Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

miR-1202 acts as anti-oncomiR in myeloid leukaemia by down-modulating GATA-1S expression.

Transient abnormal myelopoiesis (TAM) is a Down syndrome-related pre-leukaemic condition characterized by somatic mutations in the haematopoietic transcription factor GATA-1 that result in exclusive production of its shorter isoform (GATA-1S). Given the common hallmark of altered miRNA expression profiles in haematological malignancies and the pro-leukaemic role of GATA-1S, we aimed to search for miRNAs potentially able to modulate the expression of GATA-1 isoforms. Starting from an in silico prediction of miRNA binding sites in the GATA-1 transcript, miR-1202 came into our sight as potential regulator of GATA-1 expression. Expression studies in K562 cells revealed that miR-1202 directly targets GATA-1, negatively regulates its expression, impairs GATA-1S production, reduces cell proliferation, and increases apoptosis sensitivity. Furthermore, data from TAM and myeloid leukaemia patients provided substantial support to our study by showing that miR-1202 down-modulation is accompanied by increased GATA-1 levels, with more marked effects on GATA-1S. These findings indicate that miR-1202 acts as an anti-oncomiR in myeloid cells and may impact leukaemogenesis at least in part by down-modulating GATA-1S levels.

Promoter-centred chromatin interactions associated with EVI1 expression in EVI1+3q- myeloid leukaemia cells.

EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.

In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice.

CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.

Prevalence and clinical expression of germ line predisposition to myeloid neoplasms in adults with marrow hypocellularity.

Systematic studies of germ line genetic predisposition to myeloid neoplasms in adult patients are still limited. In this work, we performed germ line and somatic targeted sequencing in a cohort of adult patients with hypoplastic bone marrow (BM) to study germ line predisposition variants and their clinical correlates. The study population included 402 consecutive adult patients investigated for unexplained cytopenia and reduced age-adjusted BM cellularity. Germ line mutation analysis was performed using a panel of 60 genes, and variants were interpreted per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines; somatic mutation analysis was performed using a panel of 54 genes. Of the 402 patients, 27 (6.7%) carried germ line variants that caused a predisposition syndrome/disorder. The most frequent disorders were DDX41-associated predisposition, Fanconi anemia, GATA2-deficiency syndrome, severe congenital neutropenia, RASopathy, and Diamond-Blackfan anemia. Eighteen of 27 patients (67%) with causative germ line genotype were diagnosed with myeloid neoplasm, and the remaining with cytopenia of undetermined significance. Patients with a predisposition syndrome/disorder were younger than the remaining patients and had a higher risk of severe or multiple cytopenias and advanced myeloid malignancy. In patients with myeloid neoplasm, causative germ line mutations were associated with increased risk of progression into acute myeloid leukemia. Family or personal history of cancer did not show significant association with a predisposition syndrome/disorder. The findings of this study unveil the spectrum, clinical expressivity, and prevalence of germ line predisposition mutations in an unselected cohort of adult patients with cytopenia and hypoplastic BM.

The miR-106b-25 cluster mediates drug resistance in myeloid leukaemias by inactivating multiple apoptotic genes.

Drug resistance is a major obstacle to the successful treatment of cancer. The role of the miR-106b-25 cluster in drug resistance of haematologic malignancies has not yet been elucidated. Here, we show that the miR-106b-25 cluster mediates resistance to therapeutic agents with structural and mechanistic dissimilarity in vitro and in vivo. RNA sequencing data revealed that overexpression of the miR-106b-25 cluster or its individual miRNAs resulted in downregulation of multiple key regulators of apoptotic pathways. Luciferase reporter assay identified TP73 as a direct target of miR-93 and miR-106b, BAK1 as a direct target of miR-25 and CASP7 as a direct target of all three miRNAs. We also showed that inhibitors of the miR-106b-25 cluster and BCL-2 exert synergistic effects on apoptosis induction in primary myeloid leukaemic cells. Thus, the members of the miR-106b-25 cluster may jointly contribute to myeloid leukaemia drug resistance by inactivating multiple apoptotic genes. Targeting this cluster could be a promising combination strategy in patients resistant to therapeutic agents that induce apoptosis.

RUNX1 isoform disequilibrium promotes the development of trisomy 21-associated myeloid leukemia.

Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

Splenic red pulp macrophages provide a niche for CML stem cells and induce therapy resistance.

Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs), and cure relies on their eradication. The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance. Although leukemic infiltration of the spleen is a hallmark of CML, it is unknown whether spleen cells form a niche that maintains LSCs. Here, we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression. Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model. Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen. Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs). These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state, resulting in disease progression and resistance to therapy.

Highly precise breakpoint detection of chromosome balanced translocation in chronic myelogenous leukaemia: Case series.

Chronic myelogenous leukaemia (CML) has a special phenomenon of chromosome translocation, which is called Philadelphia chromosome translocation. However, the detailed connection of this structure is troublesome and expensive to be identified. Low-coverage whole genome sequencing (LCWGS) could not only detect the previously unknown chromosomal translocation, but also provide the breakpoint candidate small region (with an accuracy of ±200 bases). Importantly, the sequencing cost of LCWGS is about US$300. Then, with the Sanger DNA sequencing, the precise breakpoint can be determined at a single base level. In our project, with LCWGS, BCR and ABL1 are successfully identified to be disrupted in three CML patients (at chr22:23,632,356 and chr9:133,590,450; chr22:23,633,748 and chr9:133,635,781; chr22: 23,631,831 and chr9:133,598,513, respectively). Due to the reconnection after chromosome breakage, classical fusion gene (BCR::ABL1) was found in bone marrow and peripheral blood. The precise breakpoints were helpful to investigate the pathogenic mechanism of CML and could better guide the classification of CML subtypes. This LCWGS method is universal and can be used to detect all diseases related to chromosome variation, such as solid tumours, liquid tumours and birth defects.

Complex karyotype with double Philadelphia chromosome and T315I mutation results in blastic phase and extensive extramedullary infiltration in a chronic myeloid leukemia patient.

Chronic myeloid leukemia (CML) is a common hematological malignancy originating from bone marrow stem cells. Chromosomal abnormalities can be seen in almost all cases, the most known anomaly being Philadelphia (Ph) chromosome, a derivative chromosome resulting from a translocation between 9. and 22. chromosome. Other chromosomal abnormalities may be present in 10% of patients at diagnosis, although they emerge frequently during the acute transformation and can be associated with unfavorable significance. Also, point mutations like T315I in BCR-ABL fusion gene may arise during the course of the disease and thereby cause tyrosine kinase inhibitors (TKI) resistance. Here, we report a BCR-ABL positive CML patient who was followed for 6 years in major molecular response (MMR), complete cytogenetic response (CCR), and complete hematological response (CHR). He had a sudden loss of hematological, cytogenetic, and molecular response with a very aggressive blastic course and extensive extramedullary infiltration, with T315I mutation, complex translocations, an extra Ph chromosome, and additional chromosomes. The patient who received intensive cytotoxic chemotherapy together with ponatinib treatment, which is effective for the T315I mutation, never went into remission, and there was no chance of transplantation because a suitable donor for HLA could not be found. Although these findings are not very rare individually, coexistence of complex karyotype and T315I mutation is not frequent and complicates clinical management. Our patient is the first case in literature with all disclosed findings together and indicates the importance of early detection of these chromosomal and molecular abnormalities.

A sensitive and inexpensive high-resolution melting-based testing algorithm for diagnosis of transient abnormal myelopoiesis and myeloid leukemia of Down syndrome.

Patients with Down syndrome (DS) are commonly affected by a pre-leukemic disorder known as transient abnormal myelopoiesis (TAM). This condition usually undergoes spontaneous remission within the first 2 months after birth; however, in children under 5, 20%-30% of cases evolve to myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS are caused by co-operation between trisomy 21 and acquired mutations in the GATA1 gene. Currently, only next-generation sequencing (NGS)-based methodologies are sufficiently sensitive for diagnosis in samples with small GATA1 mutant clones (≤10% blasts). Alternatively, this study presents research on a new, fast, sensitive, and inexpensive high-resolution melting (HRM)-based diagnostic approach that allows the detection of most cases of GATA1 mutations, including silent TAM. The algorithm first uses flow cytometry for blast count, followed by HRM and Sanger sequencing to search for mutations on exons 2 and 3 of GATA1. We analyzed 138 samples of DS patients: 110 of asymptomatic neonates, 10 suspected of having TAM, and 18 suspected of having ML-DS. Our algorithm enabled the identification of 33 mutant samples, among them five cases of silent TAM (5/110) and seven cases of ML-DS (7/18) with blast count ≤10%, in which GATA1 alterations were easily detected by HRM. Depending on the type of genetic variation and its location, our methodology reached sensitivity similar to that obtained by NGS (0.3%) at a considerably reduced time and cost, thus making it accessible worldwide.

TYRO3 Knockdown Suppresses the Growth of Myeloid Leukaemia Cells.

BACKGROUND/AIM: TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in the growth of leukaemia cells have been reported, the effect of TYRO3 has not been determined. Therefore, we examined the effects of TYRO3 knockdown on the growth of leukaemia cell lines. MATERIALS AND METHODS: Three human leukaemia cell lines (AA derived from pure erythroid leukaemia, OCI/AML2, and K562), which express TYRO3 protein were used in this study. To induce TYRO3 knockdown, small interfering RNA (siRNA) against TYRO3 was transfected using an electroporation system. Cell growth was assessed by a colorimetric assay. The expression levels and activation of various signalling proteins were examined by immunoblotting. Changes in comprehensive gene expression after TYRO3 knockdown were examined by microarray analysis. RESULTS: TYRO3 knockdown suppressed cell growth in the leukaemia cell lines tested. Additionally, the knockdown suppressed phosphorylation of signal transducer and activator of transcription-3 in AA cells, and extracellular signal-regulated kinase (ERK) 1/2 in AA and OCI/AML2 cells; both are downstream molecules of TYRO3 signalling. TYRO3 knockdown also suppressed the expression of survivin in all the cell lines. TYRO3 knockdown potently suppressed TYRO3 mRNA expression but not that of AXL and MERTK. Furthermore, TYRO3 knockdown suppressed cyclin D1 mRNA expression, which is a downstream molecule of ERK. CONCLUSION: TYRO3 plays a role in leukaemia cell growth and is a potential therapeutic target for leukaemia.

A study of Telomerase Reverse Transcriptase rare variants in myeloid neoplasia.

Telomere dysfunctions are associated with several hematopoietic stem cell (HSC) malignancies. Recent findings have indicated that the occurrence of rare variants of unknown significance (VUS) in the Telomerase Reverse Transcriptase (TERT) gene influences the outcomes of patients with myelodysplastic syndromes undergoing allogeneic HSC transplantation. However, the role of TERT variants has been historically controversial as initially considered pathogenic variants (H412Y, A202T) presenting functional consequences, were found very frequent in general population questioning their pathogenicity and risk allele significance. Herein, we show that overall TERT VUS are non-recurrent in myeloid disorders and cannot be considered risk alleles individually nor can their biological impact.

SIRT2 regulates proliferation and chemotherapy response of MLL-ENL-driven acute myeloid leukemia.

Both MLL-AF9 and MLL-ENL leukemia fusion proteins drive oncogenic transformation of hematopoietic cells through their N-terminal DNA/histone binding mixed-lineage leukemia 1 domain and C-terminal fragment of AF9 or ENL containing an unstructured linker region and the ANC1 homology domain, which recruits transcription factors. Despite of their structural similarity, acute myeloid leukemia (AML) patients bearing MLL-ENL show more adverse outcomes compared to those with MLL-AF9. We recapitulated the clinical patterns of these two MLL-fusions driven AMLs using murine models and found that MLL-ENL AML cells showed slower cell cycle progression and more resistance to standard chemotherapy than MLL-AF9 cells. These phenotypes were primarily controlled by the linker regions of ENL and a highly conserved lysine residue K469 within. Substitution of K469 with an acetylated mimic glutamine abolished the ability of MLL-ENL to suppress proliferation and promote chemo-resistance. We showed that deacetylase Sirt2 might act as an upstream regulator of MLL-ENL. Deletion of Sirt2 promoted proliferation of AML cells with either MLL fusions. Importantly, loss of Sirt2 greatly enhanced the sensitivity of the MLL-ENL AML cells to chemo-treatment. Taken together, our study uncovered a unique regulatory role of Sirt2 in leukemogenesis and suggested targeting SIRT2 as a new way to sensitize MLL-ENL AML patience for chemotherapy.

Comparison of myeloid neoplasms with nonclassic 3q26.2/MECOM versus classic inv(3)/t(3;3) rearrangements reveals diverse clinicopathologic features, genetic profiles, and molecular mechanisms of MECOM activation.

MECOM rearrangements are recurrent in myeloid neoplasms and associated with poor prognosis. However, only inv(3)(q21q26.2) and t(3;3)(q21;q26.2), the classic MECOM rearrangements resulting in RPN1-MECOM rearrangement with Mecom overexpression and GATA2 haploinsufficiency, define the distinct subtype of acute myeloid leukemia (AML), and serve as presumptive evidence for myelodysplastic syndrome based on the current World Health Organization classification. Myeloid neoplasms with nonclassic 3q26.2/MECOM rearrangements have been found to be clinically aggressive, but comparative analysis of clinicopathologic and genomic features is limited. We retrospectively studied cohorts of myeloid neoplasms with classic and nonclassic MECOM rearrangements. Cases with classic rearrangements consisted predominantly of AML, often with inv(3) or t(3;3) as the sole chromosome abnormality, whereas the group of nonclassic rearrangements included a variety of myeloid neoplasms, often with complex karyotype without TP53 mutations and similarly dismal overall survival. Immunohistochemistry revealed Mecom protein overexpression in both groups, but overexpression in cases with nonclassic rearrangements was mediated through a mechanism other than GATA2 distal enhancer involvement typical for classic rearrangement. Our results demonstrated that myeloid neoplasms with nonclassic 3q26.2/MECOM rearrangements encompass a diverse group of diseases with poor clinical outcome, overexpression of Mecom protein as a result of the nonclassic mechanism of MECOM activation.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells.

Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Decreased CD177pos neutrophils in myeloid neoplasms is associated with NPM1, RUNX1, TET2, and U2AF1 S34F mutations.

A decreased percentage of CD177pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177pos neutrophils and somatically acquired mutations involving several genes.

Treatment Failure in Acute Myeloid Leukemia: Focus on the Role of Extracellular Vesicles.

Acute myeloblastic leukemia (AML) is one of the most common types of blood malignancies that results in an AML-associated high mortality rate each year. Several causes have been reported as prognostic factors for AML in children and adults, the most important of which are cytogenetic abnormalities and environmental risk factors. Following the discovery of numerous drugs for AML treatment, leukemic cells sought a way to escape from the cytotoxic effects of chemotherapy drugs, leading to treatment failure. Nowadays, comprehensive studies have looked at the role of extracellular vesicles (EVs) secreted by AML blasts and how the microenvironment of the tumor changes in favor of cancer progression and survival to discover the mechanisms of treatment failure to choose the well-advised treatment. Reports show that malignant cells secrete EVs that transmit messages to adjacent cells and the tumor's microenvironment. By secreting EVs, containing immune-inhibiting cytokines, AML cells inactivate the immune system against malignant cells, thus ensuring their survival. Also, increased secretion of EVs in various malignancies indicates an unfavorable prognostic factor and the possibility of drug resistance. In this study, we briefly reviewed the challenges of treating AML with a glance at the EVs' role in this process. It is hoped that with a deeper understanding of EVs, new therapies will be developed to eliminate the relapse of leukemic cells.