WT1 together with RUNX1::RUNX1T1 targets DUSP6 to dampen ERK activity in acute myeloid leukaemia.

Wilms' tumour 1 (WT1) can function as an oncogene or a tumour suppressor. Our previous clinical cohort studies showed that low WT1 expression at diagnosis independently predicted poor outcomes in acute myeloid leukaemia (AML) with RUNX1::RUNX1T1, whereas it had an opposite role in AML with non-favourable cytogenetic risk (RUNX1::RUNX1T1-deficient). The molecular mechanism by which RUNX1::RUNX1T1 affects the prognostic significance of WT1 in AML remains unknown. In the present study, first we validated the prognostic significance of WT1 expression in AML. Then by using the established transfected cell lines and xenograft tumour model, we found that WT1 suppresses proliferation and enhances effect of cytarabine in RUNX1::RUNX1T1(+) AML but has opposite functions in AML cells without RUNX1::RUNX1T1. Furthermore, as a transcription factor, WT1 physically interacts with RUNX1::RUNX1T1 and acts as a co-factor together with RUNX1::RUNX1T1 to activate the expression of its target gene DUSP6 to dampen extracellular signal-regulated kinase (ERK) activity. When RUNX1::RUNX1T1-deficient, WT1 can activate the mitogen-activated extracellular signal-regulated kinase/ERK axis but not through targeting DUSP6. These results provide a mechanism by which WT1 together with RUNX1::RUNX1T1 suppresses cell proliferation through WT1/DUSP6/ERK axis in AML. The current study provides an explanation for the controversial prognostic significance of WT1 expression in AML patients.

Prophylactic therapy using epigenetic agents for RUNX1::RUNXT1-positive high-risk AML after Allo-HSCT.

Disease recurrence is the leading cause of treatment failure in patients with RUNX1::RUNXT1-positive acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Post-transplant maintenance therapy, guided by monitoring minimal residual disease (MRD), is commonly administered; however, relapse rates remain high. This prospective study aimed to assess the effectiveness and safety of epigenetic agents as prophylactic therapy in patients with RUNX1::RUNXT1-positive AML. Thirty high-risk patients received prophylactic therapy (n = 17 and n = 13 in the chidamide and AZA groups, respectively) between January 2019 and July 2023. 34 high-risk patients who received preemptive treatment due to molecular relapse were included in the analysis. The two-year relapse-free survival (RFS) and overall survival (OS) were significantly higher in the prophylactic group compared to the preemptive group (82.82% vs. 51.38%, P = 0.014; 86.42% vs. 56.16%, P = 0.025, respectively); 2-year cumulative incidence of relapse rates were 13.8% and 36.40%, respectively (P = 0.037). In conclusion, prophylactic therapy with epigenetic agents may improve long-term prognosis and is well-tolerated in patients with RUNX1::RUNXT1-positive high-risk AML. Timely post-transplant prophylactic therapy may be more effective than preemptive therapy based on positive MRD results.

Combination of eriocalyxin B and homoharringtonine exerts synergistic anti-tumor effects against t(8;21) AML.

Understanding the molecular pathogenesis of acute myeloid leukemia (AML) with well-defined genomic abnormalities has facilitated the development of targeted therapeutics. Patients with t(8;21) AML frequently harbor a fusion gene RUNX1-RUNX1T1 and KIT mutations as "secondary hit", making the disease one of the ideal models for exploring targeted treatment options in AML. In this study we investigated the combination therapy of agents targeting RUNX1-RUNX1T1 and KIT in the treatment of t(8;21) AML with KIT mutations. We showed that the combination of eriocalyxin B (EriB) and homoharringtonine (HHT) exerted synergistic therapeutic effects by dual inhibition of RUNX1-RUNX1T1 and KIT proteins in Kasumi-1 and SKNO-1 cells in vitro. In Kasumi-1 cells, the combination of EriB and HHT could perturb the RUNX1-RUNX1T1-responsible transcriptional network by destabilizing RUNX1-RUNX1T1 transcription factor complex (AETFC), forcing RUNX1-RUNX1T1 leaving from the chromatin, triggering cell cycle arrest and apoptosis. Meanwhile, EriB combined with HHT activated JNK signaling, resulting in the eventual degradation of RUNX1-RUNX1T1 by caspase-3. In addition, HHT and EriB inhibited NF-κB pathway through blocking p65 nuclear translocation in two different manners, to synergistically interfere with the transcription of KIT. In mice co-expressing RUNX1-RUNX1T1 and KITN822K, co-administration of EriB and HHT significantly prolonged survival of the mice by targeting CD34+CD38- leukemic cells. The synergistic effects of the two drugs were also observed in bone marrow mononuclear cells (BMMCs) of t(8;21) AML patients. Collectively, this study reveals the synergistic mechanism of the combination regimen of EriB and HHT in t(8;21) AML, providing new insight into optimizing targeted treatment of AML.

AML1-ETO-Related Fusion Circular RNAs Contribute to the Proliferation of Leukemia Cells.

The AML1-ETO (RUNX1-RUNX1T1) fusion gene created by the chromosome translocation t(8;21) (q21;q22) is one of the essential contributors to leukemogenesis. Only a few studies in the literature have focused on fusion gene-derived circular RNAs (f-circRNAs). Here, we report several AML1-ETO-related fusion circular RNAs (F-CircAEs) in AML1-ETO-positive cell lines and primary patient blasts. Functional studies demonstrate that the over-expression of F-CircAE in NIH3T3 cells promotes cell proliferation in vitro and in vivo. F-CircAE expression enhances the colony formation ability of c-Kit+ hematopoietic stem and progenitor cells (HSPCs). Meanwhile, the knockdown of endogenous F-CircAEs can inhibit the proliferation and colony formation ability of AML1-ETO-positive Kasumi-1 cells. Intriguingly, bioinformatic analysis revealed that the glycolysis pathway is down-regulated in F-CircAE-knockdown Kasumi-1 cells and up-regulated in F-CircAE over-expressed NIH3T3 cells. Further studies show that F-CircAE binds to the glycolytic protein ENO-1, up-regulates the expression level of glycolytic enzymes, and enhances lactate production. In summary, our study demonstrates that F-CircAE may exert biological activities on the growth of AML1-ETO leukemia cells by regulating the glycolysis pathway. Determining the role of F-CircAEs in AML1-ETO leukemia can lead to great strides in understanding its pathogenesis, thus providing new diagnostic markers and therapeutic targets.

Mutated KIT Tyrosine Kinase as a Novel Molecular Target in Acute Myeloid Leukemia.

KIT is a type-III receptor tyrosine kinase that contributes to cell signaling in various cells. Since KIT is activated by overexpression or mutation and plays an important role in the development of some cancers, such as gastrointestinal stromal tumors and mast cell disease, molecular therapies targeting KIT mutations are being developed. In acute myeloid leukemia (AML), genome profiling via next-generation sequencing has shown that several genes that are mutated in patients with AML impact patients' prognosis. Moreover, it was suggested that precision-medicine-based treatment using genomic data will improve treatment outcomes for AML patients. This paper presents (1) previous studies regarding the role of KIT mutations in AML, (2) the data in AML with KIT mutations from the HM-SCREEN-Japan-01 study, a genome profiling study for patients newly diagnosed with AML who are unsuitable for the standard first-line treatment (unfit) or have relapsed/refractory AML, and (3) new therapies targeting KIT mutations, such as tyrosine kinase inhibitors and heat shock protein 90 inhibitors. In this era when genome profiling via next-generation sequencing is becoming more common, KIT mutations are attractive novel molecular targets in AML.

[A favorable clinical course of acute myeloid leukemia with t (6;21;8)(p23;q22;q22)].

Variants of the t (8;21) (q22;q22) involving chromosome 8, 21, and other chromosomes account for about 3% of all t (8;21) (q22;q22) in patients with acute myeloid leukemia (AML). However, the prognosis of AML with variant t (8;21) remains unknown due to the scarcity of reported cases. Herein we report a case of AML with t (6;21;8) (p23;q22;q22). Fluorescence in situ hybridization confirmed a RUNX1-RUNX1T1 fusion signal on the derivative chromosome 8. This is the first report on a variant of t (8;21) involving the breakpoint 6p23. After induction chemotherapy, our patient achieved complete remission and has been stable for four years.

Characteristics and prognosis of pediatric myeloid sarcoma in the cytogenetic context of t(8;21).

The prognosis of myeloid sarcoma (MS) is controversial. Many reports indicated that orbital-MS has a good prognosis and is closely related to t(8;21), but the prognostic role of MS in pediatric t(8;21) AML is unclear. We retrospectively analyzed data from 127 patients with pediatric t(8;21) AML diagnosed between January 2010 and June 2018. We compared patients with (n = 30) and without MS (n = 97). The median follow-up time was 52.6 months. The proportion of t(8;21) AML patients with MS was 23.6%. Males were more likely to have MS than females. The complete remission rate after the first course of induction chemotherapy and the 3-year relapse-free survival (RFS) among patients with MS were lower than those among patients without MS (60% vs. 78.4%, p = 0.045) (68.8 ± 8.8% vs. 88.0 ± 3.4%, p = 0.004). The female sex and a higher level of RUNX1/RUNX1T1 transcripts after consolidation were risk factors for poor RFS among patients with MS. Our data showed that MS was an independent risk factor in pediatric t(8;21) AML. Close monitoring of measurable residual disease of the bone marrow and extramedullary lesions is needed to guide stratified treatment.

Allogeneic hematopoietic stem cell transplantation can improve the prognosis of high-risk pediatric t(8;21) acute myeloid leukemia in first remission based on MRD-guided treatment.

BACKGROUND: Pediatric acute myeloid leukemia (AML) with t(8;21) (q22;q22) is classified as a low-risk group. However, relapse is still the main factor affecting survival. We aimed to investigate the effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on reducing recurrence and improving the survival of high-risk pediatric t(8;21) AML based on minimal residual disease (MRD)-guided treatment, and to further explore the prognostic factors to guide risk stratification treatment and identify who will benefit from allo-HSCT. METHODS: Overall, 129 newly diagnosed pediatric t(8;21) AML patients were included in this study. Patients were divided into high-risk and low-risk group according to RUNX1-RUNX1T1 transcript levels after 2 cycles of consolidation chemotherapy. High-risk patients were divided into HSCT group and chemotherapy group according to their treatment choices. The characteristics and outcomes of 125 patients were analyzed. RESULTS: For high-risk patients, allo-HSCT could improve 5-year relapse-free survival (RFS) rate compared to chemotherapy (87.4% vs. 61.9%; P = 0.026). Five-year overall survival (OS) rate in high-risk HSCT group had a trend for better than that in high-risk chemotherapy group (82.8% vs. 71.4%; P = 0.260). The 5-year RFS rate of patients with a c-KIT mutation in high-risk HSCT group had a trend for better than that of patients with a c-KIT mutation in high-risk chemotherapy group (82.9% vs. 75%; P = 0.400). Extramedullary infiltration (EI) at diagnosis was associated with a high cumulative incidence of relapse for high-risk patients (50% vs. 18.4%; P = 0.004); allo-HSCT can improve the RFS (P = 0.009). CONCLUSIONS: allo-HSCT can improve the prognosis of high-risk pediatric t(8;21) AML based on MRD-guided treatment. Patients with a c-KIT mutation may benefit from allo-HSCT. EI is an independent prognostic factor for high-risk patients and allo-HSCT can improve the prognosis.

Negative CD19 expression is associated with inferior relapse-free survival in children with RUNX1-RUNX1T1-positive acute myeloid leukaemia: results from the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study.

We performed a retrospective analysis of leukaemic surface antigen expression and genomic data from a total of 100 RUNX1-RUNX1T1-positive paediatric acute myeloid leukaemia (AML) patients enrolled in the Japanese Paediatric Leukaemia/Lymphoma Study Group (JPLSG) AML-05 protocol to determine risk factors for relapse. In univariate analysis, the KIT exon 17 mutation (n = 21) and CD19 negativity (n = 59) were significant risk factors for relapse (P = 0·01). In multivariate analysis, CD19 negativity was the sole significant risk factor for relapse (hazard ratio, 3·09; 95% confidence interval, 1·26-7·59; P < 0·01), suggesting that biological differences between CD19-positive and CD19-negative RUNX1-RUNX1T1 AML patients should be investigated.

Different prognostic effects of core-binding factor positive AML with Korean AML registry data.

Core-binding factor acute myeloid leukemia (CBF-AML) data in Asian countries has been rarely reported. We analyzed 392 patients with CBF-AML [281 with t(8;21), 111 with inv.(16)/t(16;16)] among data from 3041 patients with AML from the Korean AML Registry. Interestingly, del(9q) was less frequently detected in Korean than in German patients with t(8;21) (7.5% vs. 17%), and del(7q) was more frequently detected in Korean patients with inv(16). Overall survival (OS) was similar between patients in the first complete remission (CR) who received allogeneic (alloSCT) and autologous stem cell transplantation (ASCT) for CBF-AML. OS of t(8;21) patients was poor when undergoing alloSCT in second/third CR, while OS of inv(16) patients in second/third CR was similar to that in first CR. Patients with > 3-log reduction of RUNX1/RUNX1T1 qPCR had improved 3-year event-free survival (EFS) than those without (73.2% vs. 50.3%). Patients with t(8;21) AML with D816 mutation of the c-Kit gene showed inferior EFS and OS. These poor outcomes might be overcome by alloSCT. Multivariate analysis for OS in patients with t(8;21) revealed older age, > 1 course of induction chemotherapy to achieve CR, loss of sex chromosome, del(7q), and second/third CR or not in CR before SCT as independent prognostic variables. Especially, del(7q) is the most powerful prediction factor of poor outcomes, especially in patients with t(8;21) (hazard ratio, 27.23; P < 0.001). Further study is needed to clarify the clinical effect of cytogenetics and gene mutation in patients with CBF-AML, between Asian and Western countries.

Elucidation of Novel Therapeutic Targets for Acute Myeloid Leukemias with RUNX1-RUNX1T1 Fusion.

The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1, fusion protein is difficult to target, as it lacks kinase activities. Here, we used bioinformatic tools to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AML cases from The Cancer Genome Atlas (TCGA) database, we found expression of 293 genes that correlated to the expression of the RUNX1-RUNX1T1 fusion gene. Based on these 293 genes, the cyclooxygenase (COX), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) pathways were predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion, when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve clinical outcomes.

CSF3R Mutations are frequently associated with abnormalities of RUNX1, CBFB, CEBPA, and NPM1 genes in acute myeloid leukemia.

BACKGROUND: Mutations in the colony-stimulating factor 3 receptor (CSF3R) gene occur frequently in chronic neutrophilic leukemia and are rare in de novo acute leukemia. The objective of this study was to assess the incidence of CSF3R mutations in acute leukemia and their association with other genetic abnormalities. METHODS: Amplicon-targeted, next-generation sequencing of 58 genes was performed retrospectively on 1152 patients (acute myeloid leukemia [AML], n = 587; acute lymphoid leukemia [ALL], n = 565). Reverse transcriptase-polymerase chain reaction analysis was used to detect 35 leukemia-specific gene fusions. RESULTS: CSF3R mutations (26 patients) were detected in 3.6% (13 of 364 patients), 4.6% (8 of 175 patients), and 8.3% (4 of 48 patients) of those with de novo, relapsed, and secondary AML, respectively, and in 0.2% (1 of 565 patients) of those with ALL. In total, 9 distinct CSF3R mutations were detected. Membrane-proximal missense mutations and cytoplasmic truncations were identified as mutually exclusive. The proportion of patients who had French-American-British subtypes M2 and M4 in the CSF3R-mutated group was significantly greater than that in the CSF3R wild-type group for both the de novo AML cohort (P = .001) and the relapsed AML cohort (P = .024). All de novo and relapsed AMLs with CSF3R mutations were associated with genetic alterations in transcription factors, including RUNX1-RUNX1T1, CBFB-MYH11, double-mutated CCAAT/enhancer binding protein α (CEBPAdm), and NPM1 mutations; and core-binding factor gene abnormalities and CEBPAdm accounted for 90.5% (19 of 21 patients). CONCLUSIONS: CSF3R mutations are uncommon in AML; however, when they occur, they are often associated with core-binding factor gene abnormalities and CEBPAdm. An in-depth understanding of the interaction between these genetic alterations could facilitate a clearer understanding of the role of CSF3R mutations in AML development and may be used for disease classification, prognosis, and the development of targeted therapy.

Interferon-α Is Effective for Treatment of Minimal Residual Disease in Patients with t(8;21) Acute Myeloid Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation: Results of a Prospective Registry Study.

BACKGROUND: RUNX1-RUNX1T1 transcript levels were established as a powerful marker for predicting relapse in patients with t(8;21) acute myeloid leukemia (AML). We aimed to identify the efficacy of minimal residual disease (MRD)-directed interferon-alpha (IFN-α) treatment in patients with t(8;21) AML who were positive for MRD after allogeneic hematopoietic stem cell transplantation (allo-HSCT; n=42). SUBJECTS, MATERIALS, AND METHODS: MRD-positive status was defined as a <4.5-log reduction from diagnosis in RUNX1-RUNX1T1 transcripts and/or the loss of a ≥4.5-log reduction after 3 months after HSCT. Patients with positive MRD received six cycles of IFN-α treatment (twice or thrice weekly of every 4 weeks cycle). RESULTS: The 1-year cumulative incidence of severe acute and chronic graft-versus-host disease after MRD-directed IFN-α treatment was 7.1% and 4.8%, respectively. After the treatment, 15 (35.7%), 5 (11.9%), 3 (7.1%), and 9 (21.5%) patients achieved MRD negativity at 1, 2, 3, and >3 months, respectively. Three patients relapsed after the IFN-α treatment, in which the 1-year cumulative incidence of relapse was 7.2%. One patient died of severe infection at 460 days after treatment. The 1-year probabilities of event-free survival, disease-free survival, and overall survival after treatment were 76.0%, 92.4%, and 92.5%, respectively. The clinical outcomes in patients who received MRD-directed IFN-α treatment were significantly better than those of the MRD-positive patients without any interventions in the historical cohort. CONCLUSION: MRD-directed IFN-α treatment is effective for patients with t(8;21) AML who were MRD-positive after allo-HSCT. The study was registered at http://clinicaltrials.gov as NCT02027064. IMPLICATIONS FOR PRACTICE: In patients with t(8;21) acute myeloid leukemia (AML), the presence of post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) minimal residual disease (MRD), measured by RUNX1-RUNX1T1 transcript levels, has been established as a powerful marker for predicting relapse. Interferon-alpha (IFN-α) could exert a relatively strong graft-versus-leukemia effect, and MRD-directed IFN-α treatment is effective for patients with t(8;21) AML who were MRD-positive after allo-HSCT.

A minicircuitry of microRNA-9-1 and RUNX1-RUNX1T1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia.

MicroRNA-9-1(miR-9-1) plays an important role in the mechanism that regulates the lineage fate of differentiating hematopoietic cells. Recent studies have shown that miR-9-1 is downregulated in t (8; 21) AML. However, the pathogenic mechanisms underlying miR-9-1 downregulation and the RUNX1-RUNX1T1 fusion protein, generated from the translocation of t (8; 21) in AML, remain unclear. RUNX1-RUNX1T1 can induce leukemogenesis through resides in and functions as a stable RUNX1-RUNX1T1-containing transcription factor complex. In this study, we demonstrate that miR-9-1 expression increases significantly after the treatment of RUNX1-RUNX1T1 (+) AML cell lines with decitabine (a DNMT inhibitor) and trichostatin A (an HDAC inhibitor). In addition, we show that RUNX1-RUNX1T1 triggers the heterochromatic silencing of miR-9-1 by binding to RUNX1-binding sites in the promoter region of miR-9-1 and recruiting chromatin-remodeling enzymes, DNMTs, and HDACs, contributing to hypermethylation of miR-9-1 in t (8; 21) AML. Furthermore, because RUNX1, RUNX1T1, and RUNX1-RUNX1T1 are all regulated by miR-9-1, the silencing of miR-9-1 enhances the oncogenic activity of these genes. Besides, overexpression of miR-9-1 induces differentiation and inhibits proliferation in t (8; 21) AML cell lines. In conclusion, our results indicate a feedback circuitry involving miR-9-1 and RUNX1-RUNX1T1, contributing to leukemogenesis in RUNX1-RUNX1T1 (+) AML cell lines.

t(2;2;21;8)(p21;q37;q22;q22), a novel four-way complex translocation involving variant t(8;21) in case of acute myeloid leukemia : A case report and literature review.

Chromosomal translocation serves as a crucial diagnostic marker in the classification of acute myeloid leukemia. Among the most prevalent cytogenetic abnormalities is t(8;21)(q22;q22), typically associated with the FAB subtype AML-M2. On occasion, alternative forms of t(8;21) have been observed. This report presents a case of AML with RUNX1::RUNX1T1, wherein the karyotype revealed t(2;2;21;8)(p21;q37;q22;q22), representing the first instance of a variant t(8;21) involving both chromosomes 2. The combination of routine karyotype analysis and fluorescence in situ hybridization proves to be an effective method for identifying complex translocations of t(8;21).

[Analysis of the therapeutic effect of avatinib bridged allogeneic hematopoietic stem cell transplantation on 7 cases of recurrent/refractory RUNX1-RUNX1T1 positive acute myeloid leukemia with KIT mutations].

Objective: To evaluate the efficacy of avatinib plus allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the treatment of recurrent/refractory RUNX1-RUNX1T1 positive acute myeloid leukemia (AML) with KIT mutations. Method: A retrospective study was conducted on the clinical data of seven relapsed/refractory AML patients containing the RUNX1-RUNX1T1 fusion gene and KIT mutation who received afatinib plus allo-HSCT treatment at the First Affiliated Hospital of Soochow University from June 2019 to June 2023. Results: The seven AML patients included one male and six females with a median age of 37 (18-56) years. All seven patients had KIT mutations (five positive for D816V and two positive for D816Y) . There were two refractory patients and five relapsed patients (all of whom had bone marrow recurrence) . All patients had to complete at least one course of treatment with afatinib before transplantation. Four patients achieved complete remission (CR) after treatment with afatinib, six patients had negative KIT gene mutations, and one had a decreased KIT gene mutational burden. There were three cases of unrelated identical transplantation and four cases of haploidentical transplantation. All patients received the modified Bu/Cy pretreatment regimen. After transplantation, all patients were successfully implanted and a bone marrow examination showed CR and minimal residual disease turned negative. Five patients exhibited negative fusion genes. Two patients died from infection following transplantation. Conclusion: Afatinib plus allo-HSCT may be an effective and safe new treatment strategy for RUNX1-RUNX1T1 positive AML patients with KIT-D816 mutation.

RUNX1::RUNX1T1 Acute Myeloid Leukemia Cytogenetically Showing t(6;8)(p23;q22).

Runt-related transcription factor 1 (RUNX1)::RUNX1 partner transcriptional co-repressor 1 (RUNX1T1) acute myeloid leukemia (AML) is a subtype of acute leukemia primarily classified as French American British M2. RUNX1::RUNX1T1 transcript is formed by a reciprocal translocation between chromosomes 8q22 and 21q22. However, we encountered a case of AML that showed molecular positivity for RUNX1::RUNX1T1 fusion transcript but exhibited cytogenetically atypical translocation t(6;8). Fluorescence in situ hybridization (FISH) analysis, in combination with G-banding, clarified the three-way translocation t(6;21;8)(p25;q22;q22), which was partially cryptic. The case emphasizes the importance of employing molecular analysis alongside cytogenetics to determine disease subtypes in patients with acute leukemia.

Transformation into acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 from JAK2-mutated essential thrombocythemia: a case report.

BACKGROUND: Blast transformation is a rare but well-recognized event in Philadelphia-negative myeloproliferative neoplasms associated with a poor prognosis. Secondary acute myeloid leukemias evolving from myeloproliferative neoplasms are characterized by a unique set of cytogenetic and molecular features distinct from de novo disease. t(8;21) (q22;q22.1); RUNX1::RUNX1T1, one of the most frequent cytogenetic abnormalities in de novo acute myeloid leukemia, is rarely observed in post-myeloproliferative neoplasm acute myeloid leukemia. Here we report a case of secondary acute myeloid leukemia with t(8;21) evolving from JAK2-mutated essential thrombocythemia. CASE PRESENTATION: The patient was a 74-year-old Japanese woman who was referred because of thrombocytosis (platelets 1046 × 109/L). Bone marrow was hypercellular with increase of megakaryocytes. Chromosomal analysis presented normal karyotype and genetic test revealed JAK2 V617F mutation. She was diagnosed with essential thrombocythemia. Thrombocytosis had been well controlled by oral administration of hydroxyurea; 2 years after the initial diagnosis with ET, she presented with leukocytosis (white blood cells 14.0 × 109/L with 82% of blasts), anemia (hemoglobin 91 g/L), and thrombocytopenia (platelets 24 × 109/L). Bone marrow was hypercellular and filled with 80% of myeloperoxidase-positive blasts bearing Auer rods. Chromosomal analysis revealed t(8;21) (q22;q22.1) and flow cytometry presented positivity of CD 13, 19, 34, and 56. Molecular analysis showed the coexistence of RUNX1::RUNX1T1 chimeric transcript and heterozygous JAK2 V617F mutation in leukemic blasts. She was diagnosed with secondary acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 evolving from essential thrombocythemia. She was treated with combination chemotherapy with venetoclax and azacytidine. After the first cycle of the therapy, blasts disappeared from peripheral blood and decreased to 1.4% in bone marrow. After the chemotherapy, RUNX1::RUNX1T1 chimeric transcript disappeared, whereas mutation of JAK2 V617F was still present in peripheral leukocytes. CONCLUSIONS: To our best knowledge, the present case is the first one with JAK2 mutation preceding the acquisition of t(8;21). Our result suggests that t(8;21); RUNX1::RUNX1T1 can be generated as a late event in the progression of JAK2-mutated myeloproliferative neoplasms. The case presented typical morphological and immunophenotypic features associated with t(8;21) acute myeloid leukemia.

Deregulated Gene Expression Profiles and Regulatory Networks in Adult and Pediatric RUNX1/RUNX1T1-Positive AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous and complex disease concerning molecular aberrations and prognosis. RUNX1/RUNX1T1 is a fusion oncogene that results from the chromosomal translocation t(8;21) and plays a crucial role in AML. However, its impact on the transcriptomic profile of different age groups of AML patients is not completely understood. Here, we investigated the deregulated gene expression (DEG) profiles in adult and pediatric RUNX1/RUNX1T1-positive AML patients, and compared their functions and regulatory networks. We retrospectively analyzed gene expression data from two independent Gene Expression Omnibus (GEO) datasets (GSE37642 and GSE75461) and computed their differentially expressed genes and upstream regulators, using limma, GEO2Enrichr, and X2K. For validation purposes, we used the TCGA-LAML (adult) and TARGET-AML (pediatric) patient cohorts. We also analyzed the protein-protein interaction (PPI) networks, as well as those composed of transcription factors (TF), intermediate proteins, and kinases foreseen to regulate the top deregulated genes in each group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were further performed for the DEGs in each dataset. We found that the top upregulated genes in (both adult and pediatric) RUNX1/RUNX1T1-positive AML patients are enriched in extracellular matrix organization, the cell projection membrane, filopodium membrane, and supramolecular fiber. Our data corroborate that RUNX1/RUNX1T1 reprograms a large transcriptional network to establish and maintain leukemia via intricate PPI interactions and kinase-driven phosphorylation events.