Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

Disovery of Novel Mutations Exclusively Shared By Accelerated and Blast Crisis Phase CML Patients Using Whole Exome Sequencing:Implication in Hunt for Common Biomarker /Drug Target of CML Progression

Introduction:Chronic Myeloid Leukemia (CML) is resulted due t (9;22) which leads to fusion oncogene BCR-ABL. Although chronic phase (CP) CML is treatable, accelerated (AP) and blast phases (BP) have high degrees of drug resistance and subsequent treat failures 1. Mechanism of this disease progression in CML is poorly understood and few markers studied in this regard are found only in a subset of advanced phase CML patients 2,3. Therefore, this study was designed to find out common gene mutations (trunk mutations) associated with all advanced phase CML patients.Material and Methods:We investigated imatinib responder AP (N=13), BC (10) and CP (5) CML patients along with age-sex matched healthy controls using whole exome sequencing (WES). Illumina NGS instrument (HiSeq) generated bcl files which were converted to fastq files by using bcl2fastqtool 4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio 4. R package was employed to align specific gene mutants to disease phases 5. In order find a common CML progression biomarkers, gene mutations shared by all patients at a CML phase (so called trunk mutations) were selected and confirmed using Sanger sequencing.Results:Whole exome sequencing revealed novel gene mutations associated with different disease phases in CML. CP CML patients with long term CML response harbored mutations in TTN, PDLIM4, CELF2, ANO5, DOK2, MYO16, RAI1, LTBP3, CNNM3. On the other hand, specific genes were found mutated in AP (EV15L, FAM120B, ZNF208, PML, TMEM145, ASXL1, ABL1) and BC (ATXN3)CML patients.Discussion & Conclusions:All the genes mutated in AP and BC CML were either involved in other cancers or cellular processes like DNA repair, regulation of gene expression (for instance ATXN3 gene mutated in all BC-CML is involved in DNA repair and in repressing expression of some other genes6 which indicates role of damaged DNA repair in CML blast crisis). Mutations in long term CML responders indicate role of all are some of these genes in relation to sensitivity to tyrosine kinase inhibitors (TKIs). Very recently, it has been reported that CML patients with rs460089-GC (SLC22A4) genotype had stable major molecular response 7. Biomarkers of stable response can help identify patients who can be the candidates of cessation of TKIs which is one of the major focus of many CML trials. Identification of biomarkers of early CML progression can help identify and clinically intervene patients at high risk of transformation to advanced and blast phases of CML which can considerably minimize morbidities and mortalities associated with CML patients 2,3.

Advanced Phases at Diagnosis of Childhood Chronic Myeloid Leukemia : The Experience of the International Registry for Chronic Myeloid Leukemia (CML) in Children and Adolescents (I-CML-Ped Study)

Introduction. Chronic myeloid leukemia (CML) is a very rare disease in children and adolescents. The international registry of CML in children and adolescents (I-CML-Ped Study) established in the year 2011 has proven very beneficial to better describe this disease in pediatric cohorts.Aims: To describe the characteristics and the outcome of the pediatric population with advanced phases of CML at initial diagnosis.Patients and Methods: Patients enrolled in the international registry for CML in children and adolescents (I-CML-Ped Study) were the subjects of the study .Disease phases were defined according to the ELN criteria. Characteristics and outcome were determined in children in accelerated (AP) and blastic phase (BP). No recommendations were given regarding the treatment of the patients. Overall survival (OS) was estimated from diagnosis by the Kaplan and Meier method.Results. 479 children and adolescents less than 18 years old with CML were enrolled in the I-CML-Ped study between January 2011 and April 2017. Among them, 37 patients (7.7%) presented at initial diagnosis with CML in advanced phase according to the ELN criteria: 20 children (13 boys and 7 girls, median age 12.6 years - range 3 to 18 years) and 17 children (11 boys and 6 girls, median age 11 years - range 5 to 17 years) were diagnosed in AP and in BP, respectively. Among the 17 patients diagnosed in BP, 12 (70%) had lymphoid and 2 myeloid phenotype (not specified: 2 patients with extramedullary disease as unique criteria of BP). According to the Sokal score for patients less than 45 years, 73% and 82% of the patients in AP and BP at diagnosis were high risk patients, respectively. For the 17 children in BP, treatment consisted of tyrosine kinase inhibitors (TKI) (n=3) or a combination of chemotherapy and TKI successively and/or simultaneously (n=14); 11 of them were transplanted (sibling donor: n=2; unrelated donor n=8; unknown: n=1) with a median time after diagnosis of 7 months (range, 4.5 to 10 months). For the 20 children in AP, treatment consisted of TKI (n=17) or a combination of chemotherapy and TKI successively and/or simultaneously (n=3); 6 of them were transplanted (sibling donor: n=5; unrelated donor n=1) with a median time after diagnosis of 8 months (range, 4 to 21.5 months). With a median time from diagnosis of 3.5 years (range 8 months to 13.5 years), 13 out of 17 patients in BP at initial diagnosis are alive in at least major molecular response (MMR) (11 patients) or without MMR (2 patients) including 5 patients who were not transplanted; death occurred in 3 transplanted patients (relapses: 2 patients; chronic graft versus host disease: 1 patient) and in one patient with progression of the disease. Nineteen out of 20 patients in AP at initial diagnosis are alive (one patient died after progression) in at least MMR (15 patients) or without MMR (2 patients) or unknown status (2 patients). The 5 years OS rates were 94% (95% confidence interval (CI): 66%-99%) and 74% (95% CI: 44%-89%) for patients diagnosed in AP or BP, respectively.Conclusion: The data indicates that the frequency of advanced phases at diagnosis of CML is similar in children and in adults but with a predominance of lymphoid phenotype in children in blastic phase compared to adult population. Hematopoietic stem cell transplantation remains a curative treatment of children in blastic phase at diagnosis.Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company DisclosuresNo relevant conflicts of interest to declare.