ASXL1 Mutations Research Articles

Comprehensive analysis of clinical, pathological, and molecular features in chronic myelomonocytic leukemia: frequent ASXL1 and NRAS mutations and higher mutation burden in myeloproliferative CMML compared to myelodysplastic CMML

Various aspects of myeloproliferative chronic myelomonocytic leukemia (MP-CMML) and myelodysplastic CMML (MD-CMML) have been reported but inconsistencies remain. This study conducted a comprehensive retrospective analysis of clinical, pathological, and molecular data from a cohort of CMML. The results revealed a higher frequency of ASXL1 and NRAS mutations and a greater mutation burden in MP-CMML, characterized by more tier 1 or 2 variants and dominant mutations. Significant genotype-phenotype correlations were observed, including distinct patterns within MD-CMML subgroups. Additionally, NRAS or RUNX1 mutations and an abnormal karyotype were associated with worse overall survival or progression-free survival. These findings underscore the distinct molecular and pathological differences between MP-CMML and MD-CMML, highlighting the more aggressive nature of MP-CMML and the need for tailored treatment strategies.

Dual ASXL1 and CSF3R mutations drive myeloid biased stem cell expansion and enhance neutrophil differentiation.

Mutations in the epigenetic regulator Additional Sex Combs-Like 1 (ASXL1) are frequently observed in chronic neutrophilic leukemia (CNL). CNL is a myeloproliferative neoplasm (MPN) driven by activating mutations in the Colony Stimulating Factor 3 Receptor (CSF3R), which cause excessive neutrophil production. Despite the high rates of co-occurrence, the interplay between ASXL1 and CSF3R mutations in hematopoiesis and leukemia remains poorly understood. Here, we present a new mouse model with both Asxl1Y588X and Csf3rT621I mutations, which recapitulates features of human MPNs. Csf3r-mutant mice exhibit an age-associated depletion of hematopoietic stem cells, which is tempered by adding of Asxl1Y588X. This combination of mutations causes an expansion of myeloid-biased long-term hematopoietic stem cells. As the mice age, they develop neutrophilia, but leukemia is rare, suggesting additional mutations may be required for transformation. Using models of myeloid differentiation, we find that Asxl1 truncation enhances CSF3RT618I-driven neutrophil differentiation, activating inflammatory pathways associated with mature myeloid cell production. Moreover, cells with both mutations have increased H3K4me1 at neutrophil-associated enhancers. Mutant ASXL1 is known to decrease the genome-wide abundance of the repressive histone mark H2AK119ub. While we see the expected decrease in H2AK119ub in Asxl1-mutant cells, this effect is reversed when CSF3R is also mutated, suggesting a complex interplay between these mutations in regulating chromatin dynamics during hematopoiesis. Our findings highlight context-dependent effects of ASXL1 mutation in myeloid disorders and provide insights into the mechanisms underlying neutrophil differentiation in ASXL1 and CSF3R dual mutant MPN.

A mutant ASXL1-EHMT complex contributes to heterochromatin dysfunction in clonal hematopoiesis and chronic monomyelocytic leukemia.

Age-related clonal hematopoiesis (CH) is a premalignant condition associated with inflammatory cardiovascular disease. ASXL1 mutations are very frequent in CH. We show that ASXL1 interacts with EHMT1 and EHMT2, H3K9 methyltransferases that deposit H3K9me1 and me2. Using a mouse model of mutant ASXL1 to recapitulate CH, we found that old ASXL1-mutant mice showed marked expansion of myeloid cells in bone marrow, with decreased H3K9me2/3 and increased expression of transposable elements (TEs) in heterochromatin. In humans, ASXL1-mutant CH progresses to chronic monomyelocytic leukemia (CMML); CMML patient samples showed striking upregulation of many TE families, suggesting that ASXL1 mutations compromise heterochromatin integrity, hence causing derepression of TEs. Targeting heterochromatin-associated proteins and TEs might counter the progression of CH, CMML and other myeloid malignancies.

Disease characteristics and outcomes of acute myeloid leukemia in germline RUNX1 deficiency (Familial Platelet Disorder with associated Myeloid Malignancy).

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM, FPD/AML, RUNX1-FPD), caused by monoallelic deleterious germline RUNX1 variants, is characterized by bleeding diathesis and predisposition for hematologic malignancies, particularly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical data on FPDMM-associated AML (FPDMM-AML) are limited, complicating evidence-based clinical decision-making. Here, we present retrospective genetic and clinical data of the largest cohort of FPDMM patients reported to date. We describe 159 European patients (from 94 families) of whom 134 were evaluable for the development of malignant disease. Sixty developed a hematologic malignancy (44.8%), most frequently AML (36/134, 26.9%) or MDS (18/134, 13.4%). Somatic alterations of RUNX1 by gene mutation (48%) and chromosome 21 aberrations (14.3%) were the most common somatic genetic aberrations in FPDMM-AML, followed by FLT3-ITD mutations (24.1%). Somatic RUNX1 and FLT3-ITD mutations were not detected in FPDMM-associated MDS, suggesting important contributions to leukemic transformation. Remission-induction chemotherapy resulted in complete remission in 80% of FPDMM-AML patients with a 5-year overall survival (OS) of 50.4%. Survival outcome was non-inferior compared to a large cohort of newly diagnosed adult RUNX1-mutated AML (5-year OS 36.6%, p = 0.5), with relatively infrequent concurrent adverse risk somatic aberrations (ASXL1 mutation, monosomal karyotype, monosomy 5/del 5q) in FPDMM-AML. Collectively, data support the notion that step-wise leukemic evolution in FPDMM is associated with distinct genetic events and indicate that a substantial subset of FPDMM-AML patients achieves prolonged survival with conventional AML treatment, including allogeneic stem cell transplant. These findings are anticipated to inform personalized clinical decision-making in this rare disorder.

CSF3R Gln776X and ASXL1 mutations in multiple myeloma associated with chronic neutrophilic leukemia: case report and literature review

CSF3R Gln776X and ASXL1 mutations in multiple myeloma associated with chronic neutrophilic leukemia: case report and literature review

Proteomic and metabolomic exploration in relapse acute myeloid leukemia bone marrow supernatant combined with genetic characteristics

ObjectAim to investigate the multi-omic characteristics of the bone marrow supernatant of relapsed acute myeloid leukemia (AML) and search for proteins and metabolites associated with relapse.MethodsA total of 40 bone marrow supernatant from 7 patients with relapsed AML and 33 patients with non-relapsed AML were collected for proteomics and metabonomics analysis. Unsupervised clustering was used to discover the characteristics of proteins and metabolites. The prognostic significances of proteins were assessed concerning the relapse status(including death) and relapse-free survival.ResultTotally 996 proteins and 4,831 metabolites were identified in bone marrow supernatant, and two of 7 clusters were revealed through unsupervised clustering and were associated with ASXL1, TP53, and RUNX1 mutations, which were listed as high-risk factors in the 2022 edition of the WHO classification of tumors of the hematopoietic and lymphoid tissues. Among the identified proteins and metabolites, 57 proteins and 190 metabolites were found to be closely related to relapse.ConclusionThis study has revealed a significant correlation between protein expression in the bone marrow microenvironment of AML and three high-risk mutations: ASXL1, TP53, and RUNX1. Based on this finding, we further identified 227 differential proteins closely associated with these three mutations, as well as 57 proteins directly related to disease recurrence. Additionally, lipid metabolism plays a crucial role in the occurrence and development of AML within its bone marrow microenvironment.

ASXL1 mutation accelerates atherosclerosis by promoting activation of myeloid cells.

ASXL1 mutation accelerates atherosclerosis by promoting activation of myeloid cells.

Results of various somatic mutations detection in patients with chronic myeloid leukemia

Background. Somatic mutations in chronic myeloid leukemia (CML) patients are considered as possible factors for the failure of tyrosine kinase inhibitor (TKI) therapy, and the study of their characteristics is of interest.Aim. To evaluate the genetic profile of blood cells in CML patients using nextgeneration sequencing.Materials and methods. Retrospective study was conducted in two groups of patients: group 1 with TKI therapy failure (n = 29) and group 2 with optimal response to TKI therapy (n = 29). The target panel for nextgeneration sequencing included 19 genes: ASXL1, DNMT3A, FLT3, IDH1, IDH2, NPM1, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF2, KIT, WT1, CEBPA, ZRSR2, JAK2, GATA2, ABL1. In order to assess clonal evolution, additional samples were examined at a retrospective point in time closest to the primary CML diagnosis.Results. In group 1, mutations in 8 genes (including ABL1) were identified in 19/29 (66 %) patients. Excluding ABL1, mutations were identified in 15 (52 %) patients. In 9 (31 %) patients, >1 mutation (2 to 4) was detected. Frequency of genes mutations in group 1: ABL1 in 11 (38 %) patients, ASXL1 in 9 (31 %) patients, DNMT3A in 3 (10 %) patients, RUNX1, CEBPA in 2 patients (7 %), WT1, NPM1, TET2 in 1 patient (3.5 %). In 7 (24 %) patients there was a combination of mutations in ABL1 gene and in another gene; the most frequent combination of mutations in genes: ABL1 + ASXL1 – in 4 patients (14 %). The dynamics of mutant clones in group 1 was evaluated in 21/29 (72 %) patients. In 10/21 (48 %) patients somatic mutations in genes appeared during CML treatment, in 14/21 (67 %) patients previously detected mutations persisted, in 1 (5 %) the mutation disappeared. In group 2, somatic mutations were detected in 2/29 (7 %) patients: in DNMT3A (ariant Allele Frequency (AF) 5 %) and TP53 (AF 9 %) genes – these mutations were not detected at the diagnosis of CML. In one patient ASXL1 mutation (AF 5 %) was detected only at diagnosis, and was not detected subsequently with optimal response to therapy.Conclusion. The presence of somatic gene mutations is associated with a resistant CML course: somatic mutations in genes other than ABL1 were more common in CML patients with TKI therapy failure than in those with optimal response: 52 % vs. 7 % (p ≤0.05). Mutations in ASXL1 (31 %) and DNMT3A (10 %) were the most frequently detected. The frequency of ABL1 and ASXL1 mutations combination amounted to 14 %. uring followup, somatic mutations predominantly persisted or appeared over time in CML patients with TKI therapy resistance.

Leveraging Tumor Mutation Profiles to Forecast Immune Checkpoint Blockade Resistance in Melanoma, Lung, Head and Neck, Bladder and Renal Cancers

Immune checkpoint blockade (ICB), radiotherapy, chemotherapy and surgery are currently used as therapeutic strategies against melanoma, lung, bladder and renal cancers, but their efficacy is limited. Thus, I need to predict treatment response and resistance to address this challenge. In this study, I analyzed 350 lung cancer, 320 melanoma, 215 bladder cancer, 139 head and neck cancer and 151 renal carcinoma patients treated with ICB to identify tumor mutations associated with response and resistance to treatment. I identified several tumor mutations linked with a difference in survival outcomes following ICB. In lung cancer, missense mutations in ABL1, ASXL1, EPHA3, EPHA5, ERBB4, MET, MRE11A, MSH2, NOTCH1, PAK7, PAX5, PGR, ZFHX3, PIK3C3 and REL genes were indicative of favorable responses to ICB. Conversely, mutations in TGFBR2, ARID5B, CDKN2C, HIST1H3I, RICTOR, SMAD2, SMAD4 and TP53 genes were associated with shorter overall survival post-ICB treatment. In melanoma, mutations in FBXW7, CDK12, CREBBP, CTNNB1, NOTCH1 and RB1 genes predict resistance to ICB, whereas missense mutations in FAM46C and RHOA genes are associated with extended overall survival. In bladder cancer, mutations in HRAS genes predict resistance to ICB, whereas missense mutations in ERBB2, GNAS, ATM, CDKN2A and LATS1 genes, as well as nonsense mutations in NCOR1 and TP53 genes, are associated with extended overall survival. In head and neck cancer, mutations in genes like PIK3CA and KRAS correlated with longer survival, while mutations in genes like TERT and TP53 were linked to shorter survival. In renal carcinoma, mutations such as EPHA5, MGA, PIK3R1, PMS1, TSC1 and VHL were linked to prolonged overall survival, while others, including total splice mutations and mutations in B2M, BCOR, JUN, FH, IGF1R and MYCN genes were associated with shorter overall survival following ICB. Then, I developed predictive survival models by machine learning that correctly forecasted cancer patient survival following ICB within an error between 5 and 8 months based on their distinct tumor mutational attributes. In conclusion, this study advocates for personalized immunotherapy approaches in cancer patients.

Clonal hematopoiesis-related mutant ASXL1 promotes atherosclerosis in mice via dysregulated innate immunity.

Certain somatic mutations provide a fitness advantage to hematopoietic stem cells and lead to clonal expansion of mutant blood cells, known as clonal hematopoiesis (CH). Among the most common CH mutations, ASXL1 mutations pose the highest risk for cardiovascular diseases (CVDs), yet the mechanisms by which they contribute to CVDs are unclear. Here we show that hematopoietic cells harboring C-terminally truncated ASXL1 mutant (ASXL1-MT) accelerate the development of atherosclerosis in Ldlr-/- mice. Transcriptome analyses of plaque cells showed that monocytes and macrophages expressing ASXL1-MT exhibit inflammatory signatures. Mechanistically, we demonstrate that wild-type ASXL1 has an unexpected non-epigenetic role by suppressing innate immune signaling through the inhibition of IRAK1-TAK1 interaction in the cytoplasm. This regulatory function is lost in ASXL1-MT, resulting in NF-κB activation. Inhibition of IRAK1/4 alleviated atherosclerosis driven by ASXL1-MT and decreased inflammatory monocytes. The present work provides a mechanistic and cellular explanation linking ASXL1 mutations, CH and CVDs.

Mutation Analysis of ASXL1 in Normal Karyotype Myelodysplastic Syndromes: Experience From Pakistan

ABSTRACTBackgroundThe challenges in advancing treatment modalities for myelodysplastic syndromes (MDS) may stem from an incomplete understanding of the disease's complex pathophysiology and lack of reliable prognostic molecular markers. Advanced genomic techniques have revolutionized disease prognosis and risk stratification, particularly for cases with a normal karyotype.AimThe present study aimed to analyze ASXL1 mutations in MDS exhibiting a normal karyotype.Methods and ResultsThe study enrolled 41 MDS patients with normal karyotypes. Genomic DNA was extracted from peripheral blood samples, followed by Sanger sequencing. The Chi‐square and Mann–Whitney U tests were applied to assess the association of age and hemogram with the occurrence of mutations. Survival analysis was done via the Kaplan–Meier method. Statistical operations were performed employing the IBM SPSS Statistics version 24. The patients had a median age of 40 years comprising 28 (68.3%) males and 13 (31.7%) females. ASXL1 mutations were identified in 8 (19.5%), particularly stopgain single nucleotide variant (SNV) and frameshift insertion. The median total leucocyte count × 109/L and blast percentage among the patients with ASXL1 mutation were 3.9 and 4.5, respectively. A survival of 85 weeks was recorded for ASXL1‐mutated and nonmutated cases. The association of ASXL1 mutation status with age and studied hematological parameters was found nonsignificant.ConclusionASXL1 mutation plays a pivotal role in MDS prognosis, warranting assessment at diagnosis for risk stratification and treatment decisions. Early identification of ASXL1 mutation, particularly in low‐risk patients or those with normal karyotype, is imminent to identify patients exhibiting unfavorable prognosis. Integrating molecular insights into existing risk assessment holds significance for improved clinical outcomes, reduction in disease progression, and ultimately the improved quality of life and should be identified early in the course of disease even in the developing world.

Pretransplant Chromosome Genomic Array Testing Improves Prognosis for Myelofibrosis Patients Undergoing Transplantation.

Despite its known superior diagnostic yield for chromosomal anomalies compared with karyotype and fluorescence in situ hybridization (FISH) studies, chromosome genomic array testing (CGAT) is not used as a routine clinical test for myelofibrosis. Although many prognostic systems exist that risk stratify patients at diagnosis, limited tools are available to prognosticate transplant outcome. The current study aimed at testing whether CGAT results obtained before transplantation improves prognosis of post-transplant outcome in patients with myelofibrosis compared with current risk categorization systems, for example, DIPSS plus (Dynamic International Prognostic Scoring System). We studied patients with myelofibrosis who underwent hematopoietic cell transplantation between 2000 and 2017 at our center (N = 44). We assessed the prognostic significance of CGAT, DIPSS plus, and the total count of gene mutations for post-transplant clinical outcomes, including relapse-free survival (RFS), overall survival (OS), and graft-versus-host-disease (GVHD). Abnormal CGAT results were seen in 24 patients (55%), including 18 with copy-neutral loss of heterozygosity (cnLOH, 41%). With a median follow-up of 91 (range 2-258) months starting from the CGAT sample date, RFS was 59% and OS was 68%. The outcome analysis showed significant prognostic implication from CGAT (normal vs. abnormal), specifically for patients with intermediate risk by DIPSS-plus scores and those with 0∼2 mutations. CGAT alone significantly stratified the patients' RFS outcome (P = .03). The addition of CGAT to DIPSS-plus improved the significance from a P value of .08 to .003, whereas the addition of CGAT to mutation count improved the P value from .02 to .01. The best stratification system for RFS was achieved when CGAT, DIPSS-plus, and mutation count were all considered (P = 1e-08). The current study also confirmed individual anomalies that are prognostically significant, including U2AF1 mutation (n = 5, P = .03) and 1q gain (n = 3, P = .01), which were associated with worse RFS. ASXL1 mutations (n = 14) appeared to associate with a later onset of chronic GVHD (P =.03). Pretransplant CGAT analysis augments the existing risk stratification tools and may be considered as routine clinical testing for myelofibrosis.

Elevated serum direct bilirubin is predictive of a poor prognosis for primary myelodysplastic syndrome

BackgroundThe aim of this study was to assess the prognostic significance of serum direct bilirubin (DBIL) for patients newly diagnosed with myelodysplastic syndromes (MDS).MethodsThe clinical, laboratory, and follow-up data of MDS patients were collected, and the associations of DBIL levels with overall survival (OS) and leukemia-free survival (LFS) were analyzed.ResultIn total, 262 MDS patients were assigned to the high DBIL level group or the normal DBIL level group in the retrospective study. High DBIL was associated with older age, reduced hemoglobin, higher levels of β2-microglobin, lactate dehydrogenase, and serum ferritin, along with the number of co-mutations (> 1) and a higher frequency of ASXL1, KIT, and KRAS mutations. Multivariate analyses found that high DBIL level was an independent adverse predictor for OS (p = 0.002, hazard ratio = 2.723, 95%CI = 1.442–5.143) but not for LFS (p = 0.057, hazard ratio = 1.678, 95%CI = 0.986–2.857). A novel nomogram based on DBIL, sex, age, β2-microglobulin, lactate dehydrogenase, the Revised International Prognostic Scoring System (IPSS-R) was constructed, which demonstrated superior accuracy compared with the IPSS-R (C-index, 0.790 vs. 0.731, respectively).ConclusionAn elevated DBIL level was identified as an independent adverse prognostic factor for MDS patients. An individualized prediction model was established and validated to improve prediction of OS and LFS.

DNMT3A, TET2, and ASXL1 (DTA) Mutations Are Associated with Thrombosis and Bleeding in Patients with Myeloproliferative Neoplasms

DNMT3A, TET2, and ASXL1 (DTA) Mutations Are Associated with Thrombosis and Bleeding in Patients with Myeloproliferative Neoplasms

The Combination of Asciminib with ATP Competing Tyrosine Kinase Inhibitors Might Overcome the Negative Impact of ASXL1 Mutations on Molecular Response in Newly Diagnosed CML Patients

The Combination of Asciminib with ATP Competing Tyrosine Kinase Inhibitors Might Overcome the Negative Impact of ASXL1 Mutations on Molecular Response in Newly Diagnosed CML Patients

Prognostic impact of 'multi-hit' versus 'single-hit' TP53 alteration in patients with acute myeloid leukemia: results from the Consortium on Myeloid Malignancies and Neoplastic Diseases.

While there is clear evidence to suggest poorer outcome associated with multi-hit (MH) TP53 mutation (TP53MT) compared to a single-hit (SH) mutation in lower-risk myelodysplastic syndrome (MDS), data are conflicting in both higher-risk MDS and acute myeloid leukemia (AML). We conducted an in-depth analysis utilizing data from ten US academic institutions to study differences in molecular characteristics and outcomes of SH (N=139) versus MH (N=243) TP53MT AML. Complex cytogenetics were more common in MH than in SH TP53MT AML (P<0.001); whereas ASXL1 (P<0.001), RAS (P<0.001), splicing factor (P=0.003), IDH1/2 (P=0.001), FLT3 ITD (P<0.001) and NPM1 (P=0.005) mutations clustered significantly with SH TP53MT AML. Survival after excluding patients who received best supportive care alone was dismal but not significantly different between patients with SH or MH disease (event-free survival: 3.0 vs. 2.20 months, respectively, P=0.22; overall survival: 8.50 vs. 7.53 months, respectively, P=0.13). In multivariable analysis, IDH1 mutation and allogeneic hematopoietic stem cell transplantation as a time-dependent covariate were associated with superior event-free survival (hazard ratio [HR]=0.44, 95% confidence interval [95% CI]: 0.19-1.01, P=0.05 and HR=0.34, 95% CI: 0.18-0.62, P<0.001) and overall survival (HR=0.24, 95% CI: 0.08-0.71, P=0.01 and HR=0.28, 95% CI: 0.16-0.47, P<0.001). Complex cytogenetics (HR=1.56, 95% CI: 1.01-2.40, P=0.04) retained an unfavorable significance for overall survival. Our analysis suggests that MH TP53MT is less relevant in independently predicting outcomes in patients with AML than in those with MDS.

Prognostic Significance and Treatment Response Associations of Genetic Mutations in Chronic Myelomonocytic Leukemia: A Retrospective Cohort Study.

Background: This retrospective cohort study investigates the prognostic significance of genetic mutations in Chronic Myelomonocytic Leukemia (CMML) and their association with treatment responses among patients treated at a single institution, juxtaposed with a statewide dataset from Kentucky. Methods: The study includes 51 patients diagnosed with CMML under the World Health Organization criteria from January 2005 to December 2023. It examines their genomic profiles and subsequent survival outcomes. The analysis also categorizes patients into CMML-1 and CMML-2 subtypes and assesses survival differences between transformed and non-transformed cases. Results: Mutations in TET2, ASXL1, and SRSF2 were found to significantly influence survival, establishing their roles as critical prognostic markers. Additionally, the cohort from the University of Kentucky exhibited distinct survival patterns compared to the broader Kentucky state population, suggesting that demographic and treatment-related factors could underlie these variances. Conclusions: This research underscores the pivotal role of targeted genetic profiling in deciphering the progression of CMML and refining therapeutic strategies. The findings emphasize the necessity for advanced genetic screening in managing CMML to better understand individual prognoses and optimize treatment efficacy, thereby offering insights that could lead to personalized treatment approaches.

Cohesin RAD21 Gene Promoter Methylation in Patients with Acute Myeloid Leukemia.

Aberrant gene promoter methylation is one of the hallmarks of Acute Myeloid Leukemia (AML). RAD21 is an important gene, implicated in sister chromatids cohesion, DNA repair, the regulation of gene transcription, apoptosis and hematopoiesis. In this study, we investigate the possible implication of RAD21 promoter methylation in AML pathogenesis using a cohort of AML patients and a cohort of healthy individuals. RAD21 promoter methylation was found in 24% of patients and in none of the controls (p = 0.023), indicating a possible contribution to AML development. Interestingly, a statistically higher frequency of RAD21 methylation was observed in patients with trisomy 8 (9/21, 42.9%, p = 0.021), while none of the patients with aberrations of chromosome 11 had RAD21 gene promoter methylation (0%, 0/11, p = 0.048). Patients with monosomal and complex karyotypes showed low frequencies of RAD21 methylation (7.7% and 15.4%, respectively) without reaching statistical significance. Moreover, ASXL1 mutations were not found to be associated with RAD21 methylation. This is the first study which provides evidence for a possible pathogenetic role of RAD21 promoter methylation in AML development and especially in AML with trisomy 8. Further studies of RAD21 promoter methylation in large series of different AML genetic subgroups may contribute to the elucidation of AML pathogenesis and to the identification of new epigenetic biomarkers with diagnostic and prognostic value.

Myeloid Sarcoma of Conjunctiva in a patient with JAK2-positive myeloid neoplasm: two distinct myeloid neoplasms sequentially arising from a common precursor

Abstract Introduction/Objective Myeloid sarcoma (aka granulocytic sarcoma or chloroma), a rare type of myeloid neoplasm is characterized by the presence of myelogenous blastic cells outside of the bone marrow either before, during or after the development of leukemia. Presentation in the orbit is extremely rare, unlike other body parts. We report a case of myeloid sarcoma of conjunctiva in a patient with JAK2-positive myeloid neoplasm. Methods/Case Report An 84-year-old man presented with bilateral conjunctival masses which on biopsy showed diffuse involvement by blasts, immunohistochemically expressing positivity for myeloperoxidase, CD33, CD68, CD34 (partial), CD117 (partial) but were non-reactive to CD15, CD3 and PAX5. Next generation sequencing (NGS) revealed mutations in NRAS, ASXl1 and TET2 genes, consistent with the diagnosis of myeloid sarcoma (MS) of the conjunctiva. While patient’s past medical history included status post radiation and chemotherapy for prostate cancer and colonic adenocarcinoma respectively. it was also significant for a JAK 2 positive myeloproliferative neoplasm (MPN), six-years prior to current presentation. At the time he was treated with hydroxyurea for 5 years but developed neutrophilic leukocytosis and transfusion-dependent anemia. Subsequently a treatment regimen including ruxolitinib and pacritinib was initiated but without clinical and hematological improvements. NGS of the initial JAK2-positive MPN revealed ASXL1, JAK2 and TET2 mutations. Of note are the similarities and variations in the molecular signatures of patient’s initial MPN and current conjunctival MS. While the two neoplasms carried an identical TET2 mutation with highest variant allelic frequency, they are different in other mutations. Results (if a Case Study enter NA) NA Conclusion Our case illustrates an unusual manifestation of myeloid sarcoma localized to the conjunctiva. The identification of clonal relatedness on NGS with distinct mutations, adds complexity to our understanding of tumor origin. It highlights a series of events which may have contributed to this phenomenon, including consequence of therapy, clonal evolution with molecular discordance or origin as a totally independent entity. This report underscores the importance of genetic profiling in guiding prognosis and treatment strategies in such settings.

Generation and Characterization of Induced Pluripotent Stem Cells Carrying An ASXL1 Mutation.

Additional sex combs-like 1 (ASXL1) is an epigenetic modulator frequently mutated in myeloid malignancies, generally associated with poorprognosis. Current models for ASXL1-mutated diseases are mainly based on the complete deletion of Asxl1 or overexpression of C-terminal truncations in mice models. However, these models cannot fully recapitulate the pathogenesis of myeloid malignancies. Patient-derived induced pluripotent stem cells (iPSCs) provide valuable disease models that allow us to understand disease-related molecular pathways and develop novel targeted therapies. Here, we generated iPSCs from a patient with myeloproliferative neoplasm carrying a heterozygous ASXL1 mutation. The iPSCs we generated exhibited the morphology of pluripotent cells, highly expressed pluripotent markers, excellent differentiation potency in vivo, and normal karyotype. Subsequently, iPSCs with or without ASXL1 mutation were induced to differentiate into hematopoietic stem/progenitor cells, and we found that ASXL1 mutation led to myeloid-biased output and impaired erythroid differentiation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that terms related to embryonic development, myeloid differentiation, and immune- and neural-related processes were most enriched in the differentially expressed genes. Western blot demonstrated that the global level of H2AK119ub was significantly decreased when mutant ASXL1 was present. Chromatin Immunoprecipitation Sequencing showed that most genes associated with stem cell maintenance were upregulated, whereas occupancies of H2AK119ub around these genes were significantly decreased. Thus, the iPSC model carrying ASXL1 mutation could serve as a potential tool to study the pathogenesis of myeloid malignancies and to screen targeted therapy for patients.