SETBP1 Mutations Research Articles

High Mobility Group A1 Chromatin Remodeling Proteins: Potential Therapeutic Targets Involved in Polycythemia Vera Transformation to Acute Leukemia in Humans and JAK2 V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although the mechanisms driving disease progression remain elusive. Polycythemia vera (PV) is the most common clinical subtype caused by acquired mutations of JAK2. While PV generally presents as an indolent process, ~25% of patients will progress to MF or AML and JAK2 mutations alone do not account for MPN transformation.HMGA1 / 2 genes encode oncogenic chromatin binding proteins that are overexpressed in acute leukemia and drive clonal expansion in murine leukemia models. In preliminary studies, we found up-regulation in gene expression of both HMGA1/2 in HSC with disease progression in MPN. We therefore sought to test the hypothesis that HMGA proteins are critical drivers of transformation and rational therapeutic targets to treat or prevent PV progression.Methods: We examined clonal evolution in HSC from 49 JAK2V617F -positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes linked to myeloid cancers. HSC clonal burden was assessed via JAK2V617F genotype analysis on single cells. We also measured HMGA1 / 2 in JAK2V617F -positive human AML cell lines from MPN patients (PV-AML cells), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F / MPLSV ; Blood 2015;126:484) using real-time quantitative RT-PCR. To elucidate the role of HMGA1/2, we silenced HMGA1 or HMGA2 via short hairpin RNA and assessed proliferation in human PV-AML cells. Finally, to investigate Hmga1 function in vivo, we crossed mice deficient in Hmga1 onto murine models of PV and PV-AML. We also compared leukemic engraftment in JAK2V617F -positive PV-AML cells with or without HMGA1/2 silencing.Results: Both HMGA1/2 mRNA were up-regulated in all JAK2V617F -positive contexts. In primary human PV CD34+ cells, HMGA1 and HMGA2 were increased by 7- and 100-fold, respectively, compared to controls. There was a dramatic up-regulation in both HMGA1/2 in HSC of patients who transformed from PV to MF or AML compared to chronic phase PV when analyzed in cross-sectional samples from different patients or prospectively in selected patients. Overexpression of HMGA1 /2 also correlated with clonal dominance of JAK2V617F -homozygous stem cells, and additional mutations of epigenetic regulators, including EZH2 and SETBP1 . Similarly, when assessed in unfractionated bone marrow or tumor samples in transgenic mouse models for PV and PV-AML, Hmga1 / 2 were overexpressed compared to control littermates, with highest levels in the PV-AML mouse model. Silencing HMGA1 or HMGA2 in human PV-AML cells (DAMI, SET-2) dramatically halted proliferation. Moreover, leukemic engraftment was prevented in PV-AML cells after silencing HMGA1 or HMGA2. In preliminary studies with the transgenic PV-AML murine model, Hmga1 deficiency ameliorates the fulminant phenotype of aggressive leukemia and early mortality. PV-AML mice survived a median of 5 weeks (range 3-10; n=40) whereas PV-AML in Hmga1 deficient backgrounds survived a median of 8 weeks (range 7-16; n=7). The leukemic burden was decreased in mice with Hmga1 deficiency.Conclusions:HMGA1 and HMGA2 are overexpressed in MPN, and higher levels associate with disease progression to MF and AML, in both human PV and in transgenic murine models of PV. Strikingly, silencing HMGA1 or HMGA2 halted proliferation and prevented leukemic engraftment in vivo . Preliminary data in Hmga1 deficient models suggest that Hmga1 is required for PV progression to AML. These data highlight a key role for HMGA proteins as critical drivers of PV transformation and suggest that HMGA1 /2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1 /2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. DisclosuresNo relevant conflicts of interest to declare.

Mutational Pattern and Clonal Hierarchy in MDS with Monosomy 7

Background: Monosomy 7 (-7) is a typical abnormality in MDS with a frequency of ~ 10%. -7 either occurs as the sole abnormality (~4%), accompanied by one additional abnormality (~1%) or in a complex karyotype (~5%). A detailed molecular analysis of MDS with -7 as the sole abnormality (-7 sole) has not been performed to date.Aims: Evaluation of molecular mutations in MDS with -7 sole and analysis of the clonal hierarchy.Patient cohorts and methods: 100 patients (pts) with MDS at diagnosis harboring -7 sole in chromosome banding analysis (59 male, 41 female) with available cytomorphology and material for mutation screening were selected. Median age was 70 years (range: 21-89 years). 86 pts had de novo and 14 pts therapy-related MDS. Blast count was <5% in 29 MDS and ≥5% in 71 MDS pts. 97 pts were also analyzed by interphase FISH using a probe for the centromeric region of chromosome 7. Targeted sequencing of 29 genes commonly mutated in myeloid malignancies was performed in all 100 pts (Illumina, San Diego, CA). Variants of unknown significance were excluded from statistical analysis.Results: The most frequently mutated genes were: ASXL1 (42%), SETBP1 (31%), U2AF1 (29%), DNTM3A (27%), RUNX1 (17%), ETV6 (14%), EZH2 (14%), TET2 (12%), SRSF2 (10%), CBL (9%), PTPN11 (9%), NF1 (6%), CSF3R (4%), JAK2 (4%), TP53 (3%), KRAS (3%), NRAS (3%), and MPL (3%). Genes mutated in 2% or less of pts were: GATA2, IDH1, IDH2, KIT, CALR, ETNK1, NPM1, SF3B1, and WT1 . No mutations were observed in CSNK1A1 and GATA1 (figure 1).RUNX1 mutations were significantly more frequent in t-MDS as compared to de novo MDS (43% vs 13%, p=0.02), while ASXL1 mutations were more frequent in MDS with blast count <5% (60% vs 41%, p=0.04).A median of 2 mutations per case were observed (range 0-6), with 94 pts harboring at least 1 mutation. No association between age and the number of mutations was observed. However, mutations in ETV6, SETBP1, U2AF1 and PTPN11 were associated with a higher number of mutations, while this was not the case for other gene mutations. In pts with 0-2 mutations ETV6, SETBP1, U2AF1 and PTPN11 were mutated in 4%, 15%, 14%, and 2%, respectively, while the respective mutation frequencies were 27%, 49%, 47%, and 19% in pts with >2 mutations (for all comparisons p<0.01).The DNMT3A mutation frequency was low in ASXL1 mutated pts as compared to ASXL1 wild type (wt) pts (8% vs 42%; p<0.0001), while RUNX1 mutations were more frequent in ASXL1 mutated pts (28% vs 10%, p=0.03). U2AF1 and ETV6 mutations frequently co-occurred as 67% of ETV6 mutated pts also harboured a U2AF1 mutation (p=0.002) while only 21% of ETV6 wt pts showed U2AF1 mutations. In addition, the following mutation combinations were more frequent than expected U2AF1 / ETV6 (p=0.002), RUNX1 / EZH2 (p=0.04), EZH2 / TET2 (p=0.04), SETBP1 / U2AF1 (p=0.01), SETBP1 / PTPN11 (p=0.02), and U2AF1 / TP53 (p=0.02), while the following mutation combinations co-occurred less frequently than expected: EZH2 / SETPB1 (p=0.05), RUNX1 / SETBP1 (p=0.02), and SRSF2 / U2AF1 (p=0.02).For clonal architecture analysis the clone size of cells harboring -7 was related to the mutation loads of the 11 most frequently mutated genes. The proportion of cells harboring -7 determined by FISH analyses varied between 6% and 95% (median: 57%). The -7 clone size was significantly correlated to the mutation loads of DNMT3A (r=0.67, p<0.001), RUNX1 (r=0.65, p=0.003), PTPN11 (r=0.71, p=0.02), and NF1 (r=0.81, p=0.01) indicating that -7 and the respective mutation were present together in the same clone. The mutation loads of the following mutations were significantly correlated: ASXL1 / PTPN11 (r=0.95, p=0.01), ASXL1 / U2AF1 (r=0.58, p=0.04), ETV6 / SETBP1 (r=0.91, p=0.005), and ETV6 / U2AF1 (r=0.87, p=0.03). Estimated based on clone size and mutation load -7 was present in the main clone (MC) in 61% of cases and in the subclone (SC) in 39%. Most mutations occurred in the MC as well as in the SC at comparable frequencies (ASXL1, EZH2, RUNX1, SETBP1, SRSF2). Mutations in ETV6 and PTPN11 were present slightly more frequently in a SC only (64% and 60%), while mutations in CBL, DNMT3A, TET2 and U2AF1 were found rarely in a SC only (33%, 18%, 18% and 17%) (figure 2).Conclusions: A broad spectrum of molecular mutations is present in MDS with -7 sole with the highest mutation frequencies in ASXL1, SETBP1, U2AF1, DNMT3A and RUNX1 . No clear hierarchy of -7 and mutations was identified, however, in most cases if mutated DNMT3A, TET2 and U2AF1 are present in the main clone. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Targeted Deep Sequencing in Blast Phase Myeloproliferative Neoplasms

Background: We have previously published our observations regarding targeted next generation sequencing (NGS) in primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV) (Blood Advances 2016 1:105; Blood Advances 2016 1:21). In the current study, we took a similar approach in order to examine the mutational repertoire in blast phase myeloproliferative neoplasms (MPN-BP) and explore its clinical correlates, including phenotype, treatment response and survival.Methods:Diagnoses of MPN, including PMF, PV, ET and MPN-BP were according to WHO criteria (Blood 2016;127:2391). The study population was selected on the basis of the availability of sufficient DNA and was not otherwise biased by any other selection criteria. Targeted capture assays were carried out on bone marrow or whole blood DNA, according to previously published methods (Blood Advances 2016 1:105). Statistical analyses considered clinical and laboratory data collected at the time of blast transformation. Survival was calculated from the date of leukemic transformation to the date of death or last contact. Treatment response in MPN-BP was assessed according to conventional criteria (JCO 2003;21:4642).Results:72 consecutive cases of MPN-BP underwent target capture NGS using a panel of 39 genes. •Mutational landscape, frequencies and co-segregation patternsDriver mutations were detected in 62 (86%) patients, including JAK2 only in 39 (54%), JAK2 + CALR 3 (4%), JAK2 + MPL 3 (4%), CALR only 10 (14%), CALR + MPL 2 (3%) and MPL only 5 (7%). Sixty-six (92%) patients displayed at least one mutation/DNA variant, other than JAK2 / CALR / MPL ; the number (%) of patients with 0, 1, 2, 3, 4, 5, 6 or 7 such mutations was 6 (8%), 9 (13%), 19 (26%), 19 (26%), 13 (18%), 5 (7%), 0 and 1 (1.4%). The most frequent mutations (incidence), other than JAK2 / CALR / MPL were ASXL1 (46%), RUNX1 (22%), TET2 (21%), TP53 (17%), EZH2 (15%), IDH1 (14%), SRSF2 (14%), FLT3 (13%), and SH2B3 (11%). JAK2 mutations clustered with ASXL1 (p=0.02); MPL with IDH1 (p=0.0004) and SF3B1 (p=0.008); ASXL1 with EZH2 (p=0.009); RUNX1 with SRSF2 (p=0.02) and SH2B3 (p=0.04); SRSF2 with IDH2 (p=0.002); IDH2 with SH2B3 (p=0.04); and EZH2 with DNMT3A (p=0.0007). •Clinical course and managementAt the time of this writing, complete clinical and follow-up data were available in 66 patients with MPN-BP (median age 65 years; 65% males). Induction chemotherapy for MPN-BP was given to 38 patients, resulting in complete remission (CR) or CR with incomplete recovery of counts (CRi) in 14 (37%) patients. Treatment for MPN-BP included allogenic stem cell transplant (ASCT) in 6 patients. Median follow-up from time of leukemic transformation was 3.9 months with 64 documented deaths (97%). •Impact of mutations on survival and treatment response ratesIn univariate analysis, adverse impact on survival was demonstrated for TET2 (p=0.03), DNMT3A (p=0.05), PTPN11 (p=0.008) and SETBP1 (p=0.01) mutations, which were subsequently grouped as “adverse mutations”. Twenty-one (32%) patients harbored at least one adverse mutation. Patients with or without adverse mutations were similar in terms of their age and gender distribution, clinical characteristics at time of leukemic transformation and karyotype (P>0.05 in all instances). Spectrum and frequency of adverse mutations were similar among specific driver mutations, including triple-negative cases (p=0.97).The presence of adverse mutations was strongly predictive of shortened survival (HR 2.8, 95% CI 1.6-5.0; p=0.0003; Figure 1) and the effect was independent of several other clinical and laboratory variables including age, karyotype, specific MPN-BP morphologic category, blast burden and treatment undertaken for MPN-BP. In multivariable analysis, survival was independently affected by presence of adverse mutations (HR 4.8, 95% CI 2.0-11.8) and achievement of CR/CRi (HR 0.4; 95% CI 0.2-0.8), but not by ASCT. The presence of adverse mutations did not affect response rate to induction chemotherapy (p=0.6).Conclusions:The current study identifies the presence of prognostically-adverse mutations in approximately a third of patients with blast phase MPN (MPN-BP). The survival effect of adverse mutations was independent of other disease features and treatment strategies, including whether or not patients achieved CR/CRi or received ASCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinicopathologic correlates and natural history of atypical chronic myeloid leukemia.

There are limited data on the clonal mechanisms underlying leukemogenesis, prognostic factors, and optimal therapy for atypical chronic myeloid leukemia (aCML). The authors evaluated the clinicopathologic features, outcomes, and responses to therapy of 65 patients with aCML. The median age was 67 years (range, 46-89 years). The most frequently mutated genes included ASXL1 (83%), SRSF2 (68%), and SETBP1 (58%). Mutations in SETBP1, SRSF2, TET2, and GATA2 appeared at variant allele frequencies (VAFs) greater than 40%, whereas other RAS pathway mutations were more likely to appear at low VAFs. The acquisition of new, previously undetectable mutations at transformation was observed in 63% of the evaluable patients, with the most common involving signaling pathway mutations. Hypomethylating agents (HMAs) were associated with the highest response rates but with a short duration of response (median, 2.7 months). Therapy with ruxolitinib was not associated with clinically significant responses as a single agent or in combination with an HMA. Allogeneic stem cell transplantation was the only therapy associated with improved outcomes (hazard ratio, 0.144; 95% CI, 0.035-0.593; P = .007). Age, platelet counts, bone marrow blast percentages, and serum lactate dehydrogenase (LDH) levels were independent predictors of survival and were integrated in a multivariable model that allowed the prediction of 1- and 3-year survival. aCML is characterized by high frequencies of ASXL1, SRSF2, and SETBP1 mutations and is associated with a high risk of acute myeloid leukemia transformation. Response and survival outcomes with current therapies remain poor. The incorporation of age, platelet counts, bone marrow blast percentages, and LDH levels can allow survival prediction, and allogeneic stem cell transplantation should be considered for all eligible patients.

Mutations in chronic myelomonocytic leukemia and their prognostic relevance.

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy that overlaps with myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) and tends to transform into acute myeloid leukemia (AML). Among cases of CMML, > 90% have gene mutations, primarily involving TET2 (~ 60%), ASXL1 (~ 40%), SRSF2 (~ 50%), and the RAS pathways (~ 30%). These gene mutations are associated with both the clinical phenotypes and the prognosis of CMML, special CMML variants and pre-phases of CMML. Cytogenetic abnormalities and the size of genome are also associated with prognosis. Meanwhile, cases with ASXL1, DNMT3A, NRAS, SETBP1, CBL and RUNX1 mutations may have inferior prognoses, but only ASXL1 mutations were confirmed to be independent predictors of the patient outcome and were included in three prognostic models. Novel treatment targets related to the various gene mutations are emerging. Therefore, this review provides new insights to explore the correlations among gene mutations, clinical phenotypes, prognosis, and novel drugs in CMML.

CSF3R T618I, SETBP1 G870S, SRSF2 P95H, and ASXL1 Q780* tetramutation co-contribute to myeloblast transformation in a chronic neutrophilic leukemia

Chronic neutrophilic leukemia (CNL) is a rare but serious myeloid malignancy. In a review of reported cases for WHO-defined CNL, CSF3R mutation is found in about 90% cases and confirmed as the molecular basis of CNL. Concurrent mutations are observed in CSF3R-mutated CNL patients, including ASXL1, SETBP1, SRSF2, JAK2, CALR, TET2, NRAS, U2AF1, and CBL. Both ASXL1 and SETBP1 mutations in CNL have been associated with a poor prognosis, whereas, SRSF2 mutation was undetermined. Our patient was a 77-year-old man and had no significant past medical history and symptoms with leukocytosis. Bone marrow (BM) aspirate and biopsy revealed a markedly hypercellular marrow with prominent left-shifted granulopoiesis. Next-generation sequencing (NGS) of DNA from the BM aspirate of a panel of 28 genes, known to be pathogenic in MDS/MPN, detected mutations in CSF3R, SETBP1, and SRSF2, and a diagnosis of CNL was made. The patient did not use a JAK-STAT pathway inhibitor (ruxolitinib) but started on hydroxyurea and alpha-interferon and developed pruritus after 4 months of diagnosis and nasal hemorrhage 1 month later. Then, the patient was diagnosed with CNL with AML transformation and developed intracranial hemorrhage and died. We repeated NGS and found that three additional mutations were detected: ASXL1, PRKDC, MYOM2; variant allele frequency (VAF) of the prior mutations in CSF3R, SETBP1, and SRSF2 increased. The concurrence of CSF3RT618I, ASXL1, SETBP1, and SRSF2 mutation may be a mutationally detrimental combination and contribute to disease progression and AML transformation, as well as the nonspecific treatment of hydroxyurea and alpha-interferon, but the significance and role of PRKDC and MYOM2 mutations were not undetermined.

Identification of SETBP1 Mutations by Gene Panel Sequencing in Individuals With Intellectual Disability or With "Developmental and Epileptic Encephalopathy".

SETBP1 mutations are associated with the Schinzel-Giedion syndrome (SGS), characterized by profound neurodevelopmental delay, typical facial features, and multiple congenital malformations (OMIM 269150). Refractory epilepsy is a common feature of SGS. Loss of function mutations have been typically associated with a distinct and milder phenotype characterized by intellectual disability and expressive speech impairment. Here we report three variants of SETBP1, two novel de novo truncating mutations, identified by NGS analysis of an Intellectual Disability gene panel in 600 subjects with non-specific neurodevelopmental disorders, and one missense identified by a developmental epilepsy gene panel tested in 56 pediatric epileptic cases. The three individuals carrying the identified SETBP1 variants presented mild to severe developmental delay and lacked the cardinal features of classical SGS. One of these subjects, carrying the c.1765C>T (p.Arg589*) mutation, had mild Intellectual Disability with speech delay; the second one carrying the c.2199_2203del (p.Glu734Alafs19*) mutation had generalized epilepsy, responsive to treatment, and moderate Intellectual Disability; the third patient showed a severe cognitive defects and had a history of drug resistant epilepsy with West syndrome evolved into a Lennox-Gastaut syndrome. This latter subject carries the missense c.2572G>A (p.Glu858Lys) variant, which is absent from the control population, reported as de novo in a subject with ASD, and located close to the SETBP1 hot spot for SGS-associated mutations. Our findings contribute to further characterizing the associated phenotypes and suggest inclusion of SETBP1 in the list of prioritized genes for the genetic diagnosis of overlapping phenotypes ranging from non-specific neurodevelopmental disorders to “developmental and epileptic encephalopathy” (DEE).

Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia.

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

Oncogenic SETBP1 Mutations Combine with Activating Mutations in CSF3R to Produce a Highly Proliferative, Lethal Leukemia through Aberrant Myc Signaling

SETBP1 (SET Binding Protein 1) mutations are associated with exceptionally poor prognosis in myeloid neoplasms. Despite this, SETBP1's role in oncogenesis remains incompletely understood. In this study, we find that SETBP1 leads to a marked upregulation of the Myc oncogene and associated pro-stem and progenitor programs through epigenetic dysregulation. We further identify an epigenetic modulatory drug that normalizes SETBP1-driven Myc overexpression and synergizes with disease-relevant therapy. SETBP1 has documented roles in both the regulation of tumor suppressor pathways and modulation of transcription. To understand how SETBP1 modulates leukemia biology and leverage this mechanistic insight to develop novel therapeutic strategies, we turned to a genetically well-defined model system, chronic neutrophilic leukemia (CNL). SETBP1 is mutated in approximately half of all cases of CNL, a myeloproliferative neoplasm characterized by the presence of signaling-activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R). By expressing SETBP1 and CSF3R mutations in mouse hematopoietic progenitors, we have generated models of CNL that can be leveraged for mechanistic studies and drug development. In a hematopoietic colony forming unit (CFU) assay, murine hematopoietic progenitors co-expressing mutant SETBP1 and mutant CSF3R have a high proliferation phenotype compared to those with mutant CSF3R alone. When cells co-expressing mutant SETBP1 and CSF3R are transplanted into lethally irradiated mice, they develop a rapidly lethal disease relative to the control mice. This is associated with an expansion of the granulocyte lineage, quantified by complete blood count, flow cytometry and histology. We find that SETBP1 is essential for the induction of a pro-proliferative transcriptional program. One of the most prominent SETBP1-associated signatures is that of Myc target genes. Myc itself is one of the top differentially expressed genes driven by SETBP1. Congruent with its increased expression, we also find higher Myc transcriptional activity in cells overexpressing SETBP1. To better understand the epigenetic regulation of Myc and progenitor pathways by SETBP1, we employed a low input profiling methods called CUT&amp;Tag to assess the activation of regulatory elements. We began by profiling two epigenetic marks associated with active enhancers-H3K4me1 and H3K27Ac. We also assessed activation of the Myc promoter by measuring H3K27Ac and H3K4me3. Together this data helps us to understand the epigenetic underpinnings for dysregulation of Myc-driven programs by SETBP1. Therapeutic strategies that normalize aberrant Myc activity may be effective against SETBP1-driven disease. We found that SETBP1 and CSF3R transformed hematopoietic progenitors are highly sensitive to inhibitors of the epigenetic regulator lysine specific demethylase 1 (LSD1). LSD1 inhibition restores Myc expression to physiological levels and represses Myc promotor activity in vitro. Furthermore, LSD1 inhibitors are highly synergistic with JAK inhibitors, which block signaling downstream of CSF3R. Together these data establish the importance of Myc activation in SETBP1-driven malignancies and identify a therapeutic approach to normalize aberrant Myc activity. In future, we will use the insight gained in this genetically well defined disease to inform studies on the mechanistic basis and therapeutic vulnerabilities of other SETBP1-mutant myeloid malignancies. Disclosures Maxson: Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

Longitudinal Genomic Characterization Using Cell-Free DNA in Patients with Idiopathic Aplastic Anemia

The molecular profile of clonal hematopoiesis in patients (pts) with idiopathic aplastic anaemia (iAA) has been shown to predict response to immunosuppressive therapy (IST). Moreover, clonal evolution is associated with transformation of iAA to aggressive myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Detection of somatic mutations may help identify pts more likely to respond to IST and also those at increased risk of transformation however the predictive value of detecting mutations at a single timepoint is limited. Longitudinal detection of changes in mutation profile over time may improve prediction of transformation risk, however due to bone marrow hypocellularity DNA quality and quantity from pts with iAA is frequently suboptimal. Serial mutation analysis of cell-free DNA (cfDNA) is one approach that may overcome this problem as the majority of cfDNA originates from hematopoietic cells and mutations detected in this compartment have been shown to closely resemble those detected from peripheral blood (PB) / bone marrow (BM) analysis in other haematological malignancies. We aimed to characterise longitudinal genomic changes in pts with iAA and contrast mutation detection in the cellular compartment with those detected in cfDNA. Fifteen pts (10 female and 5 male) with a diagnosis of iAA were included (median age 35 years (range 17 - 78)); eleven were newly diagnosed (within six months of diagnosis) and four were two or more years post diagnosis. Cellular DNA (PB or BM aspirate) and cfDNA were collected upon study enrolment and cfDNA samples were collected longitudinally for each pt. Somatic mutations in cellular samples were assessed using custom targeted next generation sequencing panels with an analytical sensitivity of ~1%. cfDNA samples were assessed using a custom anchored multiplex PCR panel with molecular barcodes with an analytical sensitivity of ~0.5%. Twenty-nine genes were assessed in both cellular and cfDNA. Somatic mutations were detected in 13 of 15 (87%) pts at the first sampled timepoint, with two or more mutations detected in seven pts. Overall 27 mutations were detected in seven genes including PIGA (n=12), DNMT3A (n=5), ASXL1 (n=5), BCOR (n=2), BCORL1, NPM1 and RUNX1 (n=1 each) with a higher proportion detected in cfDNA (25 of 27) compared to cellular DNA (22 of 27). Mutations unique to cfDNA were in PIGA (n=3), ASXL1 (n=1) and DNMT3A (n=1) and those unique to cellular DNA were in NPM1 (n=1) and PIGA (n=1). The variant allele frequency (VAF) was &amp;lt;10% for 26 of 27 mutations (median cellular and cfDNA VAF 1.78% and 1.19% respectively), highlighting the need for sensitive mutation detection approaches in the setting of iAA regardless of the compartment tested. We next assessed the mutation dynamics in cfDNA over time by analysing a median of 3 (range 2 - 4) sequential samples per pt (median follow-up interval 670 days, range 249 - 923). 20 of 25 cfDNA mutations were detected in longitudinal cfDNA at similar VAF, including PIGA (n=9), BCOR (n=2), DNMT3A (n=5), ASXL1 (n=3) and RUNX1 (n=1). Five mutations in PIGA (n=2), ASXL1 (n=2) and BCORL1 (n=1) became undetectable over time. Importantly, acquisition of new mutations during follow-up was observed in four pts (total of 13 new cfDNA mutations detected). Three out of these four pts experienced disease transformation to hematological malignancy (MDS n=2, AML n=1), with acquired mutations in SETBP1, NPM1, NRAS, KRAS, WT1 and KIT. Importantly, these mutations often went from undetectable to relatively high VAF within a short interval (5 - 13 months) which is important to consider when designing molecular screening schedules to potentially pre-empt transformation. In summary, our data shows that cfDNA mutation profiling in pts with iAA is technically feasible and is able to detect additional mutations not detectable in the cellular compartment. Importantly, we demonstrate the limitations of analysis of both compartments in the context of low VAF mutations, suggesting that concurrent testing of both cellular and cfDNA may increase the sensitivity for detecting low frequency events. Our data also suggests that longitudinal changes in mutation profile may improve identification of pts at high risk of transformation to hematological malignancy. The optimal frequency of monitoring and the magnitude of increased risk requires validation in prospective trials. GLR and LCF contributed equally to this work Disclosures Szer: Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis: Consultancy; Pfizer: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Prevail Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bajel:Astellas: Honoraria; Novartis: Honoraria; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria; Abbvie: Honoraria. Blombery:Novartis: Consultancy; Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria.

Clinicopathologic Correlates and Natural History of Atypical Chronic Myeloid Leukemia

Clinicopathologic Correlates and Natural History of Atypical Chronic Myeloid Leukemia

Myelodysplastic Syndromes with 20q Deletion: Incidence, Prognostic Value and Impact on Response to Azacitidine of ASXL1 Chromosomal Deletion and Genetic Mutations

Introduction : The 20q deletion [del(20q)] is a recurrent chromosomal aberration in myelodysplastic syndromes (MDS) and, as a single abnormality, is associated according to the Revised International Prognostic Scoring System (IPSS-R) with a favorable outcome. However, the breakpoint of del(20q) is very heterogeneous and may cause deletion of the ASXL1 gene (20q11.21). This gene is an important epigenetic regulator of hematopoiesis and its mutations have been associated in MDS with a shorter overall survival (OS) and a lower response to azacitidine (AZA). Aim: To assess the incidence, prognostic value and impact on response to AZA of ASXL1 chromosomal alterations and genetic mutations in MDS patients with del(20q). Methods: We studied 153 patients diagnosed with MDS and del(20q) as a sole abnormality (n=93), with an additional chromosomal abnormality (n=27) or in a complex karyotype (n=33). Response to AZA therapy was assessed using the 2006 International Working Group (IWG) criteria. Analysis of ASXL1 chromosome alterations was performed by FISH (Empire Genomics probe). Samples with ASXL1 alterations by FISH were analyzed using the Agilent SurePrint G3 Human CGH 8x60K Microarray. Mutations of ASXL1 were detected by Sanger sequencing. SF3B1, SRSF2, U2AF1, DNMT3A, IDH1, IDH2, TP53, RUNX1, and SETBP1 mutations were screened by high-resolution melting and positives were confirmed by Sanger sequencing. An in vitro assay of the response to AZA in HAP1 and HAP1 ASXL1 knockout cell lines (Horizon) was also performed. The association of the clinical characteristics with the molecular findings was analyzed with the SPSS statistical program (v.20.0) and P values &amp;lt;0.05 were considered as statistically significant. Results: Main patient characteristics are shown in Table 1.ASXL1 chromosomal alterations were detected by FISH in 44 patients (29%; Fig. 1): 34 patients (22%) with gene deletion (ASXL1DEL) and 10 cases (6.5%) with additional gene copies (ASXL1GAIN). All 44 cases were analyzed by microarray, and alterations in centromeric KIF3B gene (20q11.21) were also identified in 24 patients (16%). Patients with ASXL1DEL showed a lower platelet count (median, 68x109/L vs. 103x109/L, P=0.046), a poorer response to AZA (response, 9% vs. 43%, P= 0.040) and a trend towards a lower OS (34 vs. 65 months, P=0.057). ASXL1 and KIF3B chromosomal gains were associated with complex karyotypes (ASXL1GAIN, 80% vs. 23%, P&amp;lt;0.001; KIF3BGAIN, 80% vs. 25%, P=0.011). Patients with ASXL1GAIN had a similar OS and response to AZA than ASXL1nonDEL/ASXL1nonMUT patients (OS, 51 vs. 73 months, P=0.86; response, 60% vs. 44%, P=0.43). Seventy patients (46%) in the series had ≥1 genetic mutation (Fig. 1). ASXL1 mutations (ASXL1MUT; 22/153, 14%) were mostly frameshift (n=17, 77%) or nonsense (n=4, 18%) and were associated with a lower hemoglobin level (median, 8.3 vs. 10.3 g/dL; P=0.007), a higher ferritin level (median, 691 vs. 286 ng/mL, P=0.014), a higher number of mutations (23% vs. 0% ≥3 mutations, P&amp;lt;0.001) and a lower OS (22 vs. 62 months, P=0.016). U2AF1 mutations were associated with a deeper neutropenia (median, 1.17x109/L vs. 1.83x109/L, P=0.026) and TP53 mutations were associated with acute myeloid leukemia (AML) transformation (55% vs. 16%, P=0.005) and a lower OS (24 vs. 57 months, P=0.037). In the global series, patients with ASXL1 altered either by chromosomal deletion or mutation (ASXL1DEL/ASXL1MUT) had a poorer response to AZA (13% vs. 47%, P=0.020) and a lower OS (32 vs. 70 months, P=0.005; Fig. 2) than the remaining patients. In the analysis of isolated del(20q) subgroup (n=93), ASXL1DEL/ASXL1MUT patients also showed a lower OS (37 vs. 80 months, P=0.026) and ASXL1DEL was associated with a higher risk of progression to AML (33% vs. 11%, P=0.029), despite 64% of patients were clustered in lower-risk categories of the IPSS-R. In the in vitro assay performed at 2 μM AZA, HAP1 ASXL1 knockout cells showed less growth inhibition (26% vs. 46%, P=0.003), lower cell apoptosis (17% vs. 34%, P&amp;lt;0.001) and less cell cycle arrest in G0/G1 phase (6% vs. 23%, P=0.009), compared to HAP1 cells. Conclusion: In MDS patients with del(20q), the alteration of ASXL1 by chromosome deletion or somatic mutation was associated with adverse clinical features and a poorer response to AZA. Its detection at diagnosis would allow the rapid identification of a subgroup of MDS patients with a poor prognosis that could benefit of earlier and more effective therapies. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees. Tormo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; MSD: Honoraria; Daiichi Sankyo: Honoraria; Servier: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees.

Molecular and Expression Characterization of Monosomy 7 and Del(7q)

The loss of chromosome 7 (monosomy 7, -7) or its long arm [del(7q)] is a common karyotypic abnormality found in MDS and are associated with a poor prognosis. However recent studies may suggest a difference in survival outcomes. These lesions occur both independently and in a complex karyotype (CK). The leukemogenic features of -7/del7q can be explained through loss of heterozygosity or haploinsufficiency (HI) of key deleted genes. In addition, the phenotype of del(7q) and -7 might be further modified by the clonal architecture determining the mode of molecular evolution. Based on the hypothesis that a deletion will exert its biologic consequences in genes with HI expression and by co-associated mutations, a comprehensive analysis of genes on chr.7 was performed. Within a cohort of 8,161 patients with myeloid neoplasms (MN), we identified 511 with -7 (6%) and 143 with del(7q) (2%). In 29% of cases, del(7q) was found as an isolated alteration, while 90% of CK had del(7q); -7 showed similar percentages of isolated cases but was less common in CK (20%, 55%; respectively) cases. Del(7q) was significantly (P&amp;lt;.01) associated with MDS, while -7 was significantly (P=.0038) associated with sAML. When we focused on antecedent diagnosis, patients with -7 were more likely to have a previous diagnosis of aplastic anemia or chemotherapy treatment (60%; 41%, P&amp;lt;.01, for both). The most common co-associated molecular lesions with -7/del(7q) were: TP53 (31%), DNMT3A (13%), TET2 (13%), RUNX1 (12%), ASXL1 (11%), del(5q) (25%) and CK (54%). Del(7q) vs. -7 differed in the frequency of TP53 (39 vs. 29%; P=.03), DNMT3A (19 vs. 11%; P=.03), and IDH1/2 (13 vs. 52%; P&amp;lt;.01). Isolated -7/del(7q) was associated with mutations of TET2 (24% vs. 9%, P&amp;lt;.001), SRSF2 (12% vs. 3%, P&amp;lt;.001), EZH2 (10% vs. 3%, P&amp;lt;.001), and SETBP1 (8% vs. 2%; P&amp;lt;.001). Complex -7/del(7q) correlated with TP53 (39% vs. 4%; P&amp;lt;.01) and PTPN11 (9% vs. 6%; P=.02). When we focused on mutations on genes located in the 7q common deleted regions (CDR1, 7q22 and CDR2, 7q35-q36), EZH2 was the most represented 7q mutant gene (6 mutants in del(7q) and 24 mutants in -7) totaling 5% of all -7/del(7q) cases. Other mutated genes on 7q were: CUX1 in 1% del(7q) and 1% -7, CUL1 in 1% of del(7q), and LUC7L2 which was only found mutated in -7. Comparing the -7/del(7q) clone (calculated using allelic imbalance bio-analytic pipeline) to co-associated mutations allow us to identify the rank of -7/del(7q) in the clonal hierarchy: -7/del(7q) was an ancestral event in 58% and a secondary hit in 42% of the patients. When -7/del(7q) was the dominant hit, patients had fewer associated mutations, while in cases with secondary -7/del7q somatic mutations of TP53 (60%), IDH1/2 (30%), and DNMT3A (20%) were the ancestral hits suggesting a different clonal architecture according to -7/del(7q) clone size. Expression data was available for 49 of -7/del(7q) and 643 diploid cases for chr.7. Of the 694 informative genes on chr.7: 147 were deleted in all patients according to CNV analysis. We restricted the analysis to 15 uniformly HI genes selected because the expression inversely correlated with the level of 7q ploidy (ACTR3B, FASTK, GSTK1, IMPDH1, LRRC4, MEST, MGAM, NUP205, PRRT4, SLC37A3, SSBP1, TMEM209, TNPO3, ZC3HC1, ZNF277). Expression for these genes was then analyzed following normalization by -7/del7q clonal size. Unsupervised clustering grouped 72% of -7/del(7q) in one expression cluster. Classical -7/del(7q) signature genes fulfilled these criteria and with uniform HI in all cases. However, when we also included CUL1, CUX1, EZH2, KMT2C, and LUC7L2 to this analysis, the precision of -7/del(7q) clustering was 75% and defined a subcluster (#6). We were also able to identify cases previously misclassified (n=9) which were dispersed into 3 other clusters (14%, 8%, 3%) while 4% (n=23) of diploid cases were also misclassified and re-assigned into cluster 6. When we restricted the analysis to -7 cases only, a signature of 6 unique genes (C7orf43, CDK14, POLM, STAG3L5P, TRIM4) were found. Interestingly only POLM was on 7p, while the rest were on 7q22.1. In sum, we describe an integrated genomic and transcriptomic analysis of -7/del(7q) and we present data showing a complex heterogeneity of genes with HI expression which suggest that the clinical picture of patients carrying -7/del(7q) might be the result of a combination of clonality and HI. Disclosures Patel: Alexion: Other: educational speaker. Sekeres:Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

A Unique Case of CSF3R-Mutated Chronic Neutrophilic Leukemia with Thrombocytosis

Chronic neutrophilic leukemia (CNL) is a well-recognized, extremely rare myeloproliferative neoplasm (MPN) with only ~200 reported cases until 2017. The current WHO diagnostic criteria include leukocytosis of ≥ 25 x 109/L with ≥ 80% neutrophils/bands, &amp;lt; 10% circulating neutrophil precursors and &amp;lt;1% blasts (&amp;lt;5% bone marrow blasts). Exclusion criteria includes dysplasia, or clinical/molecular diagnosis of other MPN. The WHO classification was updated in 2016 to include CSF3R T618I and other activating mutations in CSF3R as diagnostic. SETBP1 and ASXL1 mutations are frequent but unspecific in CNL and appear to be of prognostic significance. Hepatosplenomegaly and a hypercellular bone marrow with marked granulocytic hyperplasia are common in CNL, but thrombocytosis has not been reported previously. We report a 72-year-old man with past medical history of hypertension and hepatitis C status post-treatment, who presented with WBC 38.4k/cmm, Hgb 6.3g/dl, MCV 102 fl and platelet count of 2601k/cmm. Reactive etiology of thrombocytosis was ruled out. Peripheral blood smear revealed rare blasts. Bone marrow biopsy was hypercellular (60-80%) with M:E ratio of 10:1, granulocytic and megakaryocytic hyperplasia, clustering of dyspoietic megakaryocytes, no erythroid or myeloid dysplasia and minimal patchy reticulin fibrosis suggestive of primary MPN. Chromosome analysis showed normal karyotype of 46 XY. Molecular studies were negative for BCR-ABL, JAK-2 V617F, CALR and MPL, Next Generation Sequencing revealed mutations in the CSF3R gene (Q781* and T618I) and concurrent inactivating ASXL1-G646fs*12 and activating U2AF1-Q157P mutations. These findings were compatible with a diagnosis of CNL. We describe an unusual case of CNL confirmed by CSF3R gene mutations (T618I and Q781*) with significant thrombocytosis and without splenomegaly. Although the T618I mutation has commonly been reported in CNL, to the best of our knowledge, the Q781* mutation is novel in CNL, and has only been reported before in severe congenital neutropenia (SCN). Association of Q781* mutation in CSF3R gene with significant thrombocytosis in our patient needs further investigation. Disclosures No relevant conflicts of interest to declare.

The recurrent SETBP1 c.2608G\u2009>\u2009A, p.(Gly870Ser) variant in a patient with Schinzel-Giedion syndrome: an illustrative case of the utility of whole exome sequencing in a critically ill neonate

BackgroundSchinzel-Giedion syndrome (SGS) is a multiple malformation syndrome mainly characterized by severe intellectual disability, distinctive facial features, and multiple congenital anomalies, including skeletal abnormalities, genitourinary and renal malformations, cardiac defects, as well as an increased pediatric cancer risk. Recently, SGS has been associated with de novo heterozygous deleterious variants in the SETBP1 gene; to date, nine different variants, clustering in exon 4 of SETBP1, have been identified in 25 patients.Case presentationIn this study, by using Whole Exome Sequencing (WES), we identified a patient with a recurrent missense mutation in SETBP1, the c.2608G > A, p.(Gly870Ser) variant, previously reported as likely pathogenic. This finding allowed us to confirm the suspected clinical diagnosis of SGS. Clinical features of patients carrying the same variant, including our patient, were evaluated by a review of medical records.ConclusionsOur study confirms SGS as a severe disorder potentially presenting at birth as a critically ill neonate and demonstrates the causal role of the c.2608G > A, p.(Gly870Ser) variant in the etiology of the syndrome. Moreover, although the cohort of SETBP1-patients reported in the literature is still small, our study reports for the first time the prevalence of the variant (about 27%, 7/26). Finally, given the heterogeneity of clinical presentations of affected patients hospitalized in Neonatal Intensive Care Units (NICU) and/or Pediatric Intensive Care Units (PICU), in agreement with emerging data from the literature, we suggest that WES should be used in the diagnosis of unexplained syndromic conditions, and even as part of a standard first-line diagnostic approach, as it would allow a better diagnosis, counseling and management of affected patients and their families.

Chronic myelomonocytic leukemia genomic signature correlates with the degree of bone marrow fibrosis: A single-institutional retrospective study.

7559 Background: Chronic myelomonocytic leukemia (CMML) has features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. The median overall survival (OS) in most series is 20-40 months. CMML is a relatively rare entity, and there is limited understanding of prognostic molecular markers. CMML associated with bone marrow fibrosis grade 1, appear to have shorter progression free survival. In this study we investigated the correlation of mutations with bone marrow reticulin fibrosis in patients with CMML. Methods: We investigated a cohort of 41 consecutive patients diagnosed with CMML 0, 1, 2, and CMML-AML from 2014 to 2019 at our institute. The median age of 41 patients was 68 years (range, 34-82). 27% were females and 73% were males. This cohort consists of 8 (20%) patients with CMML0, 19 (46%) with CMML1, 8 (20%) with CMML2 and 6 (15%) with CMML-AML. Genomic DNA was extracted from the bone marrow aspirates and targeted mutation NGS libraries were prepared from 200 ng of genomic DNA using the SureSelect target enrichment system (Agilent Technologies Inc.). The gene panel consists of 73 genes focused on myeloid neoplasms. The data were curated on the basis of our molecular pathology and national databases. Additionally, clinical data and bone marrow (BM) reticulin fibrosis grades (n = 27 available) were retrieved from the patient’s medical record. Results: The mutational profile frequency in our cohort showed that the most common mutations were TET2 (31%), ASXL1 (31%), and SRSF2 (23%), with frequencies very similar to those reported in the literature. Of the 27 cases with an available reticulin stain, only low grade fibrosis (MF-0, n = 18. MF-1, n = 9) were identified in our cohort. The frequencies of mutations in ASXL1, U2AF1, TP53, JAK2 and RUNX1, positively correlated with low grade fibrosis (MF-1). Additionally, patients with higher frequencies of SRSF2, TET2, and SETBP1 mutations showed no fibrosis (MF-0). Conclusions: This study is the first to correlate the degree of fibrosis with the frequency of mutations in CMML. We found similar mutations spectrum reported in the literature in patients with CMML. The mutational profile associated with CMML cases appears to affect the degree of the bone marrow fibrosis at the time of diagnosis.

Features and clinical significance of gene mutations in patients with myelodysplastic syndromes with ring sideroblasts

目的研究伴环状铁粒幼红细胞增多（RS）的骨髓增生异常综合征（MDS-RS）患者的基因突变特征及其临床意义。方法收集2001年1月至2019年6月于中国医学科学院血液病医院新诊断的255例原发性MDS-RS患者资料。129例采用一代测序、126例采用包含112个血液肿瘤相关基因的二代测序（NGS）进行基因突变分析。结果共193例（75.7%）检出SF3B1突变，其中SF3B1 K700E突变147例（76.2%）。非SF3B1基因突变较常见的有TET2（16.7%）、ASXL1（14.3%）、U2AF1（11.1%）、TP53（7.9%）、SETBP1（6.3%）和RUNX1（6.3%）。RS 5%～<15%患者SETBP1突变率显著高于RS≥15%患者（21.4%对4.5%，P＝0.044），其余基因突变率差异无统计学意义（P值均>0.05）。在114例NGS检出SF3B1突变的患者中，RS 5%～<15%患者SF3B1等位基因突变频率（VAF）与骨髓RS比例呈正相关（r＝0.486，P＝0.078）；RS≥15%组SF3B1突变型患者骨髓RS比例显著高于野生型患者［40.0%（15.0%～80.0%）对25.5%（15.0%～82.0%），P<0.001］，且SF3B1 VAF与骨髓RS比例呈正相关（P＝0.009，rs＝0.261）。全部患者SF3B1突变型与野生型年龄、ANC、PLT、平均红细胞体积、RS比例、IPSS-R染色体核型及IPSS-R预后分组等方面差异有统计学意义（P值均<0.05）。多因素分析显示，SF3B1突变是影响总生存（OS）时间的独立良好预后因素（HR＝0.265，95% CI 0.077～0.917，P＝0.036），TP53突变是独立不良预后因素（HR＝6.272，95% CI 1.725～22.809，P＝0.005）。根据SF3B1和TP53突变状态将MDS-RS患者分为四组：SF3B1和TP53均突变组、SF3B1和TP53均野生组、SF3B1野生伴TP53突变组及SF3B1突变伴TP53野生组，四组患者OS时间差异显著（P<0.001）。组间两两比较显示SF3B1突变伴TP53野生组OS时间显著长于SF3B1野生伴TP53突变组、SF3B1和TP53野生组，而与SF3B1和TP53均突变组比较差异无统计学意义。结论SF3B1突变在MDS-RS中的发生率较高，几乎均是错义突变，以K700E突变最为常见。SF3B1突变是MDS-RS患者生存的独立良好预后因素，TP53突变是独立不良预后因素，二者联合可更精细地指导MDS-RS患者预后分层。

Analysis of Clinical Features and Prognosis of Patients with Chronic Neutrophil Leukemia

To provide clinical basis for the diagnosis and treatment of chronic neutrophilic leukemia (CNL) and to provide possible molecular targets for the treatment. By summarizing the clinical data of 14 patients with CNL, the clinical characteristics, gene mutation types and possible prognostic factors were analyzed. Among the 14 patients with CNL, males (9 cases) were more than females (5 cases), with a median age of 57 years old. The detection rate of CSF3R mutation was 92.86% (13/14), including 12 cases (85.71%) with T318I mutation and 1 case of Y799X mutation, and only 1 case was not detected for mutation of CSF3R. The ASXL1 mutation was detected in 42.86% (6/14) of the patients, all of which were nonsense mutations, including 4 cases with R693X and 2 cases with E705X, and 14.29% (2/14) of the patients was detected for SETBP1 mutation, all of which were with D868N mutation. No patients with simultaneous ASXL1 and SETBP1 mutations were found, and JAK2 and CALR mutations were not detected. All of the patients had normal karyotypes. These patients' median survival time was 30 months (95%CI 13.19-46.80), and the influence of age over 60 years old was statistically significant (21.83 months vs 35.35 months) (P＜0.05). It is difficult to diagnose CNL. CSF3R T618I mutation is its specific mutation, and ASXL1 mutation and SETBP1 mutation have auxiliary diagnostic significance for CNL. The age＞60 years old at diagnosis is a factor of unfavourable prognosis.

Analysis of Clinical Features and Prognosis of Patients with Chronic Neutrophil Leukemia

To provide clinical basis for the diagnosis and treatment of chronic neutrophilic leukemia (CNL) and to provide possible molecular targets for the treatment. By summarizing the clinical data of 14 patients with CNL, the clinical characteristics, gene mutation types and possible prognostic factors were analyzed. Among the 14 patients with CNL, males (9 cases) were more than females (5 cases), with a median age of 57 years old. The detection rate of CSF3R mutation was 92.86% (13/14), including 12 cases (85.71%) with T318I mutation and 1 case of Y799X mutation, and only 1 case was not detected for mutation of CSF3R. The ASXL1 mutation was detected in 42.86% (6/14) of the patients, all of which were nonsense mutations, including 4 cases with R693X and 2 cases with E705X, and 14.29% (2/14) of the patients was detected for SETBP1 mutation, all of which were with D868N mutation. No patients with simultaneous ASXL1 and SETBP1 mutations were found, and JAK2 and CALR mutations were not detected. All of the patients had normal karyotypes. These patients' median survival time was 30 months (95%CI 13.19-46.80), and the influence of age over 60 years old was statistically significant (21.83 months vs 35.35 months) (P＜0.05). It is difficult to diagnose CNL. CSF3R T618I mutation is its specific mutation, and ASXL1 mutation and SETBP1 mutation have auxiliary diagnostic significance for CNL. The age＞60 years old at diagnosis is a factor of unfavourable prognosis.

Decoding Bone Marrow Fibrosis in Myelodysplastic Syndromes

BackgroundBone marrow fibrosis (BMF), a poor prognostic factor in myelodysplastic syndromes (MDS), in the context of new risk stratifications of MDS has not been fully explored. We examined the relationship between BMF in MDS and survival outcomes, and explored the landscape of somatic gene mutations in the setting of BMF. Patients and MethodsWe retrospectively evaluated 2624 MDS patients for BMF who were divided into 2 groups: grade 0-2 BMF (96%) and severe/grade 3 BMF (4%) based on analysis presented. Commonly MDS tested acquired somatic mutations were also compared between those 2 groups of patients with available next-generation sequencing data. ResultsOnly grade 3 BMF was associated with worse overall survival independent from the Revised International Prognostic Scoring System (IPSS-R) (hazard ratio = 1.6; 95% confidence interval, 1.2-1.9; P < .005). More patients with severe BMF were classified as MDS-EB1 and -EB2 by the World Health Organization 2016 classification, a higher-risk International Prognostic Scoring System score, and a high/very high IPSS-R risk category than patients with grade 0-2 BMF. A complex karyotype, higher bone marrow myeloblasts, lower platelets, and higher rate of elevated lactate dehydrogenase were observed more often in patients with severe BMF. No differences in response to hypomethylating agents or lenalidomide were observed. Among somatic gene mutations tested in MDS, TP53 mutation and SETBP1 were more frequent in patients with grade 3 BMF. ConclusionThe presence of grade 3 BMF is associated with reduced overall survival independent from IPSS-R; however, BMF grade did not affect response to hypomethylating agent or lenalidomide treatment. TP53 and SETBP1 mutations occurred with greater frequency among patients with severe fibrosis.