Chronic Myeloid Leukemia Blast Crisis Research Articles

Acquired Factor VII Deficiency Associated With Chronic Myeloid Leukemia Blast Crisis.

Factor VII (FVII) is an important, vitamin K-dependent clotting factor. Acquired FVII deficiency is a rare entity that is associated with serious bleeding complications. We report a case of acquired FVII deficiency in a patient with recurrent chronic myeloid leukemia in blast crisis who developed bilateral retinal hemorrhages. The coagulopathy was corrected with the initiation of chemotherapy and subsequent reduction in peripheral blast count.

Pediatric chronic myeloid leukemia in myeloid blast crisis

Chronic myeloid leukemia (CML) accounts for 2-3% of childhood leukemias. About 5% of cases present in a blastic phase of CML which clinically and morphologically mimics more common acute leukemias of childhood. We report a case of a 3-year-old male who presented with gradual onset swelling of the abdomen and extremities along with generalized weakness. Examination revealed massive splenomegaly, pallor, and pedal edema. Initial workup showed anemia, thrombocytopenia, and leukocytosis (120,000/uL) with a blast percentage of 35%. Blasts were positive for CD13, CD33, CD117, CD34 and HLA-DR, and stained negative for Myeloperoxidase and Periodic Acid Schiff. Fluorescence in situ hybridization was positive for b3a2/e14a2 junction BCR-ABL1 transcript and negative for RUNX1-RUNX1T1/t(8;21), clinching the diagnosis of CML in myeloid blast crisis. The patient expired within 17 days of diagnosis and initiation of therapy.

Outcome of Haploidentical Hematopoietic Stem Cell Transplantation with a Donor and Recipient Infected with SARS-CoV-2 Infection

The current coronavirus disease 2019 (COVID 19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS CoV 2), has impacted many facets of hematopoietic cell transplantation (HCT) in both developed and developing countries. The full impact of the COVID 19 pandemic, caused by the SARS CoV 2, on the field of hematopoietic cell transplantation (HCT) is unknown. Here, we report a rare case of a 21-year-old male patient known to have chronic myeloid leukemia (CML) with progression to T-cell lymphoblastic lymphoma as extramedullary blast crisis of CML. The patient was treated by pediatric chemotherapy regimen then. He underwent haploidentical stem cell transplantation. Posttransplantation, on day +7, he developed SARS-CoV-2 after receiving stem cell graft from a donor who was diagnosed with SARS-CoV-2 on the day of stem cells harvesting. The case elaborates complications and outcome of a patient receiving stem cell transplant from a donor with SARS-CoV-2 infection.

Blast crisis in chronic myeloid leukemia: An immunophenotypic analysis

Background and Aim of Study: There are two different phases of untreated chronic myeloid leukemia – chronic phase, and blast crisis – according to the World Health Organization classification of Hematolymphoid tumors. The blast cells in the chronic myeloid leukemia blast crisis can express myeloid, lymphoid, bi-phenotypic, monocytic, megakaryocytic, and erythroid phenotypes. The immunophenotype of blast population determines how chronic myeloid leukemia – blast crisis patients respond to treatment, hence flowcytometric examination is required. The aim of the study: to assess immunophenotyping outcomes of flowcytometry performed on the chronic myeloid leukemia – blast crisis. Material and Methods: A five-year retrospective descriptive analysis was carried out in Pathology Department at King George’s Medical University Lucknow, India (2017-2021). The patient’s peripheral blood and bone marrow aspirate samples were analyzed. Clinical, hematological, and immunophenotypic data were retrieved. The flow cytometry samples were prepared using the standardized “lyse-stain-wash” method. Results: A total of 43 cases of chronic myeloid leukemia – blast crisis were retrieved from the departmental archive in 5 years. The mean age of study population was 39.62±14.86 years. There were 24 males and 19 females. 27 patients were diagnosed with myeloid blast crisis, 14 cases of B-lymphoid blast crisis and 2 cases of mixed phenotypic acute leukemia. Conclusions: Identification of the blast lineage of patients with chronic myeloid leukemia – blast crisis is crucial since the existence of atypical blast phenotypes influences the disease treatment and prognosis.

Molecular genetic characterization of Philadelphia chromosome-positive acute myeloid leukemia

BackgroundPhiladelphia chromosome-positive acute myeloid leukemia (Ph+ AML) is a provisional disease entity in the 2016 WHO classification, while its genetic profile of Ph+ AML remains poorly defined. In addition, the differentiating features of Ph+ AML and chronic myeloid leukemia in myeloid blast crisis (CML-MBC) remain controversial. MethodsWe conducted a retrospective study of 15 Ph+ AML patients to compare their clinical and laboratory profiles with 27 CML-MBC patients. ResultsCompared to CML-MBC, Ph+ AML patients presented with significantly higher peripheral WBC count and bone marrow blast percentage. The immunophenotypic profiles were largely similar between Ph+ AML and CML-MBC, except for CD4 expression, which was significantly enriched in CML-MBC. Ph+ AML patients less frequently harboured co-occurring additional cytogenetic abnormalities (ACA) compared to CML-MBC, and trisomy 19 (23%) and IDH1/2 (46%) were the most common ACA and mutated genes in Ph+ AML, respectively. Overall survival (OS) did not significantly differ between Ph+ AML and CML-MBC. Ph+ AML without CML-like features appeared to have a better outcome compared to Ph+ AML with CML-like features; ACA in Ph+ AML may confer an even worse prognosis. ConclusionsOur results indicate that patients with Ph+ AML share similar genetic profiles and clinical outcomes with those with CML-MBC, thus should be classified as a high-risk entity.

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.

Molecular Aberrations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome with Excess Blast II (MDS-EB II) in Kuwait

Introduction: AML is a heterogeneous disease. Different mutational profiles have been described and correlate with variable outcomes. Genetic abnormalities are not well-described in our region. Our aim is to describe the molecular aberrations observed in the first cohort of patients. We recently introduced DNA based Oncomine Myeloid Gene Panel testing 55 genes. Method: we collected clinical, cytogenetic and molecular data prospectively since we introduced the test in late 2020. We also reviewed medical charts to collect baseline clinical data. We included patients aged 16 yrs and above with AML or MDS-EB II treated at Kuwait Cancer Center. Results: fifty-seven patients were included in the study with median age of 51 yrs. Almost one third of the patients were Kuwaitis (36%). Baseline demographic described in (table 1). We have 53 patients with AML (92%), 3 patients with MDS-EB II (5%) and 1 pt with chronic myeloid leukemia in myeloid blast crisis (CML-MBC). Most of the patients had at least one mutation (91%) detected by the panel. The most common mutations (figure1) were FLT3 (ITD & D835) detected 26.3%, followed by NPM-1 detected in 21%. We have noticed an increased PTPN11 mutation, detected in 17.5% of the cases. Conclusion: This is an important study showing extensive molecular abnormalities in AML patients in the region. This will facilitate more understanding of the disease biology and potentially guide therapeutic options

Orca-Q Demonstrates Favorable GvHD-and-Relapse-Free Survival in Haploidentical Transplants without Post-Transplant Cyclophosphamide

^Salhotra, Srour, Lowsky and Abedi contributed equally Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) can be curative for a variety of malignant and nonmalignant disorders, but until recent years, access has been limited to patients with HLA-matched donors. The increased utilization of post-transplant cyclophosphamide (PTCy) has enabled patients to receive alloHSCT with haploidentical (haplo) related donors; however, high rates of cGVHD following myeloablative conditioning (MAC) regimens and high rates of relapse following reduced intensity conditioning regimens have been observed with this approach. Thus, GVHD-and-relapse free survival (GRFS) rates in this population at 1-year post-transplant remain low. Haplo alloHSCT with PTCy is also associated with high incidence of cytokine release syndrome (CRS) and infections. Orca-Q -an investigational precision engineered cell therapy- represents an alternative strategy which may be administered with single agent prophylaxis and does not require PTCy. The cellular composition of Orca-Q consists of enriched CD34+ stem cells combined with specific T- cell subsets and is intended to reconstitute the blood and immune systems. Orca-Q is hypothesized to reduce GVHD, relapse, and serious infections. Methods: Adult patients (18 to 65 years) with high-risk hematologic malignancies undergoing myeloablative conditioning (MAC) alloHSCT were enrolled between January 2019 to July 2022 on the haplo donor dose expansion arm of a multicenter phase 1 study of Orca-Q (NCT03802695). Haplo was defined as ≥ 4/8 but < 7/8 matched related donor at HLA-A, -B, -C, and -DRB1 typed using DNA-based high-resolution. Patients received only single agent tacrolimus (starting on day -1 and taper day +60 if no GVHD) as GVHD prophylaxis. PTCy was not permitted. The Orca-Q drug product was manufactured centrally at Orca Bio Manufacturing Site in Sacramento, CA from G-CSF mobilized peripheral blood apheresis. Results: The Orca-Q drug product was successfully manufactured and delivered to all subjects with a vein-to-vein time of less than 72 hours. A total of 21 patients (11 AML, 8 ALL, 2 CML in blast crisis) with > 30 days of follow up were included in this analysis. All were 4/8 HLA match at HLA-A, -B, C, and DRB1. Median age of subjects was 44 years (range 21 - 63) and median follow-up was 333 days (range 39 - 1020). Disease risk index (DRI) score was available in 20 showing 2 very high, 3 high, 13 intermediate, and 2 low risk categories. The M:F ratio was 12:9. Patients received either TBI (n=13) or non-TBI (n=8) based MAC regimens. 2 patients had Gr 1 CRS (CTCAE v5). Median neutrophil and platelet engraftment times were 13 days (range 8 - 25) and 16 days (range 12 - 25), respectively. 1 secondary engraftment failure was observed. The estimated incidence of BMT CTN MOP3 infections was 27% at 1 year. The estimated incidence of gr 2-4 aGVHD at 6 months was 14% which compares favorably with literature reports of 21% to 63% for gr 2-4 aGVHD in patients undergoing haplo transplant with PTCy-based prophylaxis.[1-5] Likewise, gr 3-4 aGVHD was rare with Orca-Q with only 1 case of gr 3 aGVHD and no gr 4 aGVHD. Of the 16 patients who had at least 3 months follow up, only 1 developed mild cGVHD. No patients had moderate-to-severe cGVHD, a notable improvement compared to historic rates of 24% to 31% for moderate-to-severe cGVHD with PTCy, despite the fact that Orca-Q patients receive only single agent GVHD prophylaxis.[1-4] 2 patients experienced disease relapse and there was a total of 5 deaths (2 due to relapse, 1 Aspergillus pulmonary infection, 1 respiratory failure associated with renal failure, and 1 COVID infection). GVHD-and-relapse free survival (GRFS) was 71% at 1 year with Orca-Q (Figure 1) which compares favorably to a GRFS of 46% reported by Sanz in the context of MAC haplo alloHSCT with PTCy.[2] Conclusions: Initial Orca-Q data demonstrates good clinical outcomes with single agent tacrolimus and no PTCy or MMF. Patients treated with Orca-Q experienced a low adverse event profile, low incidence and severity of both aGVHD and cGVHD, and improved GRFS rates, offering a potential new treatment option for patients undergoing haplo alloHSCT. This phase 1 study of Orca-Q continues to enroll patients with haplo donors across the US. References (1) Gooptu, M., et al. (2) Sanz, J., et al. (3) Wagner, J.E., et al. (4) Baker, M., et al. (5) Bashey, A., et al. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Primary Chronic Myelogenous Leukemia Blast Crisis with Precursor B Lymphoblastic Leukemia, a Case Report

Abstract Introduction/Objective Primary chronic myelogenous leukemia (CML) blast crisis at the initial disease presentation is rare. Most CML blast crisis cases present with increased myeloblasts with a minority of patients showing lymphoblastic leukemia. Differentiating primary CML lymphoblast crisis from de novo acute lymphoblastic leukemia may represent a diagnostic challenge to both pathologists and treating clinicians. This distinction is important as it has significant implications on patient management. Methods/Case Report A 15-year-old male patient was admitted to our University Hospital for hyperleukocytosis. Patient reportedly had weight loss with occasional sweats and cervical lymphadenopathy. She was found to have massive splenomegaly. Peripheral blood showed hyperleukocytosis with predominance of granulocytes at all maturation stages ranging from blast to segmented neutrophils with increased blasts. Subsequent bone marrow findings were consistent with extensive involvement by B-lymphoblastic leukemia. Ten-color Flow cytometry showed approximately 30% blasts with rare lymphocytes and monocytes. The blasts revealed precursor B-lymphoblastic immunophenotypic expression of CD45, CD10, CD19, CD20, CD22, CD34, CD38, CD200, HLA-DR and TdT expression. The results were similar to that of peripheral blood. Granulocytes showed abnormal maturation pattern with increased immature precursors and partial expression of CD4 and CD56 with no abnormalities detected in lymphocytes. In this case, while the bone marrow findings are consistent with B-lymphoblastic leukemia, the peripheral blood findings are consistent with blast phase of chronic myeloid leukemia. Further evaluation by cytogenetic and molecular studies confirmed the presence of Philadelphia chromosome, p210 transcripts, and rearrangement of BCR-ABL1, which supported the impression of precursor B-lymphoblastic leukemia in primary blast phase of CML. The patient was treated with tyrosine kinase inhibitor combined chemotherapy and went into remission. She has been followed up without significant complications for a year. Results (if a Case Study enter NA) NA. Conclusion The diagnosis of CML in primary lymphoblastic crisis is rare and needs to be systemically excluded before giving the diagnosis of de novo BCR-ABL1-positive acute lymphoblastic leukemia. If the patient does not have splenomegaly or previous leukocytosis, it needs cytological examination and extensive molecular analyses.

B-Lymphoid Blast Phase–Chronic Myeloid Leukemia: Current Therapeutics

Blast crisis (BC) is one of the most dreaded complications of chronic myeloid leukemia (CML). Fortunately, the incidence of BC has diminished markedly in the BCR-ABL tyrosine kinase inhibitor (TKI) era. The primary objective of initial treatment in BC is to achieve a second chronic phase (CP) and to proceed to an allogeneic stem cell transplantation (SCT) in eligible patients. The clinical outcome of patients with CML BC remains unsatisfactory, even with highly potent TKIs, as remissions are short lived and there is an unmet need for novel therapies. We provide a comprehensive summary reviewing the current management of Lymphoid BC.

Histone chaperone ASF1A accelerates chronic myeloid leukemia blast crisis by activating Notch signaling

The blast crisis (BC) is the final deadly phase of chronic myeloid leukemia (CML), which remains a major challenge in clinical management. However, the underlying molecular mechanism driving blastic transformation remains unclear. Here, we show that ASF1A, an essential activator, enhanced the transformation to CML-BC by mediating cell differentiation arrest. ASF1A expression was aberrantly increased in bone marrow samples from CML-BC patients compared with newly diagnosed CML-chronic phase (CP) patients. ASF1A inhibited cell differentiation and promoted CML development in vivo. Mechanistically, we identified ASF1A as a coactivator of the Notch transcriptional complex that induces H3K56ac modification in the promoter regions of Notch target genes, and subsequently enhanced RBPJ binding to these promoter regions, thereby enhancing Notch signaling activation to mediate differentiation arrest in CML cells. Thus, our work suggests that targeting ASF1A might represent a promising therapeutic approach and a biomarker to detect disease progression in CML patients.

AML-419 Molecular Aberrations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome With Excess Blast II (MDS-EB II) in Kuwait

AML is a heterogeneous disease. Different mutational profiles have been described and correlate with variable outcomes. Genetic abnormalities are not well-described in our region. Our aim is to describe the molecular aberrations observed in the first cohort of patients. We recently introduced DNA based Oncomine Myeloid Gene Panel, testing 55 genes. We collected clinical, cytogenetic and molecular data prospectively since we introduced the test in late 2020. We also reviewed medical charts to collect baseline clinical data. We included patients aged 16 yrs and above with AML or MDS-EB II treated at Kuwait Cancer Center. Fifty-seven patients were included in the study with median age of 51 yrs. Almost one third of the patients were Kuwaitis (36%). We have 53 patients with AML (92%), 3 patients with MDS-EB II (5%), and 1 patient with chronic myeloid leukemia in myeloid blast crisis (CML-MBC). Most of the patients had at least one mutation (91%) detected by the panel. The most common mutations were FLT3 (ITD & D835), detected 26.3%, followed by NPM-1, detected in 21%. We have noticed an increased PTPN11 mutation, detected in 17.5% of the cases. This is an important study showing extensive molecular abnormalities in AML patients in the region. This will facilitate more understanding of the disease biology.

Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.

In chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (PLK1), two serine-threonine kinases involved in the maintenance of genomic stability by preserving a functional G2/M checkpoint, have been implicated in BCR::ABL1-independent resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate and in leukemic stem cell (LSC) persistence. It can be speculated that the observed deregulated activity of Aurora A and Plk1 enhances DNA damage, promoting the occurrence of additional genomic alterations contributing to TKI resistance and ultimately driving progression from chronic phase to blast crisis (BC). In this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or PLK1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Danusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors from BC patients. Our results may have implications for the development of innovative therapeutic approaches aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.

Integrated genomic sequencing in myeloid blast crisis chronic myeloid leukemia (MBC-CML), identified potentially important findings in the context of leukemogenesis model

Chronic myeloid leukemia (CML) is a model of leukemogenesis in which the exact molecular mechanisms underlying blast crisis still remained unexplored. The current study identified multiple common and rare important findings in myeloid blast crisis CML (MBC-CML) using integrated genomic sequencing, covering all classes of genes implicated in the leukemogenesis model. Integrated genomic sequencing via Whole Exome Sequencing (WES), Chromosome-seq and RNA-sequencing were conducted on the peripheral blood samples of three CML patients in the myeloid blast crisis. An in-house filtering pipeline was applied to assess important variants in cancer-related genes. Standard variant interpretation guidelines were used for the interpretation of potentially important findings (PIFs) and potentially actionable findings (PAFs). Single nucleotide variation (SNV) and small InDel analysis by WES detected sixteen PIFs affecting all five known classes of leukemogenic genes in myeloid malignancies including signaling pathway components (ABL1, PIK3CB, PTPN11), transcription factors (GATA2, PHF6, IKZF1, WT1), epigenetic regulators (ASXL1), tumor suppressor and DNA repair genes (BRCA2, ATM, CHEK2) and components of spliceosome (PRPF8). These variants affect genes involved in leukemia stem cell proliferation, self-renewal, and differentiation. Both patients No.1 and No.2 had actionable known missense variants on ABL1 (p.Y272H, p.F359V) and frameshift variants on ASXL1 (p.A627Gfs*8, p.G646Wfs*12). The GATA2-L359S in patient No.1, PTPN11-G503V and IKZF1-R208Q variants in the patient No.3 were also PAFs. RNA-sequencing was used to confirm all of the identified variants. In the patient No. 3, chromosome sequencing revealed multiple pathogenic deletions in the short and long arms of chromosome 7, affecting at least three critical leukemogenic genes (IKZF1, EZH2, and CUX1). The large deletion discovered on the short arm of chromosome 17 in patient No. 2 resulted in the deletion of TP53 gene as well. Integrated genomic sequencing combined with RNA-sequencing can successfully discover and confirm a wide range of variants, from SNVs to CNVs. This strategy may be an effective method for identifying actionable findings and understanding the pathophysiological mechanisms underlying MBC-CML, as well as providing further insights into the genetic basis of MBC-CML and its management in the future.

Co-occurrence of CML Blast Crisis and Severe COVID 19 Infection: A Case Report

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affecting multiple organ systems. It can cause severe cytokine storms leading to intensive care unit admission requiring mechanical ventilation. However, there have been few studies establishing the outcomes of chronic myeloid leukemia (CML) patients on tyrosine kinase inhibitors who are infected with COVID-19. We present a 69-year-old male with a history of CML on imatinib therapy with COVID-19 who developed acute respiratory distress syndrome needing mechanical ventilatory support, shock requiring vasopressors, and worse outcome secondary to blast crisis.

Metabolic plasticity in blast crisis-chronic myeloid leukaemia cells under hypoxia reduces the cytotoxic potency of drugs targeting mitochondria

Metabolic reprogramming (MR) influences progression of chronic myeloid leukaemia (CML) to blast crisis (BC), but metabolic programs may change transiently in a second dimension (metabolic plasticity, MP), driven by environments as hypoxia, affecting cytotoxic potency (CPot) of drugs targeting mitochondria or mitochondria-related cell stress responses (MRCSR) such as mitophagy and mitochondrial biogenesis. We assessed mitochondrial membrane potential (MMP), mitochondrial mass (MM), apoptosis, glucose uptake (GU), and CPot of arsenic trioxide (ATO), CCCP, valproic acid (VPA), vincristine (VCR), Mdivi1, and dichloroacetic acid (DCA) in CML BC cells K562 (BC-K562) under hypoxia through flow cytometry, and gene expression from GEO database. About 60% of untreated cells were killed after 72 h under hypoxia, but paradoxically, all drugs but ATO rescued cells and increased survival rates to almost 90%. Blocking mitophagy either with VCR or Mdivi1, or increasing mitochondrial biogenesis with VPA enhanced cell-survival with increased MM. DCA increased MM and rescued cells in spite of its role in activating pyruvate dehydrogenase and Krebs cycle. Cells rescued by DCA, VPA and CCCP showed decreased GU. ATO showed equal CPot in hypoxia and normoxia. MP was evidenced by differential expression of genes (DEG) under hypoxia related to Krebs cycle, lipid synthesis, cholesterol homeostasis, mitophagy, and mitochondrial biogenesis (GSE144527). A 25-gene MP-signature of BC-K562 cells under hypoxia identified BC cases among 113 transcriptomes from CML patients (GSE4170). We concluded that hypoxic environment drove a MP change evidenced by DEG that was reflected in a paradoxical pro-survival, instead of cytotoxic, effect of drugs targeting mitochondria and MRCSR.

S154: IMATINIB-RESISTANT CLONES ISOLATED FROM A MODEL OF BLAST CRISIS OF CHRONIC MYELOID LEUKAEMIA DIFFER IN MUTATIONS IN BCR::ABL1 AND OTHER CANCER RELATED GENES AND IN THEIR SENSITIVITY TO BH3-MIMETICS

Background: The treatment of blast crisis of chronic myeloid leukaemia (BC-CML) remains difficult and the outcome of patients is poor. A dysregulated apoptosis leads to survival of malignant cells. BH3-mimetics inhibit antiapoptotic proteins of BCL-2 family. These drugs have been recently introduced in the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. This work assumed that these drugs may have a treatment potential for myeloid and/or lymphoid BC-CML. Aims: The aim is to test the sensitivity of imatinib-resistant (IR) clones of KCL-22, model of BC-CML, and primary blast cells to BH3-mimetics and describe a mechanism of sensitivity/resistance using protein analysis of apoptotic pathways. Methods: Isolated IR-clones (n=10) of KCL-22 were characterized by NGS and cultivated with 4µM imatinib (IM) and dilution series of BH3-mimetics (venetoclax – anti-BCL-2, S63845 – anti-MCL-1, A-1155463 - anti-BCL-XL) for 72 hours. IC50 was determined based on proliferation. Protein expression of BCL-2 family (n=10) was conducted in 4 IR-clones cultivated with IM and with/without BH3-mimetics. For in vivo experiments NOD-SCID-gamma mice (n=16) were subcutaneously injected with 106 cells of BCR::ABL1-T315I clone and divided into control group and treated groups. Primary cells of patients with BC-CML (n=4) were exposed to IM and BH3-mimetics for 7 days and LC50 was estimated. Results: IR-clones of KCL-22 differ in their sensitivity to BH3-mimetics (Table 1). The majority of clones (8/10) including 4 clones with T315I were sensitive to MCL-1 inhibitor. A reduced sensitivity was observed in the T315I clone carrying mutated GATA2 and other clone with mutation Y253H and mutated BCOR. Moreover, two T315I clones were sensitive to venetoclax, but other T315I clones with mutations in other cancer related genes showed insensitivity (n=2) or decreased sensitivity (n=1). Interestingly, two T315I clones with mutations in other cancer related genes insensitive to venetoclax were sensitive to BCL-XL inhibitor and in general, it seems that clones insensitive to venetoclax were sensitive to BCL-XL inhibitor and vice versa. Protein analysis of parental KCL-22IR cells and 4 clones showed a high expression of BCL-2 and BCL-XL and undetectable MCL-1 expression. Except for the T315I clone (B8, Table 1), other analysed clones expressed MCL-1 after exposure to BH3-mimetics. The sensitivity of T315I clone to anti-MCL-1 can be explained by detection of cBAX (an active form of apoptotic effector) after exposure to the inhibitor. The parental cell line showed dissociation of antiapoptotic complex MCL-1/BIM after exposure to MCL-1 inhibitor. The released BIM accelerates the apoptosis as an apoptotic activator. In vivo analysis revealed a decreased tumour growth of xenografted T315I clone (B8) in IM, venetoclax or combined treatment compared to control. An additive effect of dual therapy against monotherapy was not observed. This can be explained by IM inhibition of non-mutated BCR::ABL1 in KCL-22IR carrying 2 Ph chromosomes. LC50 analysis of primary blasts (4 patients) showed anti-MCL-1 to be the most potent inhibitor. Mutations in other cancer related genes and/or changes in karyotype increased LC50 for venetoclax and BCL-XL inhibitor or venetoclax, respectively. Image:Summary/Conclusion: The preliminary data of this study showed that the MCL-1 inhibitor S63845 seems to be the most potent BH3-mimetic to induce apoptosis in BC-CML. The combined therapy of TKI and BH3-mimetics should reflect mutation status of BCR::ABL1 and other cancer related genes.

Extramedullary Chronic Myeloid Leukemia – A Rare Cause of Ascites

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease characterized by anemia, extreme blood granulocytosis and granulocytic immaturity, basophilia, often thrombocytosis, and splenomegaly. Extramedullary disease (EMD) occurs in CML blast crisis. In literature, peritoneal involvement in CML chronic phase is reported very rarely to the best of our knowledge. A 60-year-old male patient presented with complaints of significant weight loss over 1-month duration and abdomen distension for 7 days. The patient was pale and had massive ascites on presentation. Blood investigations showed a total count of 61.9 × 109 L, and liver disease was ruled out on imaging. Ascitic fluid analysis showed high protein, elevated adenosine deaminase, ascitic fluid acid-fast bacillus staining was negative, and GeneXpert negative for tuberculosis. Upper gastrointestinal endoscopy and colonoscopy were normal. Ascitic fluid cytology, though negative for malignant cells, showed myeloid precursor cells. Peripheral smear showed a leukemoid reaction. Bone marrow aspiration showed blast &lt;3%. BCR/ABL1 was detected in leukocytes of peripheral blood. The patient was diagnosed with CML – chronic phase with peritoneal involvement as massive ascites – and was started tablet imatinib 400 mg OD and had favorable response to treatment after 3 months with the resolution of ascites and on follow-up. Extramedullary CML such as peritoneal disease serves as a predictor of future blast crisis, and close monitoring is needed for patients with EMD at initial presentation.