SRSF2 Genes Research Articles

Clonal hematopoiesis in cardiovascular aging: Insights from the verona heart study.

Clonal hematopoiesis of indeterminate potential (CHIP), marked by the accumulation of somatic mutations in hematopoietic stem cells, significantly elevates the risk of all-cause mortality, mainly due to cardiovascular events. Therefore, investigating this pathophysiological phenomenon is crucial for understanding cardiovascular aging and enhancing both health span and lifespan. In the present study, we examined samples of subjects enrolled within the angiographically controlled Verona Heart Study (VHS), which provides a robust model for cardiovascular aging, particularly regarding coronary artery disease (CAD). We analyzed 44 older subjects diagnosed with coronary artery disease (CAD) and 42 healthy, sex- and age-matched controls (CAD-FREE). Employing deep sequencing and an amplicon-based approach, we focused on 11 key genetic regions in ASXL1, DNMT3A, IDH1, IDH2, JAK2, PPM1D, SF3B1, SRSF2, TET2, TP53, and U2AF1 genes to investigate clonal hematopoiesis. Subjects in the CAD group exhibited a significantly higher variant burden than those in the CAD-FREE group, both in terms of the total number of somatic variants and disruptive variants affecting protein function. This increased mutational load was notably influenced by six specific genetic regions: ASXL1, DNMT3A, IDH2, JAK2, TET2, and U2AF1, which displayed elevated variant rates in the CAD subjects. Moreover, ASXL1, DNMT3A, IDH2, JAK2, SF3B1, TET2, and TP53 exhibited substantially higher levels of disruptive variants in the CAD group. In summary, our findings highlight a correlation between clonal hematopoiesis and the accumulation of disruptive variants in specific genomic regions in the VHS cohort, thereby shedding light on their potential role in cardiovascular aging.

Influence of genetic co-mutation on chemotherapeutic outcome in NPM1-mutated and FLT3-ITD wild-type AML patients.

Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.

Poor prognosis of SRSF2 gene mutations in patients treated with VEN-AZA for newly diagnosed acute myeloid leukemia

Mutations in spliceosome genes (SRSF2, SF3B1, U2AF1, ZRSR2) correlate with inferior outcomes in patients treated with intensive chemotherapy for Acute Myeloid Leukemia. However, their prognostic impact in patients treated with less intensive protocols is not well known. This study aimed to evaluate the impact of Spliceosome mutations in patients treated with Venetoclax and Azacitidine for newly diagnosed AML. 117 patients treated in 3 different hospitals were included in the analysis. 34 harbored a mutation in at least one of the spliceosome genes (splice-mut cohort). K/NRAS mutations were more frequent in the splice-mut cohort (47% vs 19%, p=0.0022). Response rates did not differ between splice-mut and splice-wt cohorts. With a median follow-up of 15 months, splice mutations were associated with a lower 18-month LFS (p=0.0045). When analyzing splice mutations separately, we found SRSF2 mutations to be associated with poorer outcomes (p=0.034 and p=0.037 for OS and LFS respectively). This negative prognostic impact remained true in our multivariate analysis. We believe this finding should warrant further studies aimed at overcoming this negative impact.

Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

Discovery of U2AF1 neoantigens in myeloid neoplasms

BackgroundMyelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome...

Poor Prognosis of SRSF2 Gene Mutations in Patients Treated with Venetoclax-Azacitidine (VEN-AZA) for Newly Diagnosed Acute Myeloid Leukemia. a Multicentric Real-Life Study of 117 Patients

Introduction Spliceosomes are complexes composed of small nuclear RNA that remove introns in protein-encoding genes. Spliceosome mutations ( SRSF2, SF3B1, U2AF1, ZRSR2), are encountered in ∼50% of secondary AML cases. Splicing mutations (splice-mut), in particular SRSF2, correlate with inferior outcomes to standard induction therapy. A recent report in a cohort of 119 patients, of whom 33 had splice mutations and 24 were SRSF2 mutated, reported the lack of impact of splice-mut on prognosis of AML patients treated upfront with Hypomethylating agents (HMA) + VEN in clinical trials (Lachowiez, C. A. et al Blood Adv. 2021). Aims We aimed to assess the impact of splice-mut in a population of patients with newly diagnosed acute myeloid leukemia (ND-AML), treated with VEN-AZA. Methods We performed a retrospective multicentric study including patients treated in three French centers in Marseille (Institut Paoli-Calmettes, CHU La Conception) and Nice (Hôpital L'Archet). Inclusion criterias were ND AML adult patients treated with VEN (7 to 28 days per cycle) and AZA at conventional doses. Next Generation Sequencing (NGS) was performed at diagnosis and was available for all patients. Data were collected from patients' electronic medical records. Statistical analyses were performed using GraphPad Prism v9.5.1 Results We included 117 ND-AML patients (median age = 75 yo; range, 32-89) treated with VEN-AZA between October 2019 and March 2023. The five most frequently mutated genes in the whole cohort were TET2, ASXL1, TP53 and RUNX1in thirty-seven (32%), 36 (31%), 32 (27%) and 31 (26%) patients, respectively. Thirty-four patients (29%) had a mutation in at least one of the spliceosome genes (=Splice-mut), including 20 (17%), 11 (9%), 4 (3%) and 1 (1%) mutations in SRSF2, U2AF1, SF3B1 and ZRSR2, respectively (Fig.1). Median variant allele frequency (VAF) was 42%, 43% and 26% for SRFS2, U2AF1 and SF3B1 respectively. Secondary AML was found in 18 (53%) of the splice-mut AML and 21 (25.3%) of the splice-wt AML groups of patients. Prior HMA was given in seven (20.6%) patients in the splice-mut versus 6 (7.2%) in the splice-wt cohort. Complex cytogenetics was identified in 7 (20.6%) patients in the splice-mut cohort and 28 (33.7%) in the splice-wt cohort. We found a TP53 mutation in 17.6% and 31.3% of splice-mut and wt cohorts, respectively. Best overall response rate (= CR, CRi and MLFS) was 72.6%; with CR, CRi and MLFS observed in 63 (54%), 16 (14%) and 6 (5%) patients, respectively. We did not find any difference in response rate between the splice-mut (=73.5%) and splice-wt (72.3%) AML. Prior treatment with hypomethylating agents and TET2 mutation were the only factors significantly associated with lower response rates (42% vs 82%, p=0.004, and 64% vs 85%, p=0.025, respectively), while IDH2 mutation was predictive of a better response (100%vs 75%, p=0.037, OR: 9.9 [CI 95% 0.57-173]). Median overall survival (OS) and leukemia-free survival (LFS) for the whole cohort was 10.8 and 7.1 months, respectively. In multivariate analysis taking into account TP53 and/or del17p, NRASmut and/or FLT3-ITD, IDH2mut, complex karyotype, and SRSF2, the only factors predicting OS and LFS were mutations in TP53 and SRSF2 genes. Patients mutated for SRSF2 had a 4.8 months OS and a 5 months LFS compared to 11.3 and 8 months, respectively (p=0.034 and p=0.037), comparable with TP53mut AML patients ( Fig.2). Conclusions SRSF2mut seems to be predictive of a worse survival in ND AML treated with VEN-AZA. This finding warrants further exploration in larger cohorts.

Oral Decitabine/Cedazuridine Vs Intravenous Decitabine for Acute Myeloid Leukemia: Final Results of a Randomized, Crossover, Registration-Enabling, Pharmacokinetics Study

Background: Parenterally administered hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), are approved in Europe for adult patients with acute myeloid leukemia (AML) who are not candidates for standard induction chemotherapy as single agent or in combination with venetoclax. Cedazuridine(C) is a novel, potent, and safe inhibitor of cytidine deaminase (CDA) and when given in combination with decitabine, cedazuridine enables efficient oral availability of decitabine.ASTX727 (DEC-C) is a fixed-dose combination (FDC) tablet of 35 mg DEC and 100 mg cedazuridine, DEC-C has been approved for MDS in the US, Canada, and Australia. Here we present final results using DEC-C in an AML population appropriate for single-agent decitabine treatment. Aims: Describe the final clinical outcomes of patients with AML with additional analysis of genetic profiles. Methods: The ASCERTAIN study used a randomized two-period, two-sequence, two-treatment, crossover study design. Patients were randomized 1:1 to either Sequence A: DEC-C (35 mg DEC/100 mg cedazuridine) in Cycle 1 followed by IV-DEC at 20 mg/m 2 in Cycle 2, or Sequence B: receiving IV-DEC in Cycle 1 followed by DEC-C on Cycle 2 to compare pharmacokinetics (PK) [primary endpoint Area Under the Curve (AUC) equivalence over 5 days of dosing]. All patients received DEC-C from Cycle 3 until treatment discontinuation to assess safety and clinical efficacy. Patients were eligible as per the EMA-approved decitabine label (newly diagnosed AML who are not candidates for standard induction chemotherapy). Clinical responses were assessed according to modified International Working Group (IWG) 2003 response criteria. Pre-treatment peripheral blood was used for DNA isolation from leukocytes, and molecular abnormalities identified using a NGS hematologic malignancy panel. Cox-regression analysis on the various factors (eg binary mutational status, CR, etc.) was used for analyses of risk-factors for overall survival (OS). Results: 89 patients were randomized, of whom 87 were treated. The median age of patients was 78.0 years (range, 61 to 92) with 31 (35.6%) males and 56 (64.4%) females. Cytogenetic risk classification was poor-risk in 33 (37.9%), intermediate-risk in 45 (51.7%), and 9 (10.4%) not evaluated patients. For the primary endpoint, preliminary PK data was available from 69 patients who successfully completed PK assessments for both IV-DEC and DEC-C cycles, and the DEC AUC 0-24 (h*ng/mL) 5-Day geometric mean estimate was 904 for DEC-C and 907 for IV-DEC resulting in an oral/IV geometric LSM AUC ratio of 99.64% (90% CI of 91.23-108.8%). Safety findings were consistent with those anticipated for IV-DEC (related Grade ≥ 3 AEs in more than 10% were thrombocytopenia, neutropenia, anaemia, and febrile neutropenia). As of the database lock (25May2023), median follow up was 24.5 (20.2, 25.0) months (25% of 19.7 and 75% of 25.2 months). The best response was complete response (CR) in 19/87 (21.8%, 95% CI: 13.7, 32.0%); CR with incomplete blood cell count recovery (CRi) in 5 patients (5.7%); and CR with incomplete platelet recovery (CRp) in 2 (2.3%); resulting in composite response rate [CR + CRi] of 27.6%. The median overall survival was approximately 8.9 months (95% CI: 6.0, 13.1).These results obtained with DEC-C are consistent with those observed for IV-DEC. Multiple genes, including ASXL1, TP53, RUNX1, and SRSF2 genes were present in more than 20% of the AML patients. TP53 mutation was associated with worse survival. Summary/Conclusion: This randomized phase 3 study in AML patients not candidates for standard induction chemotherapy demonstrates that Daily X 5 dosing of the oral FDC of DEC-C resulted in an equivalent DEC AUC exposure to IV-DEC at 20 mg/m 2 over 5 days. Pharmacodynamic data showed similar demethylation rates (≤1.1% difference). In addition, safety findings and observed clinical activity (response rates and OS) are consistent with published data from IV-DEC, suggesting that DEC-C has the potential to be an oral alternative to the standard IV-DEC Daily×5 regimen. These findings support the use of DEC-C in patients with AML who are not candidates for standard induction chemotherapy.

BRAF-Mutated Acute Myeloid Leukemia (AML) Represents a Distinct, Prognostically Poor Subgroup Enriched in Myelodysplasia-Related (MR-)AML

Background RAS signaling pathway mutations, mainly NRAS, KRAS, and/or PTPN11 variants, are common somatic events in de novo and MR-AML. BRAF mutations occur with lower frequency in AML and are reported to be associated with poor treatment outcome. However, no comprehensive analysis exists that firmly delineates clinical and molecular characteristics of BRAF-mutated AML, including insights into clonal architecture and co-occurring mutations. Methods We identified 42 patients (pts) with BRAF-mutated AML among pts with molecularly profiled disease in the setting of large comprehensive cancer centers [enriched for relapsed/refractory (R/R) and secondary AML] and cooperative group frontline trials (Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology, enriched for de novo AML). All pts had NGS-based targeted sequencing data, cytogenetic information, and detailed clinical annotations available. To fully evaluate the molecular landscape, 6 pts underwent integrated genomic profiling including paired tumor/normal whole exome sequencing and total transcriptome sequencing. We also analyzed pt samples using simultaneous single-cell molecular profiling and cell surface protein expression (DNA+Protein) sequencing to assess cell states and clonal make-up at single-cell resolution in a subset of pts (n=8). Results The 42 pts with BRAF-mutated AML had a median age of 67 years (range, 19-84); 57% were male. BRAF mutations were most common in cases of MR-AML (as defined by WHO 5th edition), accounting for 32 (76%) pts. BRAF mutations were found in newly diagnosed (n=19, 45%), R/R (n=8, 19%) and secondary AML (n=15, 36%) pts. As detected by bulk NGS sequencing, BRAF mutations were found both as dominant clonal and subclonal events, present at variant allele fractions (VAF) of 1-83% ( Figure 1, BRAF VAF indicated via color scale). Most (n=30, 71%) pts had non-V600 BRAF mutations (including G469, n=12; D594, n=7 and other, n=12). The most frequent co-occurring mutations were in the TET2 (36%), ASXL1 (33%), NRAS (29%), KRAS (26%), RUNX1 (19%), DNMT3A, FLT3-ITD/TKD, NPM1 and SRSF2 (all 17%) genes. By whole exome sequencing, BRAF-mutated AML samples carried a median mutational burden of 15 non-synonymous coding variants (range, 9-30), including 6 RAS-pathway and 3 MR-AML mutations. In 2 pts with paired relapse material available, the BRAF mutation was either stable at relapse or lost and the pt instead acquired a IQGAP3 mutation, supporting the RAS-pathway “addiction” of this leukemia. Single-cell multi-omic sequencing studies uncovering unique genotype-immunophenotype relationships and confirming clonal hierarchies are ongoing and will be presented at the meeting. Clinically, the BRAF-mutated pts had extremely poor survival, regardless of therapy. Pts were treated with low intensity [LI, n=10 (29%); LI+venetoclax (VEN, n=10 (29%)], high-intensity [HI, n=12 (34%) or HI+VEN, n=3 (9%)] regimens. The overall composite remission (CR+CRi) rate was 39%. Median overall survival (OS) for the entire cohort was 7 months, with 31 (89%) of pts deceased at the time of last follow-up. There were no significant differences in OS based on treatment regimens ( Figure 2). Importantly, there was no impact of BRAF variant allele burden on OS, with pts wiith dominant clonal and those with subclonal mutations having similar outcomes. Given the enrichment of BRAF mutations in MR-AML, we assessed whether RAS pathway mutations generally represented poor outcome prognosticators in this subgroup. A comparison of a control cohort of MR-AML pts with (n=129) and without (n=403) RAS pathway mutations treated on cytarabine/daunorubicin-based frontline protocols, revealed no survival difference between RAS-mutated and wild-type pts, suggesting a negative survival impact specific to BRAF-carrying leukemic clones. Conclusions BRAF mutations are rare, but recurrent molecular alterations in AML that are enriched in MR-AML. They associate with distinctly poor prognosis regardless of their clonal burden, without significant therapeutic advantage of currently available treatment regimens. This suggests the need to assess the utility of BRAF inhibitors and/or RAS pathway-targeting regimens such as MEK inhibitors in pts with AML carrying BRAF mutations. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202)

Secondary-Type Mutations Do Not Impact the Favorable Outcome of NPM1-Mutated Acute Myeloid Leukemia Patients - Results from a Large Cohort of Intensively Treated Patients

Introduction: In 2022, the ELN risk classification for AML was updated for the second time. One of the major novelties of the ELN2022 is that all secondary-type mutations (STMs, i.e., mutations in the genes SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2) were now added to the adverse risk characteristics. However, a pertinent question also raised by the ELN expert panel is whether STMs abrogate the positive prognostic value of co-occurring, favorable NPM1 mutations. Aim: The aim of this study was to analyze the prognostic value of STMs in AML patients (pts) who also harbor an NPM1 mutation. Methods: We investigated a pooled cohort of 936 NPM1-mutated AML pts who were treated in previously reported multicenter trials of the Study Alliance Leukemia or the AML Cooperative Group. Eligibility was determined based on diagnosis of non-APL, age ≥ 18 years, NPM1 mutation detected in targeted sequencing, curative treatment intent, and available biomaterial at diagnosis. Standard techniques for chromosome banding and fluorescence-in-situ-hybridization (FISH) were used for karyotyping. Next-generation panel sequencing was performed to detect genetic alterations that are recurrently found in myeloid neoplasms. Results: In our multicenter cohort of 936 NPM1-mutated AML pts, median follow-up for the entire cohort was 8.0 years. We found 125 patients (13.4%) harboring at least one STM ( SRSF2 [n=48; 5.1%], STAG2 [n=32; 3.2%], EZH2 [n=22, 2.4%], BCOR [n=16; 1.7%], SF3B1 [n=13; 1.4%], ASXL1 [n=12; 1.3%], ZRSR2 [n=5; 0.5%], and U2AF1 [n=4; 0,4%]). A comparison of pretreatment clinical and genetic features revealed that pts with a STM were significantly older ( p=.003, median 59 vs. 55 years), had lower white blood cell counts ( p&amp;lt;.001, 22.2*10 9/L vs. 39.7*10 9/L,) and platelet counts ( p&amp;lt;.001, 46.5*10 9/L vs. 65.0 10 9/L). The strongest pair-wise associations between gene mutations were observed between U2AF1 and RUNX1 ( p&amp;lt;.001) as well as SRSF2 and IDH2 ( p&amp;lt;.001). With respect to outcome, complete remission (CR) rate did not differ significantly between NPM1-mutated patients with or without additional STMs ( p=.41, 74.4% vs. 77.7%, OR 0.83 [95%-CI 0.54-1.29]). Median RFS for NPM1-mutated pts with STMs was 32.9 months (95%-CI: 13.0-46.0) while patients without STMs had a median RFS of 24.3 months (95%-CI: 18.7-33.3) corresponding to a HR of 1.04 ( p=0.80, 95%-CI 0.79-1.37; Figure A). Median OS for NPM1-mutated pts with or without STMs was 27.2 months (95%-CI: 14.2-49.0) and 29.1 months (95%-CI: 23.5-41.4), respectively, corresponding to a HR of 1.11 ( p=.37, 95%-CI 0.88-1.41; Figure B). To focus solely on the impact of STMs, we subsequently excluded patients with co-occurring mutations in TP53 or myelodysplasia-related cytogenetics, which all define an ELN adverse risk. Again, we observed no differences in CR rate ( p=.54, 78% vs. 75.4%), RFS ( p=.59, median 33.2 months vs. 26.6 months), or OS ( p=.33, median 27.4 month vs. 32.6 month) between NPM1-mutated patients with or without STMs. Next, we restricted our analysis to pts who are classified favorable risk according to ELN2022. Again, we found no significant outcome differences based on the STM status (unmutated vs. mutated: CR rate, 80% vs. 70.7% [ p=.072]; RFS, median 49.7 months vs. 46.0 months [ p=.702]; OS, median 45.3 months vs. 59.8 months [ p=.092]). Conclusion: NPM1 mutations rank as the second most frequent mutations in AMLand the most common in patients with a normal karyotype and serve as an established favorable prognostic marker. Our data from a large cohort demonstrate that additional STMs have no adverse effect on the clinical outcome of NPM1-mutated patients. As a result, these patients should still be considered ELN favorable risk.

Molecular Classification of Chronic Myelomonocytic Leukemia: Results of the Analysis of an International Cohort of 2,471 Patients

Background. Chronic myelomonocytic leukemia (CMML) is a rare myeloid neoplasm that shares both dysplastic and proliferative features and includes patients with highly heterogeneous clinical manifestations and prognosis. Despite known co-mutation patterns that are suggesting of a CMML diagnosis such as TET2 and SRSF2 mutations, the current diagnosis and classification schemes rely solely on morphological criteria that fail to capture the genomic heterogeneity of the disease. Here we perform an unsupervised analysis using only genomic features to generate a novel classification schema of CMML patients with unique clinical features. Methods. A cohort of 2,471 CMML patients defined according to WHO 2016 criteria from European and US centers was analyzed. Bayesian network analysis and hierarchical Dirichlet processes were used to identify genomic associations and subgroups as a basis to define a molecular classification of CMML. Bayesian networks allow to infer the structure of conditional dependencies among mutations. Dirichlet processes were applied to define clusters capturing broad dependencies among all gene mutations and cytogenetic abnormalities. Multivariate logistic regression analysis was applied to compare clinical and hematological characteristics among different groups. ( JCO 2021 39:11, 1223-1233) Results. Baseline clinical and molecular characteristics are summarized in Table 1. We identified 11 clusters defined according to specific genomic features (Table 2). Two clusters were primarily defined by the presence of mutations in splicing genes SRSF2 and ZRSR2, frequently associated with TET2 mutations. Patients assigned to these clusters showed myelodysplastic features according to French American British (FAB) criteria, had a low rate of peripheral blood cytopenias, and low blast counts. Additionally, two clusters were characterized by the same mutations in splicing genes (i.e., SRSF2 or ZRSR2 with associated TET2), together with co-mutations in genes that correlate with a more aggressive phenotype ( RUNX1, ASXL1, EZH2). These groups showed overlapping myelodysplastic and myeloproliferative features by FAB, intermediate-risk per prognostic scores, and decreased survival as compared with the first 2 clusters. A distinct cluster was characterized by SF3B1 gene mutations (frequently associated with TET2 or DNMT3A). The SF3B1 cluster was marked by a dysplastic phenotype and more severe anemia (with a high rate of transfusion-dependence) with respect to the other genomic groups. Four clusters (27.8% of the overall population) were characterized by the presence of mutations in signal transduction and tyrosine kinase pathways genes ( NRAS/KRAS, CBL, JAK2 and SETBP1 often associated with ASXL1/TET2, respectively). Patients within these groups showed a mostly proliferative phenotype and intermediate survival. The presence of JAK2 mutations appeared to confer a better prognosis compared with other proliferative clusters. A minority of patients (4.6%) were assigned to two high-risk clusters, defined by TP53 mutations (often biallelic) with complex karyotype, and AML-like mutations ( NPM1, IDH1/2, FLT3), respectively. Patients in these clusters were predominantly dysplastic by FAB, had a high prevalence of severe cytopenias, higher blast counts, and a dismal outcome. Finally, we identified a cluster of patients defined by the absence of gene mutations and chromosomal abnormalities (4.5%). The majority of patients assigned to this cluster were characterized by a dysplastic phenotype, had low absolute monocyte counts, and favorable outcome. A subset of patients (18.9%) could not be assigned to a specified cluster in this preliminary analysis. An independent validation of this molecular classification is currently being conducted on a US prospective cohort (n=650). Conclusion. Our results provide proof of concept for a molecular classification of CMML based on the identification of homogeneous genomic signatures and associated clinical features. 15% of patients showed clear overlapping features with other myeloid neoplasms, thus providing the rational to refine the boundaries among different clinical entities. A molecular classification of CMML may provide a basis to define a next-generation prognostic tool and improve clinical decision-making.

Drivers and high-molecular-risk mutations in Argentine patients with primary myelofibrosis

Primary myelofibrosis (PMF), a BCR-ABL negative myeloproliferative neoplasm, is a heterogeneous clinical and genetic disorder with a poor prognosis. We aimed to study the mutational profile of driver genes (JAK2, CALR, and MPL) and high-molecular-risk (HMR) genes (ASXL1, EZH2, IDH1/2, and SRSF2) and their prognostic impact in 65 Argentine patients with PMF. Mutually exclusive driver mutations were identified in 88% of the cases and HMR mutations were detected in 37% of patients, including 37.5% of triple negative cases. ASXL1 and SRSF2 were the most frequently mutated HMR genes, and a significant association between these mutations was observed (P = 0.04). Additionally, age &gt;65 years, lower hemoglobin level, constitutional symptoms, male sex, and bone marrow (BM) fibrosis grade ≥ 2 were correlated with ASXL1 mutations, and circulating blasts with SRSF2 mutations. HMR mutations were significantly associated with inferior overall survival (P &lt; 0.001). These mutations were more frequent with increasing risk of DIPSS score and allowed us to identify patients with poor survival in intermediate DIPSS groups. The application of the MIPSS70 score stratified our patients into three risk groups, with significant differences in overall survival (P &lt; 0.001). Interestingly, 85% of the patients who were upgraded to a higher risk category compared to the DIPSS score showed a HMR profile. In conclusion, our study contributes to the understanding of the mutational landscape in MFP, supports the integration of molecular data to improve prognostic models and reinforces the adverse prognostic impact of HMR mutations, particularly in ASXL1 and SRSF2 genes.

The clinical relevance of broad mutational screening of myeloproliferative neoplasms at diagnosis.

Myeloproliferative neoplasm (MPN) is a heterogenous group of hematological malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F is the most frequent driver mutation in all three entities, but in PMF and ET mutations in CALR and MPL are also frequent. Mutations seen in additional genes are also often the same regardless of subtype of MPN. The aim of this study was to analyze a population based MPN cohort for genetic variants with prognostic value that can guide clinical decisions. MPN patients from Western Sweden diagnosed between 2008-2013 (n=248) were screened for mutations in 54 genes associated with myeloid malignancy. Mutations in the genes SRSF2 and U2AF1 correlated significantly with impaired overall survival but did not correlate to increased risk for vascular events, neither before nor after diagnosis. Rather, mutations in these genes showed an association with disease transformation. Several recurrent gene variants with allele frequency close to 50% were confirmed to be germline. However, none of these variants was found to have an earlier onset of MPN. In conclusion, we identified gene mutations to be independent markers of impaired survival in MPN. This indicates the need for more individualized assessment and treatment of MPN patients and a wider gene mutation screening already at diagnosis. This could ensure the identification of patients with high-risk mutations early on. In addition, several genetic variants were also identified as germline in this study but gave no obvious clinical relevance. To avoid conclusions from non-informative genetic variants, a simultaneous analysis of normal cell DNA from patients at diagnosis should be considered.

Venetoclax abrogates the prognostic impact of splicing factor gene mutations in newly diagnosed acute myeloid leukemia

AbstractMutations in splicing factor (SF) genes SRSF2, U2AF1, SF3B1, and ZRSR2 are now considered adverse risk in the European LeukemiaNet 2022 acute myeloid leukemia (AML) risk stratification. The prognostic impact of SF mutations in AML has been predominantly derived from younger patients treated with intensive (INT) therapy. We evaluated 994 patients with newly diagnosed AML, including 266 (27%) with a SFmut. Median age was 67 years overall, with patients with SFmut being older at 72 years. SRSF2 (n = 140, 53%) was the most common SFmut. In patients treated with INT, median relapse-free survival (RFS) (9.6 vs 21.4 months, P = .04) and overall survival (OS) (15.9 vs 26.7 months, P = .06) were shorter for patients with SFmut than without SFwt, however this significance abrogated when evaluating patients who received venetoclax with INT therapy (RFS 15.4 vs 20.3 months, P = .36; OS 19.6 vs 30.7 months, P = .98). In patients treated with LI, median RFS (9.3 vs 7.7 months, P = .35) and OS (12.3 vs 8.5 months, P = .14) were similar for patients with and without SFmut , and outcomes improved in all groups with venetoclax. On multivariate analysis, SFmut did not affect hazards of relapse and death for INT arm but reduced both these hazards in LI arm. In a large AML data set with >60% of patients receiving venetoclax with LI/INT therapy, SFmut had no independent negative prognostic impact. Newer prognostic models that consider LI therapy and use of venetoclax among other factors are warranted.

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.

Specific Nucleotide Substitutions and Stressors Erode Highly Conserved Genomic Regions in Acute Myelogenous Leukemia

Background: "Leukemia stressors” refer to extrinsic factors that may facilitate clonal progression. We previously reported that smoking initiates unique leukemogenic signatures in acute myelogenous leukemia (AML) characterized by a higher proportion of C>A and G>A single base substitutions (SBS) [Hoang. ASH. 2021]. These G>A SBS are known to be particularly prevalent in CpG islands due to cytosine methylation. However, toxic smoking metabolites could potentially induce specific genomic signatures in patients with AML that resemble COSMIC SBS4 in lung cancer [i.e. enrichment in C>A transversion], and spontaneous C>T SBS1 signatures within specific genomic clusters or nodes. In this study, we investigate how these SBS1/SBS4 signatures distribute within hot-spot regions and evaluate whether G>A mutations (mut), commonly observed in methylated CpG islands, are differentially expressed in AML patients who are older and/or are smokers. In our study, these hot-spot regions or nodes were referenced as a genomic pool 1 (GP1). Methods: After IRB approval, the incidence of specific SBS densities were investigated in vulnerable hot-spot regions affected by exogenous [SBS4] and endogenous [SBS1] mutagenesis. Next generation sequencing (NGS) was used in patients to obtain data regarding specific mutation types and locations. In known gene exons, we examined genomic coordinates corresponding to individual mut. COSMIC browser allowed genomic coordinate examination to "aggregate” mut within putative "vulnerable exon neighborhoods". "Hot-spot neighborhoods” were described as genomic pool 1 (GP1). Descriptive and inferential statistics were used to analyze data, which was obtained from a single institution. Results: 80 patients (pt) diagnosed with hematopoietic malignancies with available next generation sequencing (NGS) were selected for analysis. 73/80 (91%) were AML pt. The median age was 63 years (y) (range, 22-88). 42% and 64% of pt were smokers and older than 63 y, respectively. With respect to specific hot spots or nodes, AML pt showed increased G>A mutagenicity in GP1 cluster 1 (Fig 1). Specifically, for G>A, smoking increased the rate from 18.4% to 37.5% (p=0.01) (Fig 1 A and B). Age > 63 y led to an increased mutagenicity rate of 17.1% to 30.5% (p=0.03) (Fig 1 C and D). A synergistic effect in mutagenicity was observed when combining age > 63 y plus smoking (10.3% to 41.9%, p=0.01) (Fig 1. E and F). In regards to hotspots in specific genes, smoking has the ability to increase NRAS (G>A), P53 (G>A) and SRSF2 (C>A) frequencies within GP1. Interestingly, smoking increased C>A mutagenicity in the SRSF2 gene in GP1 from 50% vs 82%. Conclusions: Our data suggests that aging and smoking globally and locally erode the AML genome by potentially accelerating cytosine methylation and thus G>A mutagenicity, most likely in CpG islands within genes like NRAS and P53. These genes likely contain hot spots aggregated in GP1 that, when mutated, can de-stabilize the genome and increase the risk of AML. Importantly, we observed similar COSMIC SBS4 substitutions seen in lung cancer associated with C>A transversions in older AML pt who had a history of smoking, which is specifically found in the SRSF2 gene. Overall, our data advances our understanding on how risk factors for AML affect the human AML genome. These have strong implications for AML prevention, pathogenesis and therapy. Future studies should be aimed at identifying other intrinsic and extrinsic risk factors for AML seeking to explore their specific mutational patterns and locations within the human genome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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).

Disease-related thrombocytopenia in myelofibrosis is defined by distinct genetic etiologies and is associated with unique prognostic correlates.

Thrombocytopenia in patients with myelofibrosis (MF) is prognostically detrimental and poses a therapeutic challenge. MF patients with thrombocytopenia are considered high-risk by most prognostic models and their distinct phenotype has given rise to the emerging concept of cytopenic MF. Yet, the mechanisms underlying thrombocytopenia in MF are poorly understood. This study aimed to highlight the genetic mechanisms driving low platelet counts in treatment-naive MF patients, establish their phenotypic correlates, and assess prognostic factors specific to this group of patients. The authors found that most patients presenting with low platelets had a clear thrombocytopenia-specific genetic abnormality involving a U2AF1 Q157 mutation, deletion 20q, molecular complexity (three or more mutations), or high-risk karyotype. Etiologic clustering did not correlate with prognosis; however, thrombocytopenic patients were found to have unique prognostic variables including low serum albumin and mutations of SRSF2 and TP53. This led to the proposal of a prognostic model (SRSF2, albumin, TP53 score) that stratifies thrombocytopenic patients as low, intermediate, or high-risk with corresponding median survivals of 93.5, 29.5, and 7.2 months, respectively. This study demonstrates that thrombocytopenia in MF is driven by different genetic mechanisms and is not uniformly high-risk. As novel agents with improved hematologic safety profiles enter the treatment landscape, thoughtful, risk-adapted therapeutic decisions will be required for MF patients with thrombocytopenia. A significant minority of patients with myelofibrosis (MF) present with low platelets. Historically, these patients have been viewed as having "high-risk" disease, but this may not be uniformly true. Our study shows that there are various different causes for low platelets in MF, some of which represent high-risk disease whereas others do not. Additionally, our study shows that genetic mutations affecting the genes SRSF2 and TP53 are uniquely problematic in this group, as is a low serum albumin level. This study helps to risk-stratify MF patients with thrombocytopenia, thereby providing more information to guide informed and individualized treatment decisions.

White cell deficiency and TET2 and SRSF2 gene mutations

No abstract in English Цагаан эсийн дутагдал ба TET2, SRSF2 генийн мутаци

Comparative Outcomes and Molecular Response Predictors of IDH1/2-Mutated Adult Acute Myeloid Leukemia (AML) Patients (Pts) after Frontline Treatment with Intensive Induction Chemotherapy (IC), Targeted Inhibitors, or Hypomethylating Agents (HMA) (Alliance)

Comparative Outcomes and Molecular Response Predictors of IDH1/2-Mutated Adult Acute Myeloid Leukemia (AML) Patients (Pts) after Frontline Treatment with Intensive Induction Chemotherapy (IC), Targeted Inhibitors, or Hypomethylating Agents (HMA) (Alliance)