ASXL1 Research Articles

ASXL3 gene mutations inhibit cell proliferation and promote cell apoptosis in mouse cardiomyocytes by upregulating lncRNA NONMMUT063967.2

Congenital heart disease (CHD) is a serious condition with unknown etiology. In a recent study, a compound heterozygous mutation (c.3526C > T [p.Arg1176Trp] and c.4643A > G [p.Asp1548Gly]) in the ASXL3 gene was identified, which is associated with CHD. This mutation was overexpressed in HL-1 mouse cardiomyocyte cells, leading to increased cell apoptosis and decreased cell proliferation. However, whether this effect is mediated by long noncoding RNAs (lncRNAs) is yet to be determined. We identified the differences among lncRNA and mRNA profiles in mouse heart tissues using sequencing to explore this issue. We detected HL-1 cell proliferation and apoptosis through CCK8 and flow cytometry. Fgfr2, lncRNA, and Ras/ERK signaling pathway expressions were evaluated using quantitative real time polymerase chain reaction (qRT-PCR) and western blot (WB) assays. We also conducted functional investigations by silencing lncRNA NONMMUT063967.2. The sequencing revealed significant changes in lncRNA and mRNA profiles, with the expression of lncRNA NONMMUT063967.2 being significantly promoted in the ASXL3 gene mutations group (MT) while the expression of Fgfr2 being downregulated. The in vitro experiments showed that ASXL3 gene mutations inhibited the proliferation of cardiomyocytes and accelerated cell apoptosis by promoting the expression of lncRNAs (NONMMUT063967.2, NONMMUT063918.2, and NONMMUT063891.2), suppressing the formation of FGFR2 transcripts, and inhibiting the Ras/ERK signaling pathway. The decrease in FGFR2 had the same effect on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes as ASXL3 mutations. Further mechanistic studies revealed that suppression of lncRNA NONMMUT063967.2 and overexpression of FGFR2 reversed the effects of the ASXL3 mutations on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes. Therefore, ASXL3 mutation decreases FGFR2 expression by upregulating lncRNA NONMMUT063967.2, inhibiting cell proliferation and promoting cell apoptosis in mouse cardiomyocytes.

Hypoplastic form of myelodysplastic neoplasm.

Hypoplastic myelodysplastic neoplasm (MDS-h) is a rare hematopoietic disorder characterized by peripheral cytopenia, hypoplasia (cellularity ≤ 25%) and dysplastic changes in the bone marrow. Compared to normo- /hypercellular MDS, in addition to hypocellularity, MDS-h patients have more profound neutropenia and thrombocytopenia, a lower percentage of blasts, and less frequent abnormal karyotype. It is difficult to distinguish MDS-h from aplastic anemia in differential diagnosis. Abnormal karyotype is found in 15-50% of MDS-h patients and the most common chromosomal aberrations include -5/del (5q), -7/del (7q), +8, 17pLOH, del (20q), UPD at 4q, 11q, 13q, and 14q. Approximately 35% of MDS-h patients harbour somatic mutations that are most often detected in PIGA, TET2, DNMT3A, RUNX1, NPM1, ASXL1, STAG2, and APC genes. An autoimmune destruction of hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) mediated by abnormally activated T cells plays a key role in the pathophysiology of MDS-h. Expanded T cells overproduce proinflammatory cytokines (IFN- g and TNF-a), which inhibit proliferation and induce apoptosis of HSC/HPCs. The antigens that trigger the immune response are not known, but potential candidates have been suggested such as WT1 protein and HLA class I molecules. MDS-h does not represent a phenotypically homogeneous subtype of MDS, but rather it is a mixed entity comprising both patients showing features similar to myelodysplastic neoplasm and patients with features of non-malignant bone marrow failure. Determining the prevailing phenotype in MDS-h is important for choosing the optimal treatment and prognosis prediction. The aim of this article is to point out an interesting hypoplastic MDS, the diagnosis of which is difficult, and to provide an overview of its main clinical-pathological features, genetic background, and mechanisms of aberrant immune response.

Co-mutation of ATM and ASXL1 and relationship to durable response of a novel PARP1/2 inhibitor, venadaparib, in patients with pancreatic cancer.

e15019 Background: Homologous recombination repair (HRR) alterations such as BRCA mutation (BRCAm) and ATM mutation (ATMm) are established predictive biomarkers for poly (adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitor in pancreatic cancer (PC). Mutations in the ASXL1 gene (ASXL1m) have been associated with hematologic malignancies. In solid cancers, however, associations between HRR and non-HRR mutation such as ASXL1m as well as treatment response in patients with those are not well known. The aim of this analysis was to explore genetic alterations co-existing with HRR mutation to better predict efficacy from a monotherapy of venadaparib, a PARP inhibitor under development, in patients with metastatic PC. Methods: Patients with HRR mutation and no standard treatment or prior treatment failure were enrolled in a phase Ib basket trial (NCT04174716) to receive venadaparib monotherapy. Tumor response to treatment was evaluated according to RECIST 1.1. Exploratory blood samples were collected to analyze genetic alterations using ctDNA next-generation sequencing (NGS) (GuardantOMNI Gene Panel version 1.0, Redwood City, CA) on Day 1 pre-dose. Prevalence of co-occurring mutations was estimated and mutual exclusivity between HRR and non-HRR mutations were statistically tested by Fisher’s exact test using the cBioPortal for Cancer Genomics ( http://cbioportal.org ) in The Cancer Genome Atlas (TCGA) Pan-cancer and AACR Project Genie v13.0 registries. Efficacy between patients with co-mutations and single mutation only were presented. Results: In public dataset, the prevalence of ATMm and ASXL1m was 3.0% and 0.9% in TCGA pan-cancer study (n = 10,967), 3.0% and 1.7% in Genie v13.0 (n = 131,996), respectively. In both datasets, co-mutation of ATM and ASXL1 was tested significant (Log2 Odds Ratio 2.663 and 1.176 respectively; q-value 0.001 and 0.006 respectively). In the basket trial, 8 patients were enrolled, with 50% male, and a median age 66 (range 47-72). 6 patients had 2 prior palliative chemotherapy. 6 patients had ATMm and 2 had BRCA2m confirmed by enrolling institution at baseline. All patients had responded to prior platinum containing regimen. ctDNA NGS samples were available in 6 patients. Patients with ATMmASXL1m (n = 3) had a 33% overall response rate (1 partial response, 2 stable disease, range of progression-free survival (PFS), 23-113 weeks), while those with ATMmASXL1wt (n = 1) had a 0% (1 progressive disease (PD), PFS 9 weeks) and others (ATMwtASXL1wt, n = 2, 1 PD; ctDNA not available, n = 2, 2 PD) had a 0% response rate (range PFS 3-16 weeks). Two patients with BRCA2m only had PD on weeks 3 and 8. Conclusions: In this exploratory analysis, patients with co-occurring ATMm and ASXL1m showed higher tumor response. This signals potential value from ATM and ASXL1 co-mutation predicting efficacy of Venadaparib in PC and warrants further validation. Clinical trial information: NCT04174716 .

Clinical Analysis of Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm

To explore the clinical characteristics, treatment, and prognosis of patients with blastic plasmacytoid dendritic cell neoplasm(BPDCN). Clinical data of 5 patients diagnosed with BPDCN in Wuhan First Hospital and Wuhan Tongji Hospital from June 2016 to November 2021 were retrospectively analyzed. Among the 5 patients, 3 were male and 2 were female, with a median age of 28(10-52) years old. Four patients showed obvious skin damage at the initial diagnosis; the other one showed clinical manifestations of acute leukemia rather than obvious skin damage at the initial diagnosis, but infiltrated skin when the disease relapsed after treatment. Other infiltration sites of lesions included bone marrow (2/5), peripheral blood (2/5), lymph nodes (3/5), liver and spleen (2/5). All patients had no clinical manifestation of central nervous system infiltration. Tumor cell specific immune markers CD4, CD56, CD123 were all positive, and the median Ki-67 index was 70%. TET2, ASXL1 and NRAS gene mutations were found respectively in 3 patients by next-generation sequencing technique (NGS). ALL-like, AML-like and invasive NK/T cell lymphoma-like first-line induction chemotherapy regimens were used for the patients. One patient died of severe complications during the early stage of chemotherapy, 3 patients were evaluated as CR, and 1 patient was evaluated as PR. 2 patients were recurred and progressed after induction of chemotherapy, and one of them was evaluated as CR after re-treatment. One patient received autologous hematopoietic stem cell transplantation (auto-HSCT) and got long-term survival (OS 87 months). 3 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), of which one died of transplantation related complications, and 2 cases survived. The median follow-up time of 4 patients with evaluable efficacy was 28.5(9-84) months, the median OS time was 31.5(10-87) months. BPDCN is a highly heterogeneous malignant tumor with a poor prognosis. HSCT, especially allo-HSCT can significantly improve the prognosis of BPDCN patients.

Some results of a pilot study of mutations in ASXL1 and DNMT3A genes in myelodysplastic syndrome

Оbjective : was to determine the prevalence of the c.1934dupG mutation in the ASXL1 gene and the R882H mutation in the DNMT3A gene in MDS and their influence on significant clinical characteristics. Materials and methods : 33 men and 17 women with a median age of 57 years (18 –83) and a verified diagnosis of myelodysplastic syndrome were included in the study. Twenty-two volunteers without hematologic pathology were taken as controls, of them: 8 men and 14 women aged from 22 to 65 years. PCR examination of venous blood was performed in all study groups to detect c.1934dupG and R882H with Sanger reference sequencing. Results : the R882H mutation was not detected in any of the study groups. The c.1934dupG mutation was not detected in individuals without hematologic pathology. Due to a lack of concentration of isolated DNA from venous blood cells, no analysis of amplification efficiency could be performed in 7 patients. The c.1934dupG mutation was found in 46% of patients and was found in all IPSS-R, WPSS, and MDS-CI risk groups. No differences were found in the analysis of survival in the presence and absence of the c.1934dupG mutation. Conclusions : the study demonstrated that the developed method for the detection of the c.1934dupG mutation in venous blood cells enables optimization of diagnosis. A limitation of the adapted amplification and restriction assays was the degree of blood leucopenia. No effect of the c.1934dupG mutation on the clinical course of myelodysplastic syndrome was detected.

Analysis of somatic mutations in the <i>JAK2</i>, <i>CALR</i>, <i>MPL</i> and <i>ASXL1</i> genes and evaluation of their impact on the survival of patients with myelofibrosis

Background. The development of myelofibrosis (MF) is driven by complex molecular genetic events that include driver somatic mutations responsible for the constitutive activation of the JAK/STAT signaling pathway (JAK2, CALR, and MPL), additional mutations affecting epigenetic regulators (TET2, ASXL1, IDH1/2, etc.) and RNA splicing (SRSF2, U2AF1, SF3B1, etc.), as well as genetic aberrations that contribute to genomic instability and disease progression.Aim. To analyze driver (JAK2, CALR, MPL) and prognostic (ASXL1) somatic mutations in patients with MF and evaluate their impact on survival.Materials and methods. The study included 29 patients diagnosed with MF, selected by hematologists from the City Clinical Hospital No. 7 and Regional Clinical Hospital (Krasnoyarsk).Results. 26 (89.6 %) out of 29 examined patients had some driver mutations in JAK2, CALR, MPL genes. The p.V617F mutation in the JAK2 gene was found in 20 (68.9 %) patients. Mutations in the CALR gene were detected in 4 (13.8 %) patients, mutations in the MPL gene were found in 3 patients (10.3 %). In 1 of 26 patients, 2 driver mutations were present simultaneously. 3 (10.3 %) patients were triple negative. Mutations in the ASXL1 gene were detected in 12 (41.4 %) out of 29 examined patients. Conducted targeted NGS (next generation sequencing) for 13 out of 29 patients revealed additional genetic variants that contribute to the understanding of the development mechanism and disease course. When evaluating the overall survival in the groups of patients diagnosed with MF examined by us, depending on the combination of driver (JAK2, CALR, MPL) and prognostic (ASXL1) mutations, no statistically significant differences were found (p = 0.12). This appears to be due to the small sample size. At the same time, assessment of patient survival depending on ASXL1 status showed that in the presence of mutations in the ASXL1 gene, the median survival was 45 months (range 7–120 months), while in the absence of mutations it was 48 months (range 21–359 months) (p = 0.03).Conclusion. The results obtained allow us to assume that the presence of mutations in the ASXL1 gene is an unfavorable factor in the course of the disease.

Somatic genetic alterations predict hematological progression in GATA2 deficiency.

Germline GATA2 mutations predispose to myeloid malignancies resulting from the progressive acquisition of additional somatic mutations. Here we describe clinical and biological features of 78 GATA2-deficient patients. Hematopoietic stem and progenitor cell phenotypic characterization revealed an exhaustion of myeloid progenitors. Somatic mutations in STAG2, ASXL1 and SETBP1 genes along with cytogenetic abnormalities (monosomy 7, trisomy 8, der(1;7)) occurred frequently in patients with GATA2 germline mutations. Patients were classified into three hematopoietic spectra based on bone marrow cytomorphology. No somatic additional mutations were detected in patients with normal bone marrow (spectrum 0), whereas clonal hematopoiesis mediated by STAG2 mutations was frequent in those with a hypocellular and/or myelodysplastic bone marrow without excess blasts (spectrum 1). Finally, SETBP1, RAS pathway and RUNX1 mutations were predominantly associated with leukemic transformation stage (spectrum 2), highlighting their implications in the transformation process. Specific somatic alterations, potentially providing distinct selective advantages to affected cells, are therefore associated with the clinical/hematological evolution of GATA2 syndrome. Our study not only suggests that somatic genetic profiling will help clinicians in their management of patients, but will also clarify the mechanism of leukemogenesis in the context of germline GATA2 mutations.

Molecular Genetics of Thrombotic Myeloproliferative Neoplasms: Implications in Precision Oncology.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include polycythaemia vera, essential thrombocythaemia, and primary myelofibrosis. Unlike monogenic disorders, a more complicated series of genetic mutations are believed to be responsible for MPN with various degrees of thromboembolic and bleeding complications. Thrombosis is one of the early manifestations in patients with MPN. To date, the driver genes responsible for MPN include JAK2, CALR, MPL, TET2, ASXL1, and MTHFR. Affords have been done to elucidate these mutations and the incidence of thromboembolic events. Several lines of evidence indicate that mutations in JAK2, MPL, TET2 and ASXL1 gene and polymorphisms in several clotting factors (GPIa, GPIIa, and GPIIIa) are associated with the occurrence and prevalence of thrombosis in MPN patients. Some polymorphisms within XRCC1, FBG, F2, F5, F7, F12, MMP9, HPA5, MTHFR, SDF-1, FAS, FASL, TERT, ACE, and TLR4 genes may also play a role in MPN manifestation. This review aims to provide an insightful overview on the genetic perspective of thrombotic complications in patients with MPN.

THE VALUE OF MUTATIONS IN THE ASXL1 GENE IN THE ONCOGENESIS OF ACUTE MYELOID LEUKEMIA IN ADULTS

The aim was to evaluate the significance of the ASXL1 gene mutations in the oncogenesis of acute myeloid leukemia (AML) in adults. Materials and methods. Bone marrow and peripheral blood samples of 49 patients with AML aged from 18 to 84 years, treated at the Sverdlovsk regional hematological center, were examined, including those with a morphological variant of AML M0 – 3, M1 – 4, M2 – 19, M3 – 6, M4 – 10, M5 – 1, M6 – 1, M7 – 1, blast plasmacytoid dendritic cell neoplasm – 1, acute hybrid leukemia – 2, AML after chronic myeloproliferative disease (CMPD) – 2. Mutations in the ASXL1 gene were detected in peripheral blood and bone marrow samples by direct RNA sequencing (n=26) and high-throughput DNA sequencing (n=23) on automatic genetic analyzers ABI Prism 310 and MiSeqDX, respectively. Results. It was found that pathogenetically significant mutations of the ASXL1 gene were detected in 16.3% of samples with AML M0, M2, M4, AML from CMPD and acute hybrid leukemia. The distribution of ASXL1 anomalies was as follows: deletion n. Del (1755;2007) — 4, duplication c.1934dupG — 3, non-synonymous transversion c.1348A>T — 1. Along with lesions of the ASXL1 gene, quantitative and structural chromosomal aberrations were determined in these samples, including del(5)(q13), t(8;21)(q22;q22), t(9;22)(q34;q11), as well as point mutations in the CEBPA, IDH2, NRAS, RUNX1, SRSF2, TET2 genes, internal tandem duplication in the FLT3 gene. This indicates the cooperation of two or more mutational events in the oncogenesis of ASXL1-positive AML, which can determine the different reaction of the tumor to chemotherapeutic treatment and the prognosis of the disease.

Defining the mutational profile of lower-risk myelodysplastic neoplasm patients with respect to disease progression using next-generation sequencing and pyrosequencing.

Lower-risk myelodysplastic neoplasms (LR-MDS) comprise the majority of MDS. Despite favourable prognoses, some patients remain at risk of rapid progression. We aimed to define the mutational profile of LR-MDS using next-generation sequencing (NGS), Sanger Sequencing (SSeq), and pyrosequencing. Samples from 5 primary LR-MDS (67 exons of SF3B1, U2AF1, SRSF2, ZRSR2, TET2, ASXL1, DNMT3A, TP53, and RUNX1 genes) were subjected to NGS. Next, a genomic study was performed to test for the presence of identified DNA sequence variants on a larger group of LR-MDS patients (25 bone marrow [BM], 3 saliva [SAL], and one peripheral blood [PB] sample/s). Both SSeq (all selected DNA sequence variants) and pyrosequencing (9 selected DNA sequence variants) were performed. Next-generation sequencing results identified 13 DNA sequence variants in 7 genes, comprising 8 mutations in 6 genes (ASXL1, DNMT3A, RUNX1, SF3B1, TET2, ZRSR2) in LR-MDS. The presence of 8 DNA variants was detected in the expanded LR-MDS group using SSeq and pyrosequencing. Mutation acquisition was observed during LR-MDS progression. Four LR-MDS and one acute myeloid leukaemia myelodysplasia-related patient exhibited the presence of at least one mutation. ASXL1 and SF3B1 alterations were most commonly observed (2 patients). Five DNA sequence variants detected in BM (patients: 9, 13) were also present in SAL. We suggest using NGS to determine the LR-MDS mutational profile at diagnosis and suspicion of disease progression. Moreover, PB and SAL molecular testing represent useful tools for monitoring LR-MDS at higher risk of progression. However, the results need to be confirmed in a larger group.

Pathogenic role of insufficiency of polycomb repressive complex in primary myelofibrosis

Primary myelofibrosis (PMF) is characterized by the clonal expansion of megakaryocytes and myeloid cells from stem cells with abnormal cytokine expression, resulting in bone marrow fibrosis, angiogenesis, and osteosclerosis. The use of next-generation sequencing revealed that both genetic and epigenetic changes are important in the pathogenesis of PMF. Several epigenetic regulator genes, including TET2, the polycomb-related gene ASXL1, and the polycomb-group gene EZH2, have been found to be targeted by somatic gene mutations in PMF patients. Among these, loss of Ezh2 has been demonstrated to disrupt the function of the polycomb repressive complex 2, promoting the development of JAK2V617F-induced myelofibrosis in mice. In this analysis, we highlight the role of PRC dysfunction in the pathogenesis of PMF.

Co-Existing ASXL1 and SF3B1 Mutations Carried Worse Outcome Than ASXL1 or SF3B1 Mutations Alone

Introduction: Somatic mutations in the ASXL1 gene (additional sex combs like 1) are frequently observed in myeloid malignancies, including Clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS/MPN, and acute myeloid leukemia (AML), and have been found to be independent predicative factor for poor overall survival. Mutations in SF3B1 (Splicing factor 3B subunit 1) were also frequently detected in the above hematopoietic disorders. It represents distinct molecular and clinical patient groups and shows particular association with ringed sideroblasts. Most studies have associated SF3B1 mutations with good clinical prognosis. Both ASXL1 and SF3B1 mutations have been reported to frequently coexist with the mutations in other genes. Co-mutation of ASXL1 with SF3B1 is less frequent and has even been reported to be mutually exclusive by some authors. There are very few and contradictory reports about the clinical significance of co-existing ASXL1 and SF3B1 mutations, which is the goal of this study. Previous studies were also confounded by patients with mutations in some other genes. Method: We identified 175 patients with only ASXL1 mutations, only SF3B1 mutations, or only ASXL1/SF3B1 co-mutations from our database of 8285 patients with NextGen sequencing (NGS) results, to study and compare their clinical features and outcomes. Baseline characteristics and other somatic mutation distribution were compared by chi square (categorical variables) and t-test (continuous variables). Overall survival was calculated using the Kaplan-Meier method log-rank test. Results: Approximately 8285 patients with diagnosed or suspected myeloid neoplasm were tested by NGS. There were1515 patients with ASXL1 mutations with or without other genes mutated, and 583 patients with SF3B1 mutations with or without other genes mutated. There were also 97 patients with concurrent ASXL1 and SF3B1 mutations, most with additional genes mutated. Out of these patients, we identified 69 patients with only ASXL1 mutations (ASXL1 group), 89 patients with only SF3B1 mutations (SF3B1 group), and 17 patients with only ASXL1 plus SF3B1 mutations (ASXL1/SF3B1 group). The median age of ASXL1 group (69) was much younger than SF3B1 (76) and ASXL1/SF3B1 group (p values 0.01208 and 0.0001, respectively). The female to male ratios were similar among these groups. ASXL1 mutations were more frequent in AML and CCUS (22.47% and 33.71%) than ASXL1/SF3B1 (11.76% and 0%) and SF3B1 (1.45% and 7.25%) (p values < 0.003) mutations. On the contrary, SF3B1 and ASXL1/SF3B1 mutations were more frequent in MDS patients (75.36% and 64.71%) than ASXL1 mutations. In MDS/MPN, ASXL1/SF3B1 mutations (23.53%) were more frequent than SF3B1 (15.94%) and ASXL1 mutations (5.62%, p value 0.03513). The three groups of patients showed similar cytogenetic changes, with similar percentages of patients with normal karyotype, del(5q), del(7/7q), del(17p/TP53), del(20q), or trisomy 8. Finally, we compared the overall survival of the three groups of patients with normal karyotype by Kaplan-Meier survival analysis. As expected, ASXL1 group showed worse overall survival than SF3B1 group with a hazard ratio of 5.834 (p value 0.0175). However, it is interesting to found that ASXL1/SF3B1 group showed poorer overall survival than both SF3B1 and ASXL1 groups with a hazard ratio of 9.840 (p value 0.0052) (Figure 1). Conclusion: Co-existing ASXL1 and SF3B1 mutations carried worse outcome than ASXL1 or SF3B1 mutation alone, likely due to abnormalities in both epigenetic regulatory pathway and RNA splicing pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Characterization of Adult Acute Lymphoblastic Leukemia Identifies a Subgroup with Myeloid Mutations and Pre-Existing Clonal Hematopoiesis

Acute lymphoblastic leukemia (ALL) represents 20% of adult leukemias. Genomic risk stratification of ALL relies on the data generated from large cohorts of pediatric and young adult patients, where older patients with ALL have been underrepresented. It is imperative to understand the unique leukemogenic pathways in this group in order to develop novel therapeutic approaches. Clonal hematopoiesis (CH) is a precursor lesion for development of myeloid leukemias and mature lymphoid neoplasms (Niroula, Nat Med 2021), but its impact on the biology and outcomes of ALL is unknown. Based on our observations on high incidence of myeloid mutations in older adults with ALL, we hypothesized that CH constitutes a fertile genomic background for lymphoblastic leukemogenesis, and ALL with myeloid mutations is a unique entity with distinct clinical and molecular characteristics. We studied 400 adult patients with ALL treated at the University of Chicago, Moffitt Cancer Center, and City of Hope between 2014 and 2022 (Fig 1A). Genetic profiling of pre-treatment bone marrow samples was performed with a comprehensive panel covering 147 myeloid and lymphoid genes. Overall, 33% of ALL cases had at least one myeloid CH-associated mutation with the following frequencies: TP53 (18%), DNMT3A (5%), TET2 (4%), ASXL1 (3%), RUNX1 (3%), IDH2 (2%), BCORL1 (2%), U2AF1 (1%), CUX1 (1%), BCOR (1%), CBL (1%), and SF3B1 (1%). We further investigated the association between mutations and clinical variables by calculating the odds-ratios (OR) with adjustments for multiple testing by using the Benjamini-Hochberg method. Myeloid CH mutations in TP53, DNMT3A, TET2, and ASXL1 genes were more common in older adults with ALL. Mutations in TP53 and DNMT3A were enriched in therapy-related B-ALL group (OR 2.6, 95% CI= 1.3-5.5, q= .007 and OR 4.7, CI= 1.2-18.1, q= .01, respectively), and were more commonly associated with hypodiploid (OR 7.1, 95% CI= 1.5-32.7, q=.004 and OR 5.2, 95% CI= 0.6-48, q=.1, respectively) and hyperdiploid (OR 5.9, 95% CI= 2.7-13.1, q=<0.001 and OR 3.9, 95% CI= 1.1-16.5, q=.04, respectively) cytogenetics. We stratified the ALL cohort into three groups: TP53-mutated ALL, non-TP53 CH-associated ALL, and ALL without myeloid CH mutations. Patients in both groups of CH-associated B-ALL had worse overall survival (OS) than patients without myeloid CH mutations (median OS, 23 vs 25 vs 48 months, respectively, p=.001). In a multivariable analysis of OS for B-ALL, adjusting for age and therapy-related ALL, both TP53 (HR 1.95, 95% CI=1.2-3.17, p=.006) and non-TP53 myeloid CH (HR 1.95, 95% CI=1.14-3.35, p=.01) were associated with worse OS. Based on the data indicating high allelic frequencies of myeloid CH mutations in ALL, we hypothesized that these mutations are ancestral events arising in HSCs. To gain insights into the clonal architecture, we performed single cell DNA+protein sequencing using the Mission Bio, lnc platform. Data from 4,180 cells obtained from a CH-associated B-ALL patient are shown in Fig 1B. Cells were clustered by immunophenotype, and mutations were overlayed into 8 clusters. IDH2 R140Q mutation was shared between ALL and myeloid compartments. PTPN11 mutation was more prevalent in B-lymphoblasts. We observed a similar pattern in 7 additional patients, indicating that both lymphoblasts and non-malignant myeloid cells share CH mutations in ALL patients. Next, we investigated whether CH can be detected as a precursor lesion prior to ALL. In a patient with therapy-related B-ALL, a TET2 CH clone was detectable 5 years prior to ALL, and additional SF3B1 and CDKN2A mutations were detectable at the time of B-ALL diagnosis. We confirmed the presence of these mutations in B-lymphoblasts by single cell sequencing. This patient had persistent TET2/SF3B1 mutated CH despite achieving remission from B-ALL. We observed a similar pattern in four additional patients, for whom the CH mutations were detectable several years prior to their ALL diagnosis and were present in the dominant ALL clone. Collectively, these data support a model in which ALL can arise from pre-existing clones harboring myeloid CH mutations in a significant fraction of older adults. ALL with myeloid mutations is a distinct clinicopathologic entity with inferior survival outcomes. Future studies may decipher mechanisms driving lymphoid bias of CH-mediated leukemogenesis that can be targeted to prevent and treat ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular International Prognostic Scoring System (IPSS-M) in Myelodysplastic Syndromes Arising from Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Clonal evolution to myelodysplastic syndromes (MDS) or acute myeloid leukemia represents the most serious late complication of patients with aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). The majority of secondary MDS (sMDS) cases present high-risk features with chromosome 7 abnormalities and mutations in ASXL1, RUNX1, SETBP1, and RAS pathway genes as the most frequent molecular lesions. Other cases tend to show more favorable outcomes with distinct genomic architecture mainly driven by generally considered low-risk cytogenetic alterations (e.g., del(13q), del(20q)). Such heterogeneity of genomic makeup, the rarity of the disease, and the scarcity of information make the prognostication of sMDS challenging for routine clinical practice. In recognition of the growing role of somatic mutations, the Molecular International Prognostic Scoring System (IPSS-M) has been conceived to improve the precision of prognostication of prior schemes by adding objective molecular markers. Whereas validated on de novo and secondary/therapy-related cases, its applicability specifically on post-AA/PNH MDS has not yet been explored. Here, we applied IPSS-M to 69 sMDS with complete clinico-genomic information from a large multicenter dataset of 1008 AA/PNH patients, using a comparator cohort of primary MDS (n=1281). The median age at MDS progression was 63 years (IQR, 34-70), and most patients were male (M:F ratio 1.65). Despite low bone marrow blast percentages (median, 2; 0-7), 71% of patients showed high-risk features according to IPSS-R (scores >3.5), chiefly due to the enrichment of poor risk cytogenetics, most frequently monosomy 7. Overall, 48% of patients received disease-modifying treatments (DMT) with either HMA (28%), HSCT (60%) or both (12%). Leukemia progression was registered in 14% of cases after a median of 5.4 months (1-14) from sMDS onset. With a median follow-up of 55.6 months (95%CI, 42.5-75.8), the 60-month overall (OS) and leukemia free-survival (LFS) achieved 44% (32-60) and 42% (31-58), respectively. The IPSS-R distribution was as follows: Very Low (6%), Low (23%), Intermediate (30%), High (23%) and Very High (18%). By applying IPSS-M, patients were redistributed in Very Low (5%), Low (19%), Moderate Low (14%), Moderate High (14%), High (29%), and Very High (19%) risk categories. When comparing five-to-five (merging together the Moderate Low/High IPSS-M categories), 55% of cases were re-stratified, of which 63% up- and 37% down-staged (Fig.1A). Indeed, 77% of sMDS patients harbored at least 1 mutation with 43% presenting >2, thereby substantiating the differences observed between the two prognostication systems. Although according to IPSS-M the majority (62%) of sMDS cases still clustered in higher-risk categories, a non-negligible fraction (38%) was assigned to lower-risk groups. To further explore whether IPSS-M could be used to predict OS/LFS also in sMDS cases, we analyzed survival outcomes by taking advantage of a cohort of de novo cases. However, approximately half of sMDS received DMT, potentially generating a bias when using survival as a surrogate for outcomes comparisons, even in cases falling into equivalent risk categories. Therefore, we applied a propensity score matching by using age, gender and DMT as relevant covariates to reduce any possible confounder. As reported for others MDS subtypes in the original IPSS-M study, OS/LFS outcomes of sMDS paralleled those observed in the de novo group within the various IPSS-M risk categories (Fig.1B). We applied for the first time IPSS-M in a real-world cohort of sMDS, showing its potential for refining the assignment of patients into risk categories prognostic of disease outcomes. The actuarial survival of sMDS cases was not distinct from de novo patients matched for basic clinical features in equivalent IPSS-M risk groups. The complexity of the genomic landscape typical of AA/PNH clonal evolution makes sMDS a prototypic model whereby the incorporation of molecular information is of paramount importance to ensure adequate clinical management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Adverse Effect of Myelodysplasia-Related Mutations in De Novo Acute Myeloid Leukemia Is Associated with Higher Variant Allelic Frequency (VAF): A Proposal for a Numeric Cutoff for Variant Allelic Frequency

Mutations in myelodysplasia-related genes ASXL1, BCOR, EHZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 (MRmut) are class-defining alterations in acute myeloid leukemia (AML) in the latest World Health Organization (WHO, with the exception of RUNX1) and International Consensus Classification (ICC) schemes and indicate adverse-risk per European Leukemia Net (ELN) 2022 guidelines, unless occurring in favorable-risk AML. The lower limit of detection (LOD) for most clinical-grade next generation sequencing (NGS) is ~1-2% variant allelic frequency (VAF); however, data on the prognostic significance of MRmut relative to VAF is lacking. No guidelines that provide a numeric cutoff for VAF are available for utilizing these mutations to classify AML. Our aim was to define a clinically meaningful VAF cutoff for MR mutations in de novo AML. We included patients (pts) with de novo, untreated AML with MRmut treated at our institution between 2017 to 2022. We excluded pts with acute promyelocytic leukemia, core binding factor AML, and other ELN adverse-risk categories (adverse karyotype and/or TP53 mutation). NGS at baseline was performed using a panel of 81 genes recurrently mutated in myeloid neoplasms (LOD ~1-2%). We performed a best fit classifier on individual MRmut to determine optimal VAF cutoffs using event-free survival (EFS) and overall survival (OS) as endpoints. VAF for MRmut involving X-linked genes was adjusted for men. Pts (n=145) included 92 (63%) men and 53 (37%) women with a median age of 68 years (range, 58-75). 85 (59%) pts had diploid karyotype AML, 51 (35%) had other intermediate-risk non-diploid karyotype AML, and 9(6%) had insufficient metaphases for analysis. 34 (23%) pts had ELN favorable-risk and 111(77%) had ENL adverse-risk AML. Pts were treated with low-intensity+venetoclax (VEN) (72/50%), low-intensity (26/18%), high-intensity (22/15%), and high-intensity+VEN (12/17%) regimens. Median follow-up was 26.9 months (range, 13.9 - 40.8). 72 (49%) pts had 1 MRmut; the remaining had 2-4 concurrent MRmut. MRmut had the following frequencies and VAFs: ASXL1 (28%; median VAF: 26%, [range 2-48%]); BCOR (18%; median VAF 37% [3-100%]); EHZH2 (9%; median VAF 7% [1-55%]), RUNX1 (32%; median VAF 47% [1-96%]), SF3B1 (9%; median VAF: 38% [1-55%]), SRSF2 (46%; median VAF: 37% [1-76%]), STAG2 (20%; median VAF: 44% [15-98%]), U2AF1 (12%; median VAF: 33% [2-43%]) and ZRSR2 (2%; median VAF: 94% [93-95%]). 34 (23%) pts had ELN favorable-risk AML including 28 (23%) with mutated NPM1 without FLT3-ITD and 6 (4%) with CEBPA bZIP mutations, respectively; in 12/34 pts NPM1 or CEBPA bZIP mutations were subclonal compared with clonal MR mutation(s). A summary of baseline genetic and treatment data is provided in Fig 1A. The overall composite remission rate (CR+CRi) was 88% (64% and 24%, respectively) Median OS was 30.3 months and estimated 12/24 month OS were 77%/60%. The median EFS was 24.6 months and estimated 12/24 month EFS were 65%/51%. Pts with ELN favorable-risk AML with MRmut had longer OS and EFS compared with the adverse-risk group (median OS 34.9 vs 27.5 months, median EFS 34.9 vs 22.9 months) but the difference was not significant (p=0.60 and 0.30, respectively). The VAF cutoff for MRmut associated with EFS and OS, respectively, was: ASXL1: 21%/21%, BCOR 25%/25% , EHZH2 15%/15%, RUNX1 25%/27%, SF3B1 31%/31%, SRSF2 32%/35%, STAG2 40%/21%, U2AF1 44%/44% and ZRSR2 47/47%.There was no significant difference in OS or EFS between pts with 1 MRmut vs those involving ≥2 MR genes. Pts treated with high-intensity, high intensity+ VEN and low intensity+VEN regimens had longer OS (Fig 1B) and EFS compared with pts treated with low intensity regimens without VEN (OS: not reached, 31.7 months, 31.4 months vs 8.9 months, respectively, p<0.001; EFS: not reached, 31.7 months, 29.6 months vs 3.7 months, respectively, p<0.001). Our data suggest that the poor prognostic effect of MRmut in de novo AML is associated with higher VAF and not a binary presence or absence of MRmut. The minimum VAFs associated with worse outcome for each MRmut ranged from 15-47%. Additional analysis to create predictive models is in progress to determine the optimal VAF cutoff when using MRmut to assign AML to the WHO/ICC MR categories and to ELN adverse-risk group. Importantly, our results suggest that pts with MRmut and non-adverse karyotype have significantly better outcomes when treated with high-intensity and/or venetoclax-based treatment regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Utility of Repeated NGS Testing in Myeloid Diagnostic Work Up

Abstract Introduction/Objective NGS is an integral component of diagnostic work-up of myeloid neoplasms, commonly applied at initial diagnosis and for assessment of treatment response. It is less clear how NGS should be repeated at disease follow-up when initial result is negative. In order to assess the utility of repeated NGS testing, we retrospectively analyzed results in patients with multiple tests. Methods/Case Report Myeloid NGS results were collected between 10/2017 and 4/2022 at University of Miami. The results were grouped by patients and arranged by collection date and finding of mutations. First, we identified patients with negative result or a single mutation of common CHIP gene TET2, DNMT3A, or ASXL1 (DAT) in the first available NGS test. Then we divided these patients into negative follow-up group, if all NGS results were the same as the initial test, and positive follow-up group, if any new mutations were detected. The NGS findings were correlated with clinical history and pathology diagnosis. Results (if a Case Study enter NA) Results from 876 patients were analyzed. A total of 362 patients underwent multiple NGS tests; among them, 84 were negative and 14 had a single DAT mutation in the first NGS test. Among the 98 patients, 21 (21.4%) were in the positive follow-up group, with median time-to-positive at 132 days; the other were in the negative follow-up group with median follow-up time at 258 days. Analysis of the patient history showed the positive follow-up group consists 16 patients who were in remission of high-grade myeloid neoplasm at the time of initial testing and relapsed at the time of detection of mutations, 4 patients who acquired alteration of uncertain significance after hematopoietic stem cell transplant, and 1 patient who acquired KRAS mutation corresponding to MDS developed 846 days after the initial testing. Conclusion Positive conversion in repeated NGS testing are mostly associated with relapsed myeloid neoplasm after chemotherapy.

Over expression of mTOR gene predicts overall survival in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) is defined as heterogenous disease, it contains heterogenous leukemic stem cells with various degree of cell differentiation. The perturbation of genes involved in myeloid progenitor cell growth, differentiation and proliferation lead to morphologic dysplasia, maturation arrest, ineffective hematopoiesis hence the cytopenias and propensity to develop into acute myeloid leukemia (AML). Heterogeneous subsets of MDS patients have been defined by their clinical and biologic abnormalities. These different features lead to the development of different prognostic system; however, these approaches are limited in predicting clinical course, and management of patients remains challenging given the uncertainty of the time course of disease progression. It is of importance to identify transcriptomic marker causing maturational and differentiation arrest which could help in understanding the pathogenesis of disease. We have studied differential gene expression profiles (GEPs) in CD34 + marrow cells from myelodysplastic syndrome (MDS) patients (n = 14) and control CD34 + cells using Affymetrix Human Clariom S microarray with 20,000 well annotated genes. We found 4165 genes significantly (p < 0.05) differentially expressed in MDS. Using stringent bioinformatics analyses, we were able to identify few genes (MAPK8, JUNB, mTOR) which were differentially upregulated i.e. 5.39, 73.61 and 2.7 fold change observed in MDS than control and also validated (n = 60) these genes by RT - qPCR. Kaplan - Meier survival analysis indicated that MAPK8 and JUNB could be poor prognostic marker as patients with increased expression showed poor survival, whereas surprisingly mTOR increased expression proved to be good prognostic marker. The correlation analysis showed that the level of gene (MAPK8, JUNB, mTOR) expression was significantly (p ≤ 0.05) associated with frequency of genetic lesions. Interestingly the increased expression of MAPK8 was significantly accompanied with ASXL1 gene mutation. Our study showed an elevation of TNF and AMPK signalling pathways in MDS. TNF signalling might be mediating the proliferative advantage to myeloid clonal cells (mutation carrying cells) over normal cells, whereas, AMPK signalling could be acting as protector against it (favouring normal cells). Hence it would be interesting to explore the functions and pathways associated with mTOR, AMPK, MAPK8 and JUNB in myelopoiesis related diseases like MDS.

Clinical features and survival analysis in non-M(3) acute myeloid leukemia patients with ASXL1 gene mutation

Objective: To examine the survival rates and clinical characteristics of people with newly discovered non-M(3) acute myeloid leukemia (AML) who carry the ASXL1 gene mutation. Methods: From January 2016 to April 2021, the clinical information of patients with newly diagnosed non-M(3) AML at Shandong University's Qilu Hospital was retrospectively examined, and their clinical characteristics and survival were compared and analyzed. Gene mutation was detected by next-generation sequencing. Results: ① The study included 256 AML patients who were initially diagnosed and had complete data, including 47 cases of ASXL1 gene mutation-positive (ASXL1(+)) patients and 209 cases of ASXL1 gene mutation-negative (ASXL1(-)) patients. All patients were divided into three groups: elderly (≥60 years old, n=92) , middle-aged (45-59 years old, n=92) , and young (≤44 years old, n=72) . ②WBC, and age were higher in patients with ASXL1 mutations compared to ASXL1(-) patients, while complete response after the first round of treatment (CR(1)) was lower (P<0.05) . In the elderly group, WBC and the proportion of aberrant cells in nuclear cells in ASXL1(+) patients were higher than those in ASXL1(-) patients (P<0.05) . In the young group, the WBC of ASXL1(+) patients was higher than that of ASXL1(-) patients (z=-2.314, P=0.021) . ③IDH2 mutation and ASXL1 mutation was related (P=0.018, r=0.34) . In ASXL1(+) patients, the proportion of peripheral blasts in the high VAF group (VAF>40% ) was higher than that in the low VAF group (VAF<20% ) , and the proportion of aberrant nuclear cells was higher in the duplication and replacement mutation patients than in the deletion mutation patients (P<0.05) . ④The overall survival (OS) and progression-free survival (PFS) of ASXL1(+) patients were shorter than those of ASXL1(-) patients (median, 10 months vs 20 months, 10 months vs 17 months; P<0.05) . The proportion number of aberrant cells in nuclear cells (≥20% ) , complex karyotypes, and TET2 mutation were all independent risk variables that had an impact on the prognosis of ASXL1(+) patients, according to multivariate analysis (P<0.05) . Conclusion: ASXL1-mutated non-M(3) AML patients have higher WBC in peripheral blood, a higher proportion of aberrant cells in nuclear cells, lower CR(1) rate, and shorter OS and PFS. Additionally, a poor prognosis is linked to higher VAF, duplication, and substitution mutations in the ASXL1 gene, as well as the high proportion of aberrant cells in nuclear cells, complex karyotype, and TET2 mutation.

Significance of bone marrow fibrosis in acute myeloid leukemia for survival in the real-world.

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by the proliferation of myeloid blasts. Bone marrow fibrosis (BMF), characterized by increased deposition of reticulin or collagen fibers, can occur in AML. International authoritative guidelines do not mention AML patients with BMF and the reported studies are inconsistent. Therefore, we retrospectively analyzed the clinical data of newly diagnosed AML patients in our hospital and compared the clinical characteristics, gene mutations and prognosis of AML patients with or without BMF. We found AML patients with BMF tended to be older, were more prone to hepatosplenomegaly, their level of β2-MG was higher and they often had karyotypes associated with a poor prognosis. The proportion of AML patients without BMF was high in the intermediate-risk group and low in the high-risk group. The mutation rates of ASXL1 and TET2 genes were higher and that of CEBPA was lower in the BMF group. Multivariate analysis showed BMF had independent prognostic significance. AML patients without BMF had higher CR/CRi rate, and the time of hematopoietic recovery in patients achieving CR/CRi was longer in BMF group. The degree of BMF, prognostic level and blasts in peripheral blood were independent risk factors for CR/CRi in newly diagnosed AML. AML patients in the BMF group, especially those with BMF ≥ 2, had a lower OS rate. In age<60 years old group, the higher the degree of BMF was, the shorter the median survival time and the lower the OS rate. In age ≥ 60 years old group, the median survival time in the BMF-1 and the BMF-2/3 groups was shorter. For AML with low, intermediate and high risk, there was always a lower OS rate in patients with BMF. The median survival of AML patients decreased with an increasing degree of BMF in different risk stratifications. BMF had no effect on OS of AML patients with HSCT. In conclusion, AML patients with BMF have a poor prognosis, and BMF was an independent prognostic factor for OS. The assessment of BMF was of great significance for the treatment efficacy and prognosis of newly diagnosed AML.

Mutational Landscape of Patients Referred for Elevated Hemoglobin Level.

Background: Since the identification of JAK2 V617F and exon 12 mutations as driver mutations in polycythemia vera (PV) in 2005, molecular testing of these mutations for patients with erythrocytosis has become a routine clinical practice. However, the incidence of myeloid mutations other than the common JAK2 V617F mutation in unselected patients referred for elevated hemoglobin is not well studied. This study aimed to characterize the mutational landscape in a real-world population of patients referred for erythrocytosis using a targeted next-generation sequencing (NGS)-based assay. Method: A total of 529 patients (hemoglobin levels >160 g/L in females or >165 g/L in males) were assessed between January 2018 and May 2021 for genetic variants using the Oncomine Myeloid Research Assay (ThermoFisher Scientific, Waltham, MA, USA) targeting 40 key genes with diagnostic and prognostic implications in hematological conditions (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Results: JAK2 mutations were detected in 10.9% (58/529) of patients, with 57 patients positive for JAK2 V617F, while one patient had a JAK2 exon 12 mutation. Additional mutations were detected in 34.5% (20/58) of JAK2-positive patients: TET2 (11; 19%), DNMT3A (2;3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%), and ZRSR2 (1; 1.7%). Diagnosis of PV was suspected in 2 JAK2-negative patients based on the 2016 World Health Organization (WHO) diagnostic criteria. Notably, one patient carried mutations in the SRSF2 and TET2 genes, and the other patient carried mutations in the SRSF2, IDH2, and ASXL1 genes. Three JAK2-negative patients with elevated hemoglobin who tested positive for BCR/ABL1 fusion were diagnosed with chronic myeloid leukemia (CML) and excluded from further analysis. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and mutations were found in 6% (28/466) of these cases. Conclusion: Mutations other than JAK2 mutations were frequently identified in patients referred for erythrocytosis, with mutations in the TET2, DNMT3A, and ASXL1 genes being detected in 34.5% of JAK2-positive PV patients. The presence of additional mutations, such as ASXL1 mutations, in this population has implications for prognosis. Both the incidence and mutation type identified in patients with secondary erythrocytosis likely reflects incidental, age-associated clonal hematopoiesis of indeterminate potential (CHIP).