Adoptive transfer of ex vivo expanded SARS-CoV-2-specific cytotoxic lymphocytes: A viable strategy for COVID-19 immunosuppressed patients?

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients.

Partial T Cell-Depleted Peripheral Blood Stem Cell Transplantation from HLA-Identical Sibling Donors for Patients with Severe Aplastic Anemia.

We analyzed the outcomes of 26 consecutive patients with acquired severe aplastic anemia (SAA) undergoing peripheral blood stem cell transplantation (PBSCT) with partial ex vivo T cell depletion with a targeted T cell dose from HLA-identical sibling donors. The median patient age was 37 years (range, 3 to 63 years). Four patients with uncontrolled pneumonia at the time of transplantation died, on days +1, +2, +21, and +26. All evaluable patients engrafted, with a median time to neutrophil recovery of 11 days (range, 10 to 14 days) and a median time to platelet recovery of 19 days (range, 8 to 53 days). Two patients had transient grade I acute graft-versus-host disease (GVHD) with skin involvement, but no patients developed grade II-IV acute GVHD. Two patients had mild skin chronic GVHD, and 1 patient had moderate chronic GVHD with ocular involvement. No relapse was observed after a median follow-up of 114 months (range, 4 to 233 months). The overall cumulative incidence of TRM at 10 years was 19%, whereas it was 5% for those with a Karnofsky Performance Status (KPS) score >60 at the time of transplantation. Disease-free survival, overall survival, and GVHD and relapse-free survival at 10 years were 81%, 81%, and 80%, respectively, for all patients and 95%, 95%, and 90%, respectively, for patients with a KPS score >60 at transplantation. Our data indicate that PBSCT with partial ex vivo T cell-depleted targeted cell dose grafts from an HLA-identical sibling donor is a feasible, safe, and effective approach to reduce GVHD and cure patients with SAA.

RNA Sequencing Analysis for the Identification of a PCM1/PDGFRB Fusion Gene Responsive to Imatinib.

The platelet-derived growth factor receptor ß (PDGFRB) gene translocations lead to a spectrum of chronic myeloid neoplasms, frequently associated with eosinophilia. Clinical heterogeneity is associated with a molecular one. Here, we report a novel case of a patient harboring a t(5;8)(q33;p22) translocation, resulting in the PCM1/PDGFRB fusion. Conventional cytogenetics and RNA sequencing were performed to identify the chromosomes and the genes involved in the rearrangement, respectively. This study shows that the combination of different strategies is pivotal to fine-tune the diagnosis and the clinical management of the patient. After 1 year of treatment with imatinib, the patient achieves hematological and molecular remission. We present an attractive strategy to identify novel and/or cryptic fusions, which will be relevant for clinicians dealing with the diagnosis of the patients with myelodysplastic syndrome/myeloproliferative diseases with atypical manifestations.

WHO/ICC Classification for Myelodysplastic Neoplasms/Syndromes Performs Better for Subtype Cytomorphological Diagnosis?

The International Consensus Classification of Myeloid Neoplasms and Acute Leukemias (ICC) and the 5th edition of the WHO classification (WHO 2022) have refined the diagnosis of myelodysplastic syndromes (MDS). Both classifications segregate MDS subtypes based on molecular or cytogenetic findings but rely on the subjective assessment of blast cell percentage and dysplasia in hematopoietic cell lineages. This study aimed to evaluate interobserver concordance among 13 cytomorphologists from eight hospitals in assessing blast percentages and dysplastic features in 44 MDS patients. The study found fair interobserver agreement for the PB blast percentage and moderate agreement for the BM blast percentage, with the best concordance in cases with <5% BM blasts and >10% BM blasts. Monocyte count agreement was fair, and dysplasia assessment showed moderate concordance for megakaryocytic lineage but lower concordance for erythroid and granulocytic lineages. Overall, interobserver concordance for MDS subtypes was moderate across all classifications, with slightly better results for WHO 2022. These findings highlight the ongoing need for morphological evaluation in MDS diagnosis despite advances in genetic and molecular techniques. The study supports the blast percentage ranges established by the ICC but suggests refining BM blast cutoffs. Given the moderate interobserver concordance, a unified classification approach for MDS is recommended.

A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia.

Chromosomal translocations in hematological malignancies often result in novel fusion chimeric genes. We report a case of acute myeloid leukemia with a clonal translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. The gene fused to NUP98 (nucleoporin 98) was detected by comparative genomic hybridization array as the retinoid acid receptor gamma gene (RARG). The involvement of RARG in a chimeric fusion transcript has not been reported previously in human leukemia.

Cytogenetic Assessment and Risk Stratification in Myelofibrosis with Optical Genome Mapping.

Cytogenetic assessment in myelofibrosis is essential for risk stratification and patient management. However, an informative karyotype is unavailable in a significant proportion of patients. Optical genome mapping (OGM) is a promising technique that allows for a high-resolution assessment of chromosomal aberrations (structural variants, copy number variants, and loss of heterozygosity) in a single workflow. In this study, peripheral blood samples from a series of 21 myelofibrosis patients were analyzed via OGM. We assessed the clinical impact of the application of OGM for disease risk stratification using the DIPSS-plus, GIPSS, and MIPSS70+v2 prognostic scores compared with the standard-of-care approach. OGM, in combination with NGS, allowed for risk classification in all cases, compared to only 52% when conventional techniques were used. Cases with unsuccessful karyotypes (n = 10) using conventional techniques were fully characterized using OGM. In total, 19 additional cryptic aberrations were identified in 9 out of 21 patients (43%). No alterations were found via OGM in 4/21 patients with previously normal karyotypes. OGM upgraded the risk category for three patients with available karyotypes. This is the first study using OGM in myelofibrosis. Our data support that OGM is a valuable tool that can greatly contribute to improve disease risk stratification in myelofibrosis patients.

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia.

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4 %) patients. At least one copy of the minor SNP rs16754 allele (WT1(AG) or WT1(GG)) was detected in 30 (29 %) patients. Six patients (6 %) were homozygous for the minor allele (WT1(GG)) and this genotype was associated with higher WT1 mRNA copies (p = 0.018). FLT3 mutations were found in 37 % of patients and correlated with high WT1 mRNA expression (p = 0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1(AA)) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.

Analysis of SNP rs16754 of WT1 gene in a series of de novo acute myeloid leukemia patients.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

Prognostic value of cytogenetics in adult patients with Philadelphia-negative acute lymphoblastic leukemia.

The prognostic value of cytogenetics in adult acute lymphoblastic leukemia (ALL) is not as established as in childhood ALL. We have analyzed the outcome and prognostic value of karyotype in 84 adults diagnosed with Philadelphia-negative ALL from a single institution that received induction chemotherapy and had successful karyotype performed. The most frequent finding was normal karyotype in 35 (42%) cases, followed by aneuploidies in 20 cases (24%) and t(4;11)(q21;q23)/MLL/AF4 in 5 (6%), and the remaining 24(27%) cases carried miscellaneous clonal abnormalities. The group of patients with t(4;11)(q21;q23)/MLL/AF4, hypodiploidy and low hyperdiploidy (less than 50 chromosomes) showed a worse outcome than those with normal karyotype and miscellaneous abnormalities in terms of overall survival (OS) (3 years OS; 47% vs. 13%, p = 0.014) and relapse-free survival (RFS) (3 years RFS; 44% vs. 27%, p = 0.005). Other cytogenetic prognostic classifications reported to date were tested in our series, but any was fully reproducible. In conclusion, karyotype is a useful tool for risk assessment in adult ALL. We have confirmed the bad prognosis of t(4;11)(q21;q23)/MLL/AF4 and hypodiploidy. Besides, low hyperdiploidy could also define a high-risk group of patients who might be candidates for more intensive treatment.

Panel Sequencing for Clinically Oriented Variant Screening and Copy Number Detection in Chronic Lymphocytic Leukemia Patients.

According to current guidelines, in chronic lymphocytic leukemia (CLL), only the TP53 molecular status must be evaluated prior to every treatment's initiation. However, additional heterogeneous genetic events are known to confer a proliferative advantage to the tumor clone and are associated with progression and treatment failure in CLL patients. Here, we describe the implementation of a comprehensive targeted sequencing solution that is suitable for routine clinical practice and allows for the detection of the most common somatic single-nucleotide and copy number variants in genes relevant to CLL. We demonstrate that this cost-effective strategy achieves variant detection with high accuracy, specificity, and sensitivity. Furthermore, we identify somatic variants and copy number variations in genes with prognostic and/or predictive value, according to the most recent literature, and the tool provides evidence about subclonal events. This next-generation sequencing (NGS) capture-based target assay is an improvement on current approaches in defining molecular prognostic and/or predictive variables in CLL patients.

CYP2C8 gene polymorphism and bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma.

Osteonecrosis of the jaw is an uncommon but potentially serious complication of bisphosphonate therapy in multiple myeloma. Previous studies showed that the presence of one or two minor alleles of the cytochrome P450, subfamily 2C polypeptide 8 gene (CYP2C8) polymorphism rs1934951 was an independent prognostic marker associated with development of osteonecrosis of the jaw in multiple myeloma patients treated with bisphosphonates. The aim of this study was to validate the frequency of SNP rs193451 in 79 patients with multiple myeloma. In 9 (22%) patients developing osteonecrosis of the jaw, a heterozygous genotype was found, in contrast with those who did not develop osteonecrosis of the jaw (n=4, 11%) or healthy individuals (n=6, 13%). We found no differences in the cumulative risk of developing osteonecrosis of the jaw between patients homozygous and heterozygous for the major allele. We were unable to confirm a significant association between this polymorphism and the risk of developing osteonecrosis of the jaw.

Microencapsulation Improved Fumaric Acid and Thymol Effects on Broiler Chickens Challenged With a Short-Term Fasting Period.

The first objective of this study was to demonstrate the usefulness of the microencapsulation technique to protect fumaric acid and thymol, avoiding their early absorption and ensuring their slow release throughout the gastrointestinal tract (GIT). For this purpose, the release of a lipid matrix microencapsulated brilliant blue (BB) was assessed in vitro, using a simulated broiler intestinal fluid, and in vivo. In vitro results showed that more than 60% of BB color reached the lower intestine, including 26.6 and 29.7% in the jejunum and ileum, respectively. The second objective was to determine the effects of microencapsulated fumaric acid, thymol, and their mixture on the performance and gut health of broilers challenged with a short-term fasting period (FP). One-day-old male ROSS 308 chickens (n = 280) were randomly distributed into seven treatments, with 10 replicates of four birds each. Dietary treatments consisted of a basal diet as negative control (NC), which was then supplemented by either non-microencapsulated fumaric acid (0.9 g/kg), thymol (0.6 g/kg), or a mixture of them. The same additive doses were also administered in a microencapsulated form (1.5 and 3 g/kg for the fumaric acid and thymol, respectively). At day 21, chickens were subjected to a 16.5-h short-term FP to induce an increase in intestinal permeability. Growth performance was assessed weekly. At day 35, ileal tissue and cecal content were collected from one bird per replicate to analyze intestinal histomorphology and microbiota, respectively. No treatment effect was observed on growth performance from day 1 to 21 (p > 0.05). Microencapsulated fumaric acid, thymol, or their mixture improved the overall FCR (feed conversion ratio) and increased ileal villi height-to-crypt depth ratio (VH:CD) (p < 0.001) on day 35 of the experiment. The microencapsulated mixture of fumaric acid and thymol increased cecal abundance of Bacteroidetes, Bacillaceae, and Rikenellaceae, while decreasing that of Pseudomonadaceae. These results indicate that the microencapsulation technique used in the current study can be useful to protect fumaric acid and thymol, avoiding early absorption, ensure their slow release throughout the GIT, and improve their effects on fasted broiler chickens.

Proposed global prognostic score for systemic mastocytosis: a retrospective prognostic modelling study.

BACKGROUND: Several risk stratification models have been proposed in recent years for systemic mastocytosis but have not been directly compared. Here we designed and validated a risk stratification model for progression-free survival (PFS) and overall survival (OS) in systemic mastocytosis on the basis of all currently available prognostic factors, and compared its predictive capacity for patient outcome with that of other risk scores. METHODS: We did a retrospective prognostic modelling study based on patients diagnosed with systemic mastocytosis between March 1, 1983, and Oct 11, 2019. In a discovery cohort of 422 patients from centres of the Spanish Network on Mastocytosis (REMA), we evaluated previously identified, independent prognostic features for prognostic effect on PFS and OS by multivariable analysis, and designed a global prognostic score for mastocytosis (GPSM) aimed at predicting PFS (GPSM-PFS) and OS (GPSM-OS) by including only those variables that showed independent prognostic value (p<0·05). The GPSM scores were validated in an independent cohort of 853 patients from centres in Europe and the USA, and compared with pre-existing risk models in the total patient series (n=1275), with use of Harrells' concordance index (C-index) as a readout of the ability of each model to risk-stratify patients according to survival outcomes. FINDINGS: Our GPSM-PFS and GPSM-OS models were based on unique combinations of independent prognostic factors for PFS (platelet count ≤100 × 109 cells per L, serum ß2-microglobulin ≥2·5 µg/mL, and serum baseline tryptase ≥125 µg/L) and OS (haemoglobin ≤110 g/L, serum alkaline phosphatase ≥140 IU/L, and at least one mutation in SRSF2, ASXL1, RUNX1, or DNMT3A). The models showed clear discrimination between low-risk and high-risk patients in terms of worse PFS and OS prognoses in the discovery and validation cohorts, and further discrimination of intermediate-risk patients. The GPSM-PFS score was an accurate predictor of PFS in systemic mastocytosis (C-index 0·90 [95% CI 0·87-0·93], vs values ranging from 0·85 to 0·88 for pre-existing models), particularly in non-advanced systemic mastocytosis (C-index 0·85 [0·76-0·92], within the range for pre-existing models of 0·80 to 0·93). Additionally, the GPSM-OS score was able to accurately predict OS in the entire cohort (C-index 0·92 [0·89-0·94], vs 0·67 to 0·90 for pre-existing models), and showed some capacity to predict OS in advanced systemic mastocytosis (C-index 0·72 [0·66-0·78], vs 0·64 to 0·73 for pre-existing models). INTERPRETATION: All evaluated risk classifications predicted survival outcomes in systemic mastocytosis. The REMA-PFS and GPSM-PFS models for PFS, and the International Prognostic Scoring System for advanced systemic mastocytosis and GPSM-OS model for OS emerged as the most accurate models, indicating that robust prognostication might be prospectively achieved on the basis of biomarkers that are accessible in diagnostic laboratories worldwide. FUNDING: Carlos III Health Institute, European Regional Development Fund, Spanish Association of Mastocytosis and Related Diseases, Rare Diseases Strategy of the Spanish National Health System, Junta of Castile and León, Charles and Ann Johnson Foundation, Stanford Cancer Institute Innovation Fund, Austrian Science Fund.

Cord blood transplantation from unrelated donors in adults with high-risk acute myeloid leukemia.

Clinical studies focused on disease-specific outcomes of cord blood transplant (CBT) from unrelated donors are limited. We analyzed the outcome and prognostic factors of 49 adults with high-risk acute myelogenous leukemia (AML) receiving single-unit CBT from unrelated donors after myeloablative (MA) conditioning at a single institution. Conditioning regimens were based on the combination of thiotepa, busulfan (Bu), cyclophospamide (Cy), or fludarabine (Flu), and antithymocyte globulin (ATG). Cumulative incidence of myeloid and platelet engraftment was 96% and 73% at a median time of 20 and 62 days, respectively. Engraftment was significantly faster for patients receiving higher doses of CD34(+) cells. Confidence Interval of graft-versus-host disease (GVHD), acute GVHD (aGVHD) grade II-IV, III-IV, and extensive chronic GVHD (cGVHD) were 26%, 15%, and 30%, respectively. Leukemia-free survival (LFS), nonrelapse mortality (NRM), and relapse at 2 years were 42%, 39%, and 19%, respectively. Low number of total nucleated cells (TNC) had a negative impact on NRM and LFS. Patients transplanted in first complete remission (CR1) receiving TNC above 2 x 10(7)/kg had a 4-year LFS of 75%. These results show that CBT from unrelated donors is a curative treatment for a substantial number of patients with high-risk AML, particularly if transplant is performed with highly cellular units in patients in first CR.

Targeted-Release Organic Acids and Essential Oils Improve Performance and Digestive Function in Broilers Under a Necrotic Enteritis Challenge.

An experiment was performed to evaluate the effect of four different microencapsulated blends of organic acids (OA) and nature-identical aromatic compounds (AC) on growth performance and gut health of broilers challenged with a recycled NE litter. A total of 600 one-day-old male Ross 308 broilers were randomly assigned to five treatments consisting of a basal diet (as negative control) supplemented with each of the tested microencapsulated blends: OA1 (malic and fumaric acid) + AC; 2.5 g/kg; OA2 (calcium butyrate+fumaric acid) + AC; 1.7 g/kg; MCFA (capric-caprylic; caproic and lauric acid) + AC; 2 g/kg; and MCFA + OA3(calcium butyrate + fumaric and citric acid) + AC; 1.5 g/kg. The AC used was the same for all treatments; including cinnamaldehyde, carvacrol, and thymol (8:1:1), as major compounds. Three tested blends enhanced growth performance by improving intestinal histomorphology (p < 0.001). The tested blends enhanced the abundance of some beneficial families such as Ruminococcaceae and Lachnospiraceae; while reducing that of harmful ones such as Enterobacteriaceae and Helicobacteraceae. A further dose-response experiment showed that 0.5 g/kg of the blend 2 and 2 g/kg of the blend 4 improved growth performance and intestinal histomorphology of chickens on d 42 and decreased fecal Enterobacteriaceae and C. perfringens counts. Similar effects to the previous experiment were observed for cecum microbiota.

Unravelling the Role of Rumen Microbial Communities, Genes, and Activities on Milk Fatty Acid Profile Using a Combination of Omics Approaches.

Milk products are an important component of human diets, with beneficial effects for human health, but also one of the major sources of nutritionally undesirable saturated fatty acids (SFA). Recent discoveries showing the importance of the rumen microbiome on dairy cattle health, metabolism and performance highlight that milk composition, and potentially milk SFA content, may also be associated with microorganisms, their genes and their activities. Understanding these mechanisms can be used for the development of cost-effective strategies for the production of milk with less SFA. This work aimed to compare the rumen microbiome between cows producing milk with contrasting FA profile and identify potentially responsible metabolic-related microbial mechanisms. Forty eight Holstein dairy cows were fed the same total mixed ration under the same housing conditions. Milk and rumen fluid samples were collected from all cows for the analysis of fatty acid profiles (by gas chromatography), the abundances of rumen microbiome communities and genes (by whole-genome-shotgun metagenomics), and rumen metabolome (using 500 MHz nuclear magnetic resonance). The following groups: (i) 24 High-SFA (66.9-74.4% total FA) vs. 24 Low-SFA (60.2-66.6%% total FA) cows, and (ii) 8 extreme High-SFA (69.9-74.4% total FA) vs. 8 extreme Low-SFA (60.2-64.0% total FA) were compared. Rumen of cows producing milk with more SFA were characterized by higher abundances of the lactic acid bacteria Lactobacillus, Leuconostoc, and Weissella, the acetogenic Proteobacteria Acetobacter and Kozakia, Mycobacterium, two fungi (Cutaneotrichosporon and Cyphellophora), and at a lesser extent Methanobrevibacter and the protist Nannochloropsis. Cows carrying genes correlated with milk FA also had higher concentrations of butyrate, propionate and tyrosine and lower concentrations of xanthine and hypoxanthine in the rumen. Abundances of rumen microbial genes were able to explain between 76 and 94% on the variation of the most abundant milk FA. Metagenomics and metabolomics analyses highlighted that cows producing milk with contrasting FA profile under the same diet, also differ in their rumen metabolic activities in relation to adaptation to reduced rumen pH, carbohydrate fermentation, and protein synthesis and metabolism.

Impact of azacitidine on red blood cell alloimmunisation in myelodysplastic syndrome.

BACKGROUND: The incidence of alloimmunisation in myelodysplastic syndromes (MDS) during the era of supportive treatment ranges from 9 to 56%. However, it is unknown if the widespread use of hypomethylating agents has changed the risk of immunisation. The aim of this study is to evaluate the impact of azacitidine (AZA) therapy on red blood cell (RBC) alloimmunisation in transfused patients with MDS, myelodysplastic syndromes/myeloproliferative syndromes (MDS/MPS) and secondary acute myeloid leukaemia (AML). MATERIAL AND METHODS: We have analysed retrospectively all patients with MDS, MDS/MPS and secondary AML from MDS, who received their first transfusion in our hospital between January 1995 and December 2014. We have assessed the impact of age, sex, RBC and platelets units transfused, and AZA treatment on developing alloantibodies. RESULTS: In our study, the number of RBC units transfused increased the risk of developing alloantibodies. However aging and the treatment with AZA were associated with a lower rate of alloimmunisation. DISCUSSION: Patients with MDS, MDS/MPS and secondary AML who received treatment with AZA developed RBC antibodies at a lower rate than control group. We suggest that aging and immunosuppression due to AZA therapy could develop an immunological tolerance with a weak response to allotransfusions.

Negative impact on clinical outcome of the mutational co-occurrence of SF3B1 and DNMT3A in refractory anemia with ring sideroblasts (RARS).

The incidence of SF3B1 mutations in patients with RARS is high. Recently, it has been shown that SF3B1 and DNMT3A mutations overlap more often than expected, although it is not clear how this could affect the disease. We studied SF3B1 and DNMT3A in 123 RARS patients: 101 out of 123 samples (82%) had somatic mutations in SF3B1, and 13 of them (13%) showed a co-mutation (SF3B1mutDNMT3Amut). All co-mutated patients had a normal karyotype, and 12 of them (92%) were lower-risk patients (IPSS and IPSS-R). Despite their favorable profile, SF3B1mutDNMT3Amut patients showed a higher RBC transfusion dependency (92% versus 48%, p = .007), a shorter overall survival (OS) (median, 30 versus 97 months, p = .034), and a higher risk of progression to acute myeloid leukemia (AML) at 5 years (25% versus 2%, p = .023) than SF3B1mutDNMT3Awt patients. In conclusion, DNMT3A mutations are present in a significant proportion of SF3B1mut patients with a negative clinical impact.