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SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common β-chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo., (© 2022 by The American Society of Hematology.)

Clonal cytogenetic abnormalities are a major risk factor for relapse after hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS). We determined the impact of the recently established 5-group cytogenetic classification of MDS on outcome after HCT. Results were compared with the impact of the International Prognostic Scoring System (IPSS) 3 cytogenetic risk groups, and the additional effect of a monosomal karyotype was assessed. The study included data on 1007 patients, 1-75 years old (median 45 years), transplanted from related (n = 547) or unrelated (n = 460) donors. Various conditioning regimens were used, and marrow, peripheral blood, or cord blood served as stem cell source. Both IPSS and 5-group cytogenetic risk classifications were significantly associated with post-HCT relapse and mortality, but the 5-group classification discriminated more clearly among the lowest- and highest-risk patients. A monosomal karyotype tended to further increase the rates of relapse and mortality, even after considering the IPSS or 5-group classifications. In addition, the pathologic disease category correlated with both relapse and mortality. Mortality was also impacted by patient age, donor type, conditioning regimen, platelet count, and etiology of MDS. Although mortality declined significantly in recent years, novel strategies are needed to overcome the barrier of high-risk cytogenetics.

IDH1 SNP rs11554137 was recently reported in association with poor prognosis in normal karyotype adult acute myeloid leukemia (AML). We aimed to determine the prevalence, clinical associations, and prognostic significance of SNP rs11554137 in unselected pediatric and adult AML patients. Diagnostic marrow specimens from 527 AML patients treated on the pediatric trial Children's Oncology Group-AAML03P1 (N = 253) or adult SWOG trials (N = 274) were analyzed for the presence of the SNP. SNP rs11554137 was present in 11% of all patients. SNP status had no prognostic impact on survival in pediatric patients. In adult AML, overall survival for SNP-positive patients was 10% versus 18% for SNP-negative patients (P = .44). Among the 142 adults who achieved complete remission, 5-year relapse-free survival was significantly worse for SNP-positive patients (0% vs 25%, P = .0014). However, among adults with normal cytogenetics, FLT3/ITD was present in 90% of SNP-positive patients versus 59% of SNP-negative patients (P = .0053). In multivariate analysis, adjusting for the effects of age, cytogenetics, and FLT3/ITD, the independent prognostic effect of SNP positivity was not statistically significant (hazard ratio = 1.72, P = .18). The clinical profile of SNP-positive patients suggests that SNP rs11554137 may have biologic effects that bear further investigation. The clinical trials in this study are registered at http://www.clinicaltrials.gov as #NCT000707174 and #NCT00899171.

Recent studies of WT1 mutations in acute myeloid leukemia (AML) mostly report an association with unfavorable clinical outcome. We screened 842 patients treated on 3 consecutive pediatric AML trials for WT1 zinc-finger mutations. Eighty-five mutations were detected in 70 of 842 patients (8.3%). Mutations occurred predominantly in exon 7 (n = 74) but were also found in exons 8 (n = 5) and 9 (n = 6). Normal karyotype was observed in 35.3% of WT1(mut) patients, whereas 27.5% WT1(mut) patients harbored favorable risk cytogenetics. Patients with or without mutations had similar rates of complete remission after one course of induction chemotherapy. Overall survival (OS) for patients with WT1 mutations was 41% versus 54% for those without mutations (P = .016). Corresponding event-free survival (EFS) was also significantly worse for those with WT1 mutations (28% vs 42%; P = .01). However, FLT3/ITD was present in 36% of the WT1(mut) cohort; WT1(mut) patients without FLT3/ITD had similar OS (56% vs 56%, respectively; P = .8) and EFS (35% and 44%, respectively; P = .34) to patients who were wild type for both mutations. In current risk stratification schemes incorporating cytogenetics and FLT3/ITD status, the presence of WT1 mutations has no independent prognostic significance in predicting outcome in pediatric AML. The clinical trials are registered at www.clinicaltrials.gov as #NCT00002798 and #NCT00070174.

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

CEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations-N-terminal truncating mutations and in-frame bZip-domain mutations-were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis.

Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow microenvironment confers protection from chemotherapy-induced apoptosis. One mechanism for retention of blasts within the bone marrow is adhesion via very late antigen-4 (VLA-4), the alpha(4)beta(1) integrin heterodimer that binds to its main ligands, fibronectin, and vascular cell adhesion molecule-1 (VCAM-1). To examine the relationship of functional expression of VLA-4 to prognosis in AML, we studied marrow samples from 175 adult AML patients who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group trials. The studies included flow cytometry and functional in vitro assays for ligand binding and maximal beta(1) activation. VLA-4 expression varied widely, with mean expression 60.6% for alpha(4), and was not significantly associated with response to chemotherapy, relapse-free, or overall survival (OS). However, increased binding of soluble VCAM-1 via VLA-4 was significantly associated with longer OS, corrected for age (P = .033). Estimated 5-year OS was 31% (95% confidence interval, 14%-48%) in 30 patients with soluble VCAM-1 binding greater than or equal to 40%, compared with 10% (confidence interval, 3%-17%) in 72 patients with lower binding. Adhesion and migratory properties of AML blasts thus appear to influence chemosensitivity and therefore may be therapeutic targets.

FLT3 internal tandem duplication (FLT3/ITD) is a common somatic mutation in acute myeloid leukemia (AML) with significant variation in the position, length, and number of duplications of the FLT3 gene. We evaluated these physical characteristics in FLT3/ITD-positive patients who were treated on CCG-2941/2961 and correlated them with clinical outcome. Fiftynine of 77 FLT3/ITD-positive patients (77%) had a single ITD, 16 (21%) had 2 ITDs, and 2 (3%) had 3 ITDs. The length of the duplicated region varied from 6 to 51 amino acids, and in all cases amino acid residues Y591-Y597 were duplicated. Structural analysis demonstrated that Y591-Y597 encodes the switch and zipper regions of the juxtamembrane domain of FLT3. In addition, 24 of 77 patients (31%) had duplication of the critical STAT5 docking sites Y589/591. Patients with longer ITDs had a worse relapse-free survival (19% vs 51%, P = .035), while the presence of more than 1 ITD was not clinically significant. Physical characteristics including the length of FLT3/ITD may influence FLT3 activation state by altering its structure and may impact response to therapy.

We compared the gene expression profile of adult acute lymphoblastic leukemia (ALL) to normal hematopoietic and non-ALL samples using oligonucleotide arrays. Connective tissue growth factor (CTGF) was the highest overexpressed gene in B-cell ALL compared with the other groups, and displayed heterogeneous expression, suggesting it might have prognostic relevance. CTGF expression was examined by quantitative reverse transcriptase-polymerase chain reaction (ORT-PCR) on 79 adult ALL specimens. CTGF expression levels were significantly increased in ALL cases with B-lineage (P < .001), unfavorable cytogenetics (P < .001), and blasts expressing CD34 (P < .001). In a multivariate proportional hazards model, higher CTGF expression levels corresponded to worsening of overall survival (OS; hazard ratio 1.36, for each 10-fold increase in expression; P = .019). Further studies are ongoing to confirm the prognostic value of CTGF expression in ALL and to investigate its role in normal and abnormal lymphocyte biology.

Activating mutations of the FLT3 gene occur because of an internal tandem duplication of the juxta-membrane domain (FLT3/ITD) or point mutation of the activation loop domain (FLT3/ALM). The presence of FLT3 mutations as well as the allelic ratio of FLT3/ITD (ITD-AR, mutant-wild type ratio) may have prognostic significance. FLT3 mutation status of 630 children with de novo acute myeloid leukemia (AML) treated on CCG-2941 and -2961 was determined, and ITD-AR was calculated for patients with FLT3/ITD. Clinical characteristics and outcomes for patients with FLT3/ALM and FLT3/ITD at varying ITD-ARs was determined and compared with those without FLT3 mutations (FLT3/WT). FLT3/ITD and FLT3/ALM were detected in 77 (12%) and 42 (6.7%) of the patients. Progression-free survival (PFS) was similar in patients with FLT3/ALM and FLT3/WT (51% versus 55%, P = .862). In contrast, PFS at 4 years from study entry for patients with FLT3/ITD was inferior to that of patients with FLT3/WT (31% versus 55%, P < .001). PFS decreased with increasing FLT3/ITD-AR (P < .001), and those with ITD-AR greater than 0.4 had a significantly worse PFS than those with lower ITD-AR (16% versus 72%, P = .001) or with FLT3/WT (55%, P < .001). ITD-AR defines the prognostic significance in FLT3/ITD-positive AML, and ITD-AR greater than 0.4 is a significant and independent prognostic factor for relapse in pediatric AML.

To determine whether gene expression profiling could improve risk classification and outcome prediction in older acute myeloid leukemia (AML) patients, expression profiles were obtained in pretreatment leukemic samples from 170 patients whose median age was 65 years. Unsupervised clustering methods were used to classify patients into 6 cluster groups (designated A to F) that varied significantly in rates of resistant disease (RD; P < .001), complete response (CR; P = .023), and disease-free survival (DFS; P = .023). Cluster A (n = 24), dominated by NPM1 mutations (78%), normal karyotypes (75%), and genes associated with signaling and apoptosis, had the best DFS (27%) and overall survival (OS; 25% at 5 years). Patients in clusters B (n = 22) and C (n = 31) had the worst OS (5% and 6%, respectively); cluster B was distinguished by the highest rate of RD (77%) and multidrug resistant gene expression (ABCG2, MDR1). Cluster D was characterized by a "proliferative" gene signature with the highest proportion of detectable cytogenetic abnormalities (76%; including 83% of all favorable and 34% of unfavorable karyotypes). Cluster F (n = 33) was dominated by monocytic leukemias (97% of cases), also showing increased NPM1 mutations (61%). These gene expression signatures provide insights into novel groups of AML not predicted by traditional studies that impact prognosis and potential therapy.

FLT3 internal tandem duplications (FLT3/ITDs) in the juxtamembrane domain are found in approximately 25% of acute myeloid leukemia (AML) patients, ranging in size from 3 to hundreds of nucleotides. We examined whether the sizes of FLT3/ITDs were associated with clinical outcomes in 151 AML patients enrolled in Southwest Oncology Group studies: S9333 and S9500. FLT3/ITDs were identified in 32% of patients (median ITD size = 39 nucleotides; range, 15-153 nucleotides). The CR rates were 35%, 67%, and 52% for patients with large (>or= 40), small (< 40), and no ITDs, respectively (P = .19). Increasing ITD size was associated with decreasing OS (estimated 5-year OS: large = 13%, small = 26%, and no ITD = 21%, P = .072) and RFS (estimated 5-year RFS: large = 13%, small = 27%, and no ITD = 34%, P = .017). These studies suggest that ITD size may have prognostic significance.

The AF1q gene, a mixed-lineage leukemia fusion partner, is highly expressed in hematopoietic progenitor cells but has low expression in differentiated cells. We determined the expression of the AF1q gene by reverse transcriptase-polymerase chain reaction in 64 pediatric acute myeloid leukemia (AML) patients treated on Children's Cancer Group clinical trial CCG-2891 and correlated its expression level to clinical characteristics and outcome. AF1q expression in patients varied from 0- to 154-fold compared with normal marrow, and increasing expression level was associated with worsening survival, with a hazard ratio of 1.02 per fold increase in AF1q expression (P = .032). We divided patients into tertile groups based on AF1q expression level. Patients with high AF1q expression (top tertile) had a higher predominance of French-American-British M1 compared to patients with lower 2 tertiles of AF1q expression (43% vs 9%, P = .003). High AF1q expression was associated with poor survival in univariate and multivariate models. Overall survival at 8 years for patients with the high AF1q expression was 19% versus 50% in patients with low AF1q expression, (P = .01). AF1q expression may correlate with clinical outcome in pediatric AML, although it is not clear if AF1q is simply a marker of a more primitive phenotype or contributes directly to leukemogenesis.

Activating mutations of receptor tyrosine kinases (RTKs) and their downstream affectors are common in acute myeloid leukemia (AML). We performed mutational analysis of FLT3, c-kit, c-fms, vascular endothelial growth factor (VEGF) receptors (Flt-1, KDR [kinase domain receptor]), and ras genes in a group of 91 pediatric patients with AML treated on Children's Cancer Group clinical trial CCG-2891. Forty-six percent of patients had activating mutations of FLT3 (24.5%), c-kit (3%), or ras (21%) genes. Mutation-positive patients had a higher median diagnostic white blood cell (WBC) count (71.5 vs 19.6 x 10(9)/L; P =.005) and lower complete remission rate (55% versus 76%; P =.046) than mutation-negative patients. The Kaplan-Meier estimate of overall survival (OS) for patients with and without an activating mutation was 34% versus 57%, respectively (P =.035). However, within this group, patients with FLT3/ALM (activation loop mutation) had good outcomes (OS, 86%). Exclusion of the FLT3/ALM from analysis decreased the OS for the remaining mutation-positive patients to 26% (P =.003). Ten of the 23 mutation-positive and 11 of the 34 mutation-negative patients received an allogeneic bone marrow transplant (BMT) in first complete remission (CR). In the mutation-positive group, the disease-free survival (DFS) for the allogeneic BMT recipients was 72% versus 23% for the 13 patients who received chemotherapy or autologous BMT (P =.01). DFS for the mutation-free patients with and without allogeneic BM transplantation was 55% and 40%, respectively (P =.38). Activating mutations in the RTK/ras signaling pathway are common in pediatric AML, and their presence may identify a population at higher risk of poor outcome who may benefit from allogeneic BM transplantation.

The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RAS mutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P =.0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53 mutations were associated with a worse overall survival (median 1 versus 8 months, P =.0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes. (Blood. 2001;97:3589-3595)

The Flt3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the Flt3 gene (Flt3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with poor prognosis. We analyzed diagnostic bone marrow specimens from 91 pediatric patients with AML treated on Children's Cancer Group (CCG)-2891 for the presence of the Flt3/ITD and correlated its presence with clinical outcome. Fifteen of 91 samples (16.5%) were positive for the Flt3/ITD. Flt3/ITD-positive patients had a median diagnostic white count of 73 800 compared with 28 400 for the Flt3/ITD-negative patients (P =.05). The size of the duplication ranged from 21 to 174 base pairs (bp). Nucleotide sequencing of the abnormal polymerase chain reaction products demonstrated that all duplications involved exon 11 of the Flt3 gene and also preserved the reading frame. Lineage restriction analysis revealed that Flt3/ITD was not present in the lymphocytes, suggesting a lack of stem cell involvement for this mutation. None of the Flt3/ITD-positive patients had unfavorable cytogenetic markers, and there was no predominance of a particular FAB class. The remission induction rate was 40% in Flt3/ITD-positive patients compared with 74% in Flt3/ITD-negative ones (P =.005). The Kaplan-Meier estimates of event-free survival at 8 years for patients with and without Flt3/ITD were 7% and 44%, respectively (P =.002). Multivariate analysis demonstrated that presence of the Flt3/ITD was the single most significant, independent prognostic factor for poor outcome (P =.009) in pediatric AML.