SF3B1 Mutations Research Articles

26P Impact of SF3B1 mutation on response to immunotherapy

Tumor-derived genetic alterations can regulate tumor microenvironment (TME) and host immune responses via activation of oncogenic pathways. Therefore, detection of somatic mutations associated with TME and tumor immunity may be useful in identifying patients who could benefit from immunotherapy. Immune checkpoint inhibitors (ICIs), a type of immunotherapy, are considered to be the emerging standard of care for various cancers. However, biomarkers that can help in predicting response to ICIs remain unknown.

Prognostic correlation of NOTCH1 and SF3B1 mutations with chromosomal abnormalities in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is a monoclonal malignancy of B lymphocytes. Since common mutations in NOTCH1 and SF3B1, along with other possible chromosomal alterations, change disease severity and survival of patients with CLL, we aimed to evaluate the correlation of common mutations in NOTCH1 and SF3B1 as the poor prognostic markers with chromosomal abnormalities and clinical hematology. This retrospective study was performed on the peripheral blood of 51 patients diagnosed before chemotherapy with CLL. G-banding karyotype and FISH were performed. For NOTCH1, exon 34 and for SF3B1, exons 14,15,16 were assessed using Sanger sequencing. The mutation frequency of NOTCH1 and SF3B1 with the pathogenic clinical status was 6:51 (11.76%), and variants obtained from both genes were 9:51 (17.64%). The frequency of SF3B1 mutation (K666E) was higher than in previous studies (p-value <.05). There was a significant correlation between NOTCH1 mutations and del17p13 (p-value=.068), also SF3B1 mutations with del11q22 (p-value=.095) and del13q14 (p-value=.066). Up to 90% of the specific stimuli used for the G-banding karyotype successfully identified the malignant clone. There was a significant relationship between the cluster of differentiation 38 (CD38) expression level and NOTCH1 mutations (p-value=.019) and a significant correlation between Binet classification and the SF3B1 (p-value=.096). The correlation of NOTCH1 and SF3B1 mutations with chromosomal abnormalities and CD38 expression may reveal the overall patient's survival rate. The mutations may be effective in the clonal expansion and progression of CLL, particularly in the diagnosis stage, as well as the control and management of the treatment.

Characterizing the Development of Secondary Myelodysplastic Syndrome (MDS) with Antecedent or Concurrent Chronic Lymphocytic Leukemia (CLL) in the Era of Small Molecule Inhibitors for the Treatment of CLL

Introduction Secondary MDS (sMDS) is defined by MDS arising from another bone marrow disorder or related to previous therapy [chemotherapy (CT), radiation, or immunosuppression], and comprises about 10% of MDS cases. The rate of sMDS following CLL (including treatment-naïve CLL) has been reported as high as 3.5%. Developing in elderly populations, both CLL and MDS are associated with clonal hematopoiesis (CH), and chemoimmunotherapy (CIT) treatment for CLL likely contributes to the occurrence of MDS. There is little published data on the characteristics of patients (pts) who develop sMDS with antecedent/concurrent CLL, particularly in the era where kinase inhibitor therapy has displaced CIT. Therefore, we sought to characterize a cohort of CLL pts who develop sMDS in the era of modern treatments. Methods We performed a retrospective chart review at The Ohio State University between 01/01/2016 and 02/10/2021 of pts with a diagnosis of CLL who subsequently or concurrently were diagnosed with MDS. Clinical, next generation sequencing (NGS), and outcome data were collected. NGS test refers to a 50 gene hematologic neoplasm mutation panel. Overall survival (OS) was calculated from MDS diagnosis and estimated using Kaplan-Meier method. Results We identified 27 sMDS pts with antecedent/concurrent CLL. At CLL diagnosis, median age was 58 years (range 40-86). 5 pts (18.5%) had a TP53 mutation and/or del(17p) noted during their CLL disease course prior to sMDS diagnosis. Median number of therapies for CLL prior to sMDS was 1 (range 0-10), with 4 pts diagnosed with CLL at the same time as MDS. CLL treatments, as any line of therapy, included CT/CIT in 21 pts (77.8%) and ibrutinib in 10 pts (37.0%). Of the pts that received CLL therapy, only 1 pt did not receive CT/CIT. 5 pts (18.5%) were not treated for CLL. A median of 6.9 years (range 0-31.8) elapsed between the diagnosis of CLL and sMDS. At sMDS diagnosis median age was 70 years (range 50-86), 17 pts (65.4%) were MDS IPSS-R category high/very high. 5 pts transformed to AML after a median of 9.2 months (mos) from MDS diagnosis (range 4.1-21.3). After a median follow-up of 6.3 mos (range 0.8-109.2), the median OS from MDS diagnosis was 18.8 mos (95% CI, 10.2-not reached). (Table 1) In pts who received CT/CIT for CLL (n=21) and those who did not (n=6), a TP53 mutation developed following CLL or MDS diagnosis in 10 pts (47.6%) and 1 pt, respectively. All but 1 pt who developed TP53 mutations (n=11) received prior CT. 15 pts (71.4%) who received CT/CIT for CLL therapy had MDS with complex karyotype; this was seen in 2 pts who never received CT/CIT. Twenty-six (96.3%) pts had NGS testing. 5 pts (18.5%) had 1 or more tests completed for CLL prior to sMDS: 4 pts had TP53 mutations, 2 pts each had DNMT3A and SF3B1 mutations, and 1 pt each had an ASXL1, KMT2A, BTK, or PLCG2 mutation. Of these 5 pts, 1 pt had 5 NGS tests performed on serial B-cell selected samples (as opposed to total leukocytes): mutations included 2 TP53, 1 SF3B1 and 1 BTK mutation. 25 (92.6%) pts had 1 or more tests completed at or after sMDS diagnosis (all assessed total leukocytes): 8 pts (36.4%) had TP53, 6 pts (27.3%) had DNMT3A, 5 pts (22.7%) had RUNX1, and 3 pts (13.6%) each had ASXL1, SF3B1, and TET2 mutations. Finally, 4 pts (14.8%) had NGS testing done both pre and post sMDS diagnosis (all assessed total leukocytes). Two pts had the same mutation present pre and post sMDS diagnosis: 1 ASXL1, 1 DNMT3A. A third pt had 3 identical mutations pre and post sMDS diagnosis: 1 TP53 p.G244D, 1 TP53 p.N235_Y236del, and 1 SF3B1. (Table 2) Conclusions Here we have characterized a cohort of CLL pts who developed sMDS in the modern era, where small molecule inhibitors are commonly used for CLL, and NGS is widely available for both CLL and MDS. While the majority of pts with CLL received CT, 5 pts were treatment-naive, and 1 pt only received a BTK inhibitor and an anti-CD20 antibody. The OS of this cohort following MDS diagnosis is similar to historical data of pts with comparable IPSS-R scores, despite the fact that this is a sMDS population; this may be due to lead time bias as pts likely received frequent blood work for CLL. Prior to sMDS diagnosis, complex karyotype and TP53, SF3B1 and CH-associated mutations DNMT3A and ASXL1 were prevalent. TP53, SF3B1, and CH-associated mutations DNMT3A, ASXL1, and TET2 mutations were prevalent at the time of or following a sMDS diagnosis. Further investigation into the interplay between CLL and CH in MDS development in the modern therapy era is needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SF3B1, RUNX1 and TP53 Mutations Significantly Impact the Outcome of Patients with Lower-Risk Myelodysplastic Syndrome

Introduction: Prognosis of patients (pts) with myelodysplastic syndrome (MDS), particularly the group with lower-risk disease (LR-MDS) is very heterogeneous. In the last decade, different studies have reported the role of recurrent somatic mutations involved in multiple cellular pathways and incorporated them into clinical practice and current prognostic models, including all risk categories. Recently, the incorporation of genomic data to clinical parameters defined the new Molecular International Prognostic Scoring System (IPSS-M). Materials and Methods: A retrospective study evaluating the impact of molecular profile in a series of 181 pts with LR-MDS and non-proliferative chronic myelomonocytic leukemia (CMML) focused on survival estimates and the probability of leukemic progression. Genomic DNA was isolated from bone marrow mononuclear cells at diagnosis using DNA kit from Qiagen according to the manufacturer´s instructions. A PCR-based targeted was performed sequencing of 40 genes involved in myeloid neoplasms (Oncomine Myeloid Research panel, including hotspots of ABL, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, U2AF1, WT1 and entire coding regions of ASXL1, BCOR, CALR, CEBPA, ETEV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53, ZRSR2). Sequencing was performed using the Ion Torrent S5 sequencing platform from Life Technologies. Survival (OS) was analyzed from diagnosis until death as a result of any cause or the last follow-up and calculated by the Kaplan-Meier method and log rank test. A Cox proportional hazard regression model was performed to analyze OS as endpoint. Analysis was performed by the IBMÒ SPSSÒ version 26.0.0. The probability of leukemic progression was analyzed using the Gray test in the framework of competing risk and considering death not attributable to AML as a competing event. This study was conducted at Hospital Universitario Virgen del Rocío, approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki. Results: In total, 392 mutations were detected in the 181 pts analyzed with a median of 2 mutations per sample (range: 0-8). Sixty-seven pts (37%) harbored ³3 mutations. Table shows baseline characteristics of pts. Most frequent mutated genes were TET2, SF3B1, RUNX1, ASXL1, TP53, and SRSF2 (all of them present in >10% of pts). Epigenetic regulation was the most recurrently mutated pathway, followed by mutations in splicing factors. After a median follow-up of 47 months (95% CI, 37-56 months), 93 out of 191 pts had died (48%). Median OS for the whole cohort was 41 months (95% CI, 28-53 months). TP53 mutations were detected in 22 pts, with equal distribution of mono-allelic or multi-hit TP53 state. As no significant differences in median OS was observed between groups [23 months (95% CI, 12-53 months) in mono-allelic TP53 state vs. 17 months (95% CI, 11-22 months) in multi-hit cases ( P = 0.1)], TP53 mutation was analyzed as a single parameter. Mutations in SF3B1 were detected in 34 pts and the majority of them (31 out of 34; 91%) were classified as SF3B1a according to the recent segregation of SF3B1-mutated cases by the IPSS-M. In univariate analysis, RUNX1 or TP53mutations correlated with lower median OS (Table 2). In contrast, SF3B1 mutation was associated with prolonged median OS [95 months (95% IC, 32-157) vs. 33 months (95% CI, 19-46) in unmutated pts (P < 0.01)]. In a multivariate Cox regression model, RUNX1 mutations independently associated with shorter OS, while SF3B1 mutation retained its favorable impact on outcome (HR: 0.24, 95% CI, 0.1-0.5; P = 0.001). At last follow-up, 49 patients (25%) progressed to AML. Median time to progression was 16.2 months (range: 2-105 months). Subdistribution hazard ratios (sHRs) for leukemic progression were estimated to identify predictive covariates for progression. TP53 mutation (sHR, 6.3; 95% CI, 3-14); P < 0.001) and RUNX1 mutation (sHR, 6.9; 95% CI, 3.3-14; P < 0.001) were predictive for the probability of leukemic progression (Table 2). Conclusion: Incorporation of molecular testing in LR-MDS identified a subset of pts with expected poorer outcome, either due to lower survival or probability of leukemic progression. Results herein reported may help prognosis stratification in this set of pts with low-risk disease and support the recommendation of molecular testing in pts with MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNA Replication Stress Due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

Introduction: Myelodysplastic syndromes (MDS) with SF3B1 mutation represent a subtype of hematopoietic stem cell disorders with bone marrow (BM) erythroid dysplasia and ring sideroblasts. This mutation in a splicing factor gene causes multiple alterations of mRNA maturation. Other mutated splicing factors SRSF2 or U2AF1, produce splicing alterations and formation of unscheduled R-loops, which trigger DNA damage response and genetic instability. Mechanistically, mutant SRSF2 impairs the transcription pause release downstream of TSS allowing invasion of relaxed DNA double helix by nascent RNA. Although previous studies suggested that R-loops inappropriately form in SF3B1 mutant cells, investigations are required to elucidate why MDS with SF3B1 mutation are less prone to genetic instability. Methods: A total of 96 patients including 53 MDS with SF3B1 mutation and 43 other low risk MDS was enrolled in this study. RNA-seq of BM mononuclear cells (MNC) was performed in 21 MDS with and 6 MDS w/o SF3B1 mutation. BM CD34+-derived erythroblasts were expanded from 69 MDS and 22 healthy controls to analyze transcriptome, proteome, R-loop landscape, and DNA replication stress. For global proteome analysis, after protein digestion, peptides were fractionated on SCX column and analyzed by mass spectrometry. R-loops were detected by DNA-RNA immunoprecipitation (DRIP-seq) using S9.6 antibody. Immunofluorescence labeling of pRPA32, 53BP1 and γH2AX, DNA combing, BrdU assay and metabolomics were used to investigate DNA replication stress and DNA biosynthesis in human primary erythroblasts or in murine proerythroblastic (proE) G1E-ER4 Crispr-cas9 Sf3b1K700E/+ cell line. expressing GATA1 under the control of estrogen receptor. Results: In SF3B1-mutated BM MNC samples, DeSeq2 analysis of RNA-seq data revealed 1,058 upregulated genes (log2(FC)>0.6, BH-adj P-value<0.05) involved in DNA replication, cell cycle, nucleotide biosynthesis, DNA repair, and erythroid differentiation. Analysis of splicing identified 3,937 differential splicing events (ΔPSI>0.10, BH-adj P-value<0.05) including 1,256 events of intron retention (IR). Among IR events, 384 loss of IR events correlated with altered expression of 293 genes. In SF3B1-mutated erythroblasts, loss of IR events were more frequent in polychromatophilic erythroblasts (polyE: 383) than in proE/early basophilic erythroblasts (eBasoE: 171) suggesting that gene extinction mediated by IR during normal differentiation was altered. Quantitative proteomics revealed 924 significantly deregulated proteins with little overlap with IR transcripts, consistently involved in DNA damage, DNA repair, and nucleotide biosynthesis pathways. DRIP-seq was performed in proE/eBasoE of 8 MDS w/o or with SF3B1 mutation to investigate R-loop formation. Stringent peak calling identified 2,208 shared peaks in wild-type samples compared to 159 shared peaks in SF3B1-mutated samples, suggesting a loss of R-loops. Integrative analysis and DRIP-qPCR demonstrated that R-loop losses overlapped IR loss events, consistent with the notion that intron excision prevents R-loop formation. Compared to SRSF2 or U2AF1-mutated erythroblasts, SF3B1-mutated cells exhibited a significant increase of DNA synthesis by BrdU labeling and of replication fork speed by DNA combing. Immunofluorescence analysis detected pRPA32 foci marking single strand DNA but no evidence of double strand breaks in contrast to SRSF2 mutant cells in which 53BP1 or γH2AX foci were detected. Compared to isogenic cell lines, Sf3b1K700E/+ proE G1E-ER4 cells demonstrated higher proliferation rate and fork speed in proliferation and in estradiol-induced differentiation conditions, and significantly lower amounts of deoxynucleotides (dATP, dCTP, TTP) suggesting persistence of DNA synthesis during differentiation. Targeting of DNA polymerase by aphidicolin or ribonucleotide reductase by low doses of hydroxyurea partially rescued erythroid differentiation. Conclusion: Our data demonstrated a highly specific signature of DNA replication stress at transcriptomic and proteomic level in SF3B1-mutant cells. High replication fork speed reflects the loss of R-loops in these cells, which may reduce the risk of genetic instability at the expense of normal differentiation. Pharmacological targeting of the DNA replication stress could improve erythropoiesis.

Potential Drivers of Acquired Resistance to Idelalisib in CLL Patients

Idelalisib is an isoform-specific PI3-kinase-δ inhibitor approved for the treatment of chronic lymphocytic leukemia (CLL). We seek to understand the biologic basis of the variability in depth of response among patients. We have previously reported that intrinsic resistance to idelalisib is associated with RAS/RAF/MAP2K1 mutations and constitutive ERK activation [Blood 2021; 138: 44]. Unlike these non-responder patients, some patients with initial response will acquire resistance to therapy over time, but molecular mechanisms of this acquired resistance to idelalisib remain elusive. To identify somatic mutations that fuel acquired resistance, we performed whole exome sequencing of serial PBMC samples from three CLL patients who responded to idelalisib, but later acquired resistance (average 4.1 yrs from start). We observed that all three patients acquired distinct somatic mutations during idelalisib treatment. Patient 1 harbored TP53 and BIRC3 at baseline, but at progression (5.3 yrs later) had acquired somatic mutations in RAF1, PDIA3, RB1, and ATM. Patient 2 had baseline mutations in SF3B1, BRCA2, and HIST1HIE but acquired additional mutations in PIK3R1 and IKZF3 at progression (4.4 yrs later). Patient 3 carried somatic mutations in TP53 and SF3B1 at baseline and acquired somatic mutations in DDX3X, NOTCH1, FBXW7, CD79B, NFATC1 and NFATC3 at progression (2.7 yrs later). To identify novel drivers of resistance and validate our findings from the initial three CLL patients, we sequenced progression samples from eight additional CLL patients following resistance to idelalisib treatment. In addition to patient 1, we detected mutations in BIRC3 in two additional CLL cases, and mutations in KRASand NRAS (both of which are known to aberrantly activate the MAPK pathway) in two and one CLL patients, respectively, separate from the cases with BIRC3 mutation. In addition to the PIK3R1 acquired mutation in patient 2, we also identified mutations in other PI3K pathway-related genes, such as PIK3CA, PIK3CG, andPLCG2. Since we only sequenced progression samples from these 8 patients, we cannot confirm whether the mutations were acquired or were present in the baseline samples. Overall, these results suggest that mutations in the RAS/RAF/MAP2K1 pathway, as well as in the PI3K pathway itself, and BIRC3 (NF-kB pathway), are associated with PI3K inhibitor resistance. To identify expression programs that may lead to idelalisib resistance, we performed comparative transcriptomic profiling of responding and resistant time point samples from the three initially studied CLL patients (patient 1, 2 and 3). In this analysis, we detected 214 differentially expressed genes with adjusted p-value < 0.05 and fold change of 1.5. GSEA analysis revealed enrichment of cancer related cell signaling pathways, most notably an enrichment for the MAPK pathway (FDR q-value- 0.04). We found that AICDA, LTK, and MAPK12 are overexpressed in resistant samples from the three original CLL patients with acquired resistance as compared to the matched responding time points. In a separate cohort of frontline patients treated with idelalisib, we also found that these same 3 genes were consistently overexpressed before, during and after treatment in early progressors after idelalisib discontinuation. Higher expression of AICDA in progression samples may drive resistance by inducing somatic mutations and/or altering the epigenome of the tumor cells in CLL patients. LTK receptor tyrosine kinase activates PI3K and MAPK pathways in hematopoietic cells. Previously we have reported that MAPK pathway activation plays a key role in primary resistance to idelalisib in CLL, hence overexpression of LTK is a possible mechanism of MAPK activation. We checked the phosphorylation/activation level of AKT and ERK1/2 at the responding and progression time points in the three original CLL patients with acquired resistance and observed that pERK levels are inhibited by idelalisib at baseline, but not at progression, in 2 patients (patient 1 and 2). In conclusion, we have identified potential drivers of acquired resistance to idelalisib in CLL patients, including MAPK pathway activation as in our prior study, and we continue with ongoing work evaluating the potential role of PI3K pathway mutations, BIRC3, AICDA and LTK in treatment resistance, since insights from these studies will help us guide therapy for CLL patients with refractory disease.

Impact of Spliceosome Mutations on the Prognosis of Myelodysplastic Syndrome (MDS)

Introduction: More than half of patients with myelodysplastic syndrome (MDS) have spliceosome mutations (SM) including ZRSR2, U2AF1, SF3B1 and SRSF2. Splicing is fundamental for proper mRNA processing. Mechanistic descriptions of the after-effects of SM remain elusive as improperly formed mRNA quickly decays or changes are subtle and may lack observable effects. These mutations have been implicated in multiple biological alterations including myeloid proliferation through aberrant NF-kB signaling. SRSF2P95* has been consistently shown to provide the highest fitness advantage in the bone marrow environment. This study reports the characteristics, treatment outcomes, leukemia free and overall survival of MDS with spliceosome mutations. Patients and Methods: A retrospective review of patients diagnosed with MDS older than 18 for whom genomic data was available were included at a single institution. Patient characteristics and bone marrow data, including cytogenetic and next generation sequencing (NGS) information were assessed. Genomic DNA was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a NGS platform. The IWG 2006 response assessment criteria were used. Overall and leukemia-free survival were calculated, the latter as a composite of acute myeloid leukemia diagnosis or death from any cause, using the log-rank test for group comparison. Results: First, we compared patients with SM vs no SM. We identified 427 patients with SM and 658 patients with no SM from 2017 to 2022 (Table 1). Among patients with SM, 71% were male, median age was 71. SM patients had higher incidence of IPSS Low (33.3% vs 10.3%) and IPSS Int- 1 (35.6% vs 32.2%), and a higher incidence of diploid karyotype (45% vs 21%), compared to patients with no SM. There was no difference in treatment regimens, and 66.9% of patients with SM received hypomethylating agents (HMA) alone or in combination vs 60.3% in the group without SM. MDS with SM had a lower CR/CRi rate (27% vs 36%); however, they had higher hematologic improvement (HI) (23% vs 14%) and lower chance of disease progression or death at the time of response assessment (39% vs 31% (p = 0.01)). Leukemia-free survival was longer in patients with SM vs no SM (median 4.1 years vs 1.9 years (p<0.0001), this was independent of SF3B1 mutations. Median OS was 4.5 years vs 1.9 years in patients with SM and no SM, respectively (p <0.0001). We then compared outcomes between patients with specific SM mutations. These included SF3B1 (34%), SRSF2 (26%), U2AF1 (25%), and ZRSR2 (14%). Thirty-six percent were female. Median age was 71 (range 28-93). Median percentage of blasts was 3 (range 0-19). Most frequent diagnoses were MDS-EB (35%), MDS-RS (25%) and MDS-MLD (23%). IPSS was Low, Intermediate 1, Intermediate 2 and High in 33.3%, 35.6%, 17.1% and 4.2%, respectively. Fifty-seven percent were in the favorable cytogenetic risk group, 44% with diploid karyotype. Patients with a SM mutation did not share any other SM mutation (Table 2). There were no differences in therapy selected or response rate among SM mutations. Median LFS was not reached (NR), 5.7 years, 3.4 years, and 2.7 years for patients with SF3B1, ZRSR2, U2AF1, and SRSF2 mutations, respectively. Median OS was NR, 5.7 years, 3.6 years, and 2.7 years (p = 0.005) for the same groups, respectively. Conclusions: In our study, patients with SM had a lower rate of CR/CRi with HMA-based therapy, however, leukemia free and overall survival were markedly different for each mutated gene. SM mutations were significantly more prevalent in males. Compared to MDS with diploid cytogenetics, ZRSR2 and SF3B1 mutations have a similar median LFS and OS, but SRSF2 and U2AF1 mutations carry a significantly worse median LFS and OS. While it is convenient to group these mutations together, prognostication should be individualized to specific mutations and co-mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Metabolic Dysfunctions Driven By PRPF8 Explain Cases with Ring Sideroblasts without SF3B1 Mutations: Similarity/ Differences of Genotype/ Phenotype Association

SF3B1 mutations (SF3B1MT) mostly identify a group of MDS with ringed sideroblasts (RS), isolated erythroid dysplasia and favorable prognosis. Besides SF3B1, we and others have demonstrated that other genes might be involved in RS formation either due to convergent metabolic pathways or erythroid driver genes. MDS subtypes characterized by multilineage dysplasia lacking SF3B1MT and presenting with RS are paradigmatic examples of such a possibility. In addition, RS in AML is often unrecognized by hematopathologists due to the focus on the presence of blasts as a predominant feature and/ or the reduction of erythroid precursors inherent to blast infiltration. However, when we previously studied defects in PRPF81, a central but unappreciated target of somatic lesions in MDS, we found a correlation with the RS phenotype. To follow up on this initial finding, we compiled a well-annotated cohort of patients seen at the Cleveland Clinic to assess phenotype/ genotype association of PRPF8MT and RS. NGS results including PRPF8 mutational status of 3,545 cases with myeloid neoplasia were reviewed. Calls were retained after assessment of pathogenicity by the ACMG and VarSome classifiers. Excluding cases with concomitant SF3B1MT, we found PRPF8MT (median VAF 38% [3-79]) in 78 (2%) patients. In total, 83% of the mutations were missense, whereas 15% were truncating. Homology sequence between yeast and humans denoted that hotspots mapped to highly conserved amino acids. D1598 and C1594, localizing in the U6-snRNA interacting domain (1442-1601), were mutated in 14% and 5% of the patients, respectively. T418S mapped in a central domain of several SFs (PROCN) and was found in 9% of the cases, whereas the F1099fs alteration was detected in 5% of our cohort. Prussian blue stain showed the presence of RS in 25% of PRPF8MT. Since PRPF8 maps to the proximal regions of 17p, we also noticed that three informative cases carried somatic PRPF8MT and 17p11.2 deletion or loss of chr.17 (1/2), all showing the presence of RS. We thus screened a more complete cohort of chr.17 abnormalities (88% in the context of a complex karyotype) to investigate their contribution to RS formation. While 66% had RS, the high blast count may have concealed their presence in cases lacking this feature because of their predominant AML or high risk MDS. Notably, we also found 8 additional patients in whom SNP-A showed cryptic losses such as del17p11.2 and 17p13.1 (n=7) or UPD (n=2) containing elevated RS. In this cohort, TP53 was the most commonly mutated gene (49%) followed by ASXL1 and DNMT3A (10% each), BCOR/L1 and TET2 (7.5%). In regard to other SFMT, no patient had SRSF2MT, three had U2AF1S34/Q157, two carried somatic DDX41c.139-5C>T/A225D (with -5/del5q) and one patient had a LUC7L2G48* without -7/del7p. While the coincidental presence of marrow RS in TP53MT lacking SF3B1MT has been previously demonstrated2, no study has investigated whether RS might be a result of shared metabolic feedback mechanisms common to SFMT. We thus analyzed RNA splicing and gene expression (GE)3 of patients with single PRPF8MT (n=34) and SF3B1MT (n=281) finding overlapping splicing features in the a-hemoglobin synthesis HBA2 gene. Upregulation of HBA2 has been described in SF3B1MT patients4,5 to correlate with impaired erythrocyte maturation and its accumulation in early erythrocytes. Interestingly enough, given our data on chr.17 abnormalities, we also found that cases with single TP53MT (n=52) displayed high covariance features for HBA2, a possible culprit in cases carrying RS but unmutated for SF3B1 and PRPF8. Indeed by applying gene-set overrepresentation analysis to PRPF8MT and TP53MT GE profiles, we clustered the top 5 categories (FDR< .001) in erythrocytes differentiation (KLF1, EPB42), homeostasis and development (ALAS2, GATA1, RHAG, SLC4A1) suggesting that these genes might inversely correlate with HBA2 expression and be responsible for the formation of RS. Our study demonstrates that PRPF8MT is another molecular lesion phenocopying SF3B1MT via a compensatory metabolic mechanism responsible for the failure of terminal erythroid differentiation. In analogy, given the proximal genomic location of PRPF8 and TP53 we do not exclude that this commonality might be involved in TP53MT with RS as well. These likely phenocopying mechanisms may enable therapeutic opportunities (luspatercept) in cases lacking SF3B1MT presenting with RS and transfusion-dependent anemia.

Disruption of Intracellular Metal Homeostasis As a Vulnerability in SF3B1 Mutated AML

Acute Myeloid Leukemia (AML) is a heterogeneous disease in which outcome is highly dependent on cytogenetics and mutational profile. Adverse risk AML, recently defined and updated in the 2022 European LeukemiaNet (ELN) genetic risk classification, is associated with a poor overall survival. To identify novel therapeutic approaches for AML, we screened a library of 10,000 compounds across 56 genetically diverse primary AML specimens. We then looked for active compounds with increased activity towards adverse risk AML samples and with low cytotoxicity to normal hematopoietic cells. Among the hits identified, we were able to validate that S767 molecule is preferentially active on adverse risk AML samples. Additionally, we found a strong association between SF3B1 mutations and sensitivity to S767. To gain insights into S767 mechanism of action, we performed a CRISPR/Cas9 genome-wide functional screen, and identified multiples genes involved in mitochondrial processes both in synthetic rescue and synthetic lethal genes. In particular, knock-down of enzymes involved in heme biogenesis and iron-sulfur cluster biosynthesis appear to interfere with S767 cytotoxic activity. Given the central role of iron in these two synthetic pathways, and the structure of S767, we hypothesized that our molecule binds and disrupts intracellular iron homeostasis. Using UV-spectrometry analysis, we determined that S767 has metal affinity and binds to Fe2+, but also Cu2+ and Zn2+. We then quantified intracellular metal concentrations by ICP-MS (inductively coupled plasma mass spectrometry) and found that S767 exposure significantly decreases intracellular iron concentrations while increasing that of copper. Since metals are critical cellular cofactors involved in multiple enzymatic reactions, disruption of intracellular metal homeostasis can affect many processes. Mitochondrial respiratory chain is particularly sensitive to these perturbations as copper and iron-sulfur clusters are present in complexes I-IV of the electron transport chain and are essential for mitochondrial complexes function. By measuring oxygen consumption rate in leukemic cells exposed to S767, we found that the molecule indeed disrupts mitochondrial respiration. SF3B1 is a splicing factor recurrently mutated in myeloid malignancies including myelodysplastic syndromes (MDS) and AML. Heterozygous mutations typically affect SF3B1 splicing functions and induces consequently mis-splicing of target mRNAs, including the ABCB7 iron-sulfur cluster transporter. Analysis of the Leucegene transcriptomic data of primary AML specimens revealed mis-splicing and downregulation of ABCB7 in SF3B1 mutated patients. Strikingly, ABCB7 was one of the top synthetic lethal gene identified by chemogenomic CRISPR/Cas9 screen following exposure to S767 molecule. Given that ABCB7 exports iron-sulfur clusters out of the mitochondria to be assembled by cytosolic proteins and plays a role in iron homeostasis, we hypothesized that its aberrant expression in SF3B1 mutated AML participates to the high sensitivity of these leukemias to S767. We thus identified a novel molecule with selective activity in AML. Our data suggest that S767 is not a protein-targeting molecule but rather acts through the disruption of intracellular metal homeostasis, and ultimately affects mitochondrial respiration. Its mechanism of action highlighted a vulnerability in SF3B1 mutated AML which present a defect in ABCB7 expression. Importantly, SF3B1 mutations are now recognized in the 2022 ELN risk classification as an adverse prognostic marker if they are associated with intermediate or adverse risk AML, giving a unique opportunity to develop novel therapeutic approaches for this subgroup of patients with poor clinical outcome. In addition, SF3B1 mutations are found in 25% of MDS cases, which are associated with ring sideroblasts and accumulation of mitochondrial iron, and that could also benefit from such therapeutic approach.

MDS Classification - Do We Still Have to Count Blasts?

Background: Since the definition of MDS their classification relied on cytomorphology determining dysplasia, cytopenia and blast count. The revised 4th edition of the WHO classification encompasses only one genetically defined entity (MDS with isolated del(5q)). In the 5th edition of the WHO classification 2 additional entities are defined by genetics: MDS with low blasts and SF3B1 mutation and MDS with biallelic TP53 inactivation. All other are classified still based on morphology into i) hypoplastic MDS, ii) MDS with low blasts and iii) MDS with increased blasts. Aim: As blast counting is subjective and the boundary between low and elevated blasts is arbitrary, we set out to propose a classification for MDS based solely on genetic abnormalities obviating the need for blast counting. Patients and Methods: The cohort comprised 735 de novo MDS. The diagnosis was established following WHO classifications. In all cases chromosome banding analysis and WGS (100x, 2x151bp, NovaSeq) were performed. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline. For all cases overall survival data was available (median follow-up: 9.3 years). Results: We categorized our cohort 1) according to WHO revised 4th edition, 2) 5th edition, 3) according to blast count (<5%, 5 to <10%, 10 to <20%), and 4) according to numbers of mutations. For all 4 categorizations prognostic differences between subgroups was confirmed. Next, we performed cluster analyses to identify genetic abnormalities associated with blast count. While SF3B1 mutations and isolated del(5q) were associated with low blast count, mutations in ASXL1 and RUNX1 were associated with higher blast count (for all p<0.0001). Further, we evaluated the genomic landscape for associations of genetic parameters. RUNX1 mutations and ASXL1 mutations were highly associated with spliceosome mutations. 49/62 (79%) RUNX1mut MDS and 97/153 (63%) ASXL1mut MDS harbored also spliceosome mutations. On the other hand RUNX1mut, ASXL1mut and spliceosome mutations rarely co-occurred with biallelic TP53 inactivation or complex karyotype (only 5/41 cases and 19/78 cases). 102/735 (14%) cases harbored neither a del(5q), a complex karyotype, biallelic TP53 inactivation nor a mutation in SF3B1, SRSF2, U2AF1, ZRSR2, DNMT3A, TET2, RUNX1 or ASXL1. In 20 of these at least one mutation was found in 21 additional genes known to be mutated in MDS. Based on these data 9 genetically defined non-overlapping hierarchical subgroups (definitions of the respective prior subgroups are exclusion criteria for the following ones) are proposed: 1. biallelic TP53 inactivation 2. complex karyotype, 3. RUNX1 mutated, 4. ASXL1 mutated, 5. del(5q), 6. SF3B1 mutated, 7. mutated DNMT3A, TET2, U2AF1, SRSF2, and/or ZRSR2, 8. presence of at least one mutation in one of 21 other genes recurrently mutated in MDS, 9. none of the genetic markers present that define any of the previous entities. Survival analyses revealed marked differences between these biologically defined entities (figure 1). In univariate cox regression analyses including all 9 genetic categories and blast count the categories biallelic TP53 inactivation, complex karyotype, RUNX1mut, ASXL1mut, SF3B1mut, none of the genetic markers and blast count <5% vs ≥5% and <10% vs ≥10% were significantly associated with OS (for all p<0.002). In multivariate analyses both blast count cut offs did not show a significant impact, while the categories biallelic TP53 inactivation, complex karyotype, RUNX1mut, ASXL1mut, SF3B1mut were independently associated with OS (for all p<0.05). In figure 2 the relationship of classification according to the 4th revised, the 5th edition of the WHO and the purely genetically based classification as proposed here is depicted. Conclusions: Based on karyotype and mutation status of 9 genes complemented by 21 genes in 10-15% of cases, MDS can be separated into 9 biologically distinct subgroups, reflecting biology better than blast counts. These are associated with significant differences in overall survival. RUNX1 and ASXL1 define distinct subgroups within spliceosome mutated MDS potentially driving progression. Given the poor survival of the MDS subgroups with biallelic TP53 inactivation, complex karyotype and RUNX1 mutation it has to be further evaluated whether or not these MDS subtypes should be combined with AML harboring the respective genetic abnormalities for treatment approaches. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of a Flow Cytometric Core Marker Set in the Diagnostic Workup of Patients with Suspected Myelodysplastic Syndromes

Background Flow cytometry (FC) performed according to ELN 2012 criteria (Westers et al., 2012) is recommended by ELN guidelines for diagnostic workup in suspected myelodysplastic syndromes (MDS, Malcovati et al., 2013). Recently, the ELN working group on FC in MDS identified 17 core markers independently related to MDS as well as >3% myeloid progenitor cells (MPC) to be strongly related to MDS. In parallel, during the last decade next generation sequencing (NGS) became increasingly relevant for MDS diagnostics. Aim To validate the new ELN criteria (ELN 2022) in comparison to cytomorphology (CM) in an independent cohort of patients prospectively undergoing diagnostics for suspected MDS and their correlation with MDS specific variants as detected by NGS. Methods Bone marrow samples of 4658 patients (female/male, 1833/2825; median age 72 years, range 18-96) with suspected MDS were analyzed in parallel by CM, FC and NGS (73 gene panel). Cases with non-MDS neoplasms were excluded. Results CM and FC (ELN 2012) revealed concordant diagnostic results in 80%. 1342 (29%) cases were diagnosed MDS by both CM and FC (CM+FC+), 2384 (51%) were CM-FC-. Discordant cases were 329 (7%) CM+FC- and 603 (13%) CM-FC+, respectively. Application of ELN 2022 criteria for the diagnostic of MDS by FC (either at least 3 out of 17 markers present or more than 3% MPC) resulted in a similar degree of concordance (78%). 1016 (22%) were CM+FC+ and 2626 (56%) were CM-FC-. Discordant cases were 655 (14%) CM+FC- and 361 (8%) CM-FC+, respectively. Next, we determined the portion of cases with at least one MDS-related gene mutation as analyzed by NGS in cases discordant between CM and FC. Regarding FC ELN 2012 criteria 248/329 (75%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 352/603 (58%). Considering ELN 2022 criteria 527/655 (81%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 236/361 (65%). Focusing on mutations in detail revealed similar frequencies of CHIP mutations (ASXL1, DNMT3A, TET2) regardless of CM and FC results. Concordant CM and FC cases (ELN 2012) harbor at least one CHIP mutation in 38% (CM+FC+ 506/1341, 38%; CM-FC- 901/2384, 38%). In CM+FC- cases 137/329 (39%) had at least one CHIP mutation, while in CM-FC+ cases the rate was 220/603 (37%). Application of ELN 2022 criteria resulted in a similar distribution (CM+FC+ 379/1016, 37%; CM-FC- 983/2626, 37%; CM+FC- 254/655, 39%; CM-FC+ 138/361, 38%). Analyzing cases discordant between CM and FC regarding non CHIP mutations showed a strong correlation between CM+FC- cases and SF3B1 mutations (ELN 2012: 102/324, 32%; ELN 2022: 190/651, 29%). In contrast, in CM-FC+ cases just 13/597 (2%, ELN 2012) and 11/357 (3%, ELN 2022), respectively, had an SF3B1 mutation. In line with this correlation, CM+FC- cases (ELN 2012) with mutated SF3B1 had ≥5-14% ring sideroblasts (RS) in 17/102 (17%) and ≥15% RS in 78/102 (77%). The respective figure for CM+FC- cases (ELN 2022) were ≥5-14% RS in 22/190 (12%) and ≥15% RS in 160/190 (84%). On the other hand SRSF2 mutations were found in discordant cases slightly more frequently in CM-FC+ cases compared to CM+FC- cases (ELN 2012: 95/557 (17%) of CM-FC+ and 40/315 (13%) of CM+FC- cases). This correlation was even clearer with ELN 2022 criteria (75/334 (23%) of CM-FM+ and 101/628 (16%) of CM+FC- cases). Evaluating remaining cases discordant between CM and FC after excluding those with CHIP mutations, SF3B1 mutations, and SRSF2 mutations revealed no major differences regarding presence of at least one of the remaining mutations (ELN 2012: 32/107 (30%) of CM+FC- cases and 62/275 (23%) of CM-FC+ cases; ELN 2022: 59/175 (34%) of CM+FC- cases and 43/146 (30%) of CM-FC+ cases). Conclusion FC has already proven its power in MDS diagnostics. We here confirm the newly defined criteria of ELN for diagnosing MDS (ELN 2022) to reveal results comparable to ELN 2012 criteria. Thus, a reduced core set of markers is already sufficient for MDS diagnosis by FC without loss in accuracy. Furthermore, cases with results discordant between CM and FC are associated with MDS-related mutations with particular sensitivity of CM in SF3B1 mutated cases and of FC in SRSF2 mutated cases. The higher frequency of mutations in cases discordant between CM and FC applying ELN 2022 criteria as compared to ELN 2012 criteria argues in favor of a higher specificity of the ELN 2022 criteria. Follow-up analyses are indicated to reveal the significance of discordant findings.

Long-Term Clonal Inversion in an MDS-RS Case with Dual SF3B1 Mutations

Recent large-scale studies have traced SF3B1 mutations as a driving event in clonal establishment and expansion, and a hallmark of myelodysplastic syndrome with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage allowing SF3B1-mutant (SF3B1mt) clones to thrive remain poorly understood. Thus, tracking these events in conditions of low noise is crucial to our comprehension of clonal hematopoiesis. Here we report a 12-year clinical and molecular follow-up of a unique dual SF3B1mt MDS-RS case displaying a dynamic state of clonal competition between two clones propagated by a N626D and K666N mutation, respectively (Panel A). After an initial rapid expansion of the N626D clone compared to the relatively stable K666N clone during the initial follow-up period of ~2 years, the K666N clone spontaneously expanded at expense of the N626D clone to eventually dominate the bone marrow, driving a complete inversion of clonal prevalence. Clinically, these active shifts of high clonal involvement in the bone marrow were accompanied by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in the ring sideroblast phenotype over time (from 42% to 8% RS) in the absence of any treatment. The stability of this patient's clinical profile was therefore considered as unlikely to confound further characterization. Through a combination of whole-genome sequencing (WGS) of hematopoietic stem and progenitor cell (HSPC)-generated colony-forming units (CFU), combined DNA + RNAseq (TargetSeq) of FACS-purified hematopoietic stem cells (HSCs) and megakaryocyte-erythroid progenitor cells (MEP), and high-throughput 3'-based 10X Genomics single-cell RNAseq of CD34+ HSPCs, we molecularly characterized the two distinct SF3B1mt clones in bone marrow samples acquired before, during and after the points of clonal inversion (Panel B). Using WGS of single cell-derived colonies, we established that the acquisition of either the N626D or K666N mutation had preceded the diagnosis of MDS-RS by at least 20 years. Moreover, both mutations were associated with an increased mutational rate as compared to wildtype colonies, and the two clones presented distinct patterns of additional coding mutations as potential co-drivers of expansion. Mirroring previous characterizations of SF3B1mt HSPCs, we identified a common and deleterious alternative splicing profile to both genotypes via TargetSeq which included mis-splicing events in the known SF3B1mt targets MAP3K7 and SEPTIN6. In addition, we identified mutation-specific splicing profiles as potentially responsible for distinct downstream distinctions between both clones. Through this analysis, we identified the K666N-specific loss of expression of the tumor suppressors HOXA5, RGCC and NEURL1 as potential compound factors to the known higher risk underlying SF3B1K666N mutations. Finally, we disentangled clone-driven and aging-driven transcriptomic changes to identify a significant decrease of the early hematopoietic controllers ID1, ID2 and ID3 as a potential mechanism of aging-related cellular senescence. In conclusion, this case study comprises a unique long-term follow-up of the clonal dynamics underlying two co-existing, distinct and competing SF3B1mt clones which identifies subtle molecular changes and differences underlying the expansion and progression of SF3B1mt clones. *Moura PL, Hofman IJF, Nannya Y and Aliouat A contributed equally to this work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of Clinical, Molecular, and Prognostic Features of the W.H.O. 2022 Classification System for Myelodysplastic Neoplasms (MDS)

Introduction: Myelodysplastic neoplasms (MDS) are clonal disorders of relative bone marrow failure and a variable risk of progression to acute myeloid leukemia. MDS generally exhibit genetic and/or cytogenetic abnormalities, an observation that led to the reclassification of MDS in the World Health Organization (WHO) 2022 update (Khoury et al, Leukemia 2022). In parallel, studies examining the mutational landscape of MDS have shown that specific patterns of somatic gene alterations strongly impact prognosis (Bernard et al, NEJM Evidence, 2022). In this study, we characterized the mutational patterns and clinical outcomes associated with the WHO 2022 classification schema to provide a more comprehensive view of this disease framework. Methods: This is a single-center retrospective cohort study of patients (pts) with clinicopathologically-confirmed MDS between January 2018 and December 2021. We identified cases with next-generation sequencing (NGS) performed on either bone marrow (BM) aspirate or peripheral blood (PB) using a clinically validated 164 gene panel designed to detect genes recurrently mutated in both myeloid and lymphoid neoplasms. For all cases, demographic information, clinical and molecular data, WHO 2022 diagnostic class, and survival outcomes were collected. IPSS-R and IPSS-M scores were calculated. Survival was estimated using the Kaplan-Meier method. Comparisons of mutational frequencies between groups were made via Fisher's exact test. This study was approved by Stanford University's Institutional Review Board. Results: Our inclusion criteria identified 118 MDS pts with clinicopathologic and molecular review. The median age of the cohort was 72 years, 67% were male, and 70% were Caucasian. For morphologically defined pts, MDS with low blasts (MDS-LB) was the most common subtype (36% of the entire cohort), followed by MDS with increased blasts-2 (MDS-IB2; 24%) and MDS with increased blasts-1 (MDS-IB1; 16%). For genetically defined pts, MDS with low blasts and SF3B1 mutation (MDS-SF3B1) was the most common subtype (19%) while MDS with biallelic TP53 mutations (MDS-biTP53) comprised 6% pts (Table 1). Within the latter group, 86% had a complex karyotype. A co-mutation plot to visualize the distribution of mutations among the various subgroups is shown in Figure 1. Notably,TP53 mutations (either mono- or bi-allelic) occurred in 16% of the cohort, were frequent in pts with complex karyotype (17/22; 77%), and were exclusive of ASXL1, IDH2, or SF3B1 mutations. In the morphologically defined subgroups, pts with MDS-LB had the highest prevalence of TET2 mutations (12/42 pts; 29%), while pts with MDS-IB1 had none (0/19 pts). U2AF1 mutations were significantly more common in pts with MDS-LB (13/42; 31%) relative to pts with MDS-IB2 (2/28; 7%) (p = 0.04). Conversely, patients with MDS-IB2 more frequently harbored TP53 mutations compared to pts with MDS-LB (p = 0.002). Calculation of IPSS-M scores stratified 47% of pts into the lower risk categories, with the remainder in higher risk categories (Table 1). The median IPSS-M score for the entire cohort was 0.105 [min: -2.11, max: 4.59] (Moderate High, MH). Median IPSS-M scores were highest in pts with MDS-biTP53 (3.18; [-0.480, 4.59]) and MDS-IB2 (1.65; [-0.880, 3.47]) (Very High, VH), and lowest in pts with MDS-SF3B1 (-1.13; [-2.06, 0.250]). IPSS-R scores generally paralleled IPSS-M scores, with 46% of all pts falling into the lower risk groups (Table 1). Pts with MDS-biTP53 had a median IPSS-R score of 8.0 [3.0, 9.5] (VH) while pts with MDS-SF3B1 had a median score of 2.5 [1.5, 4.0] (MH). Median overall survival (mOS) from the time of diagnosis was highest for those with MDS-LB (not reached), MDS-IB1 (not reached), and MDS-SF3B1 (7.0 years). Survival for patients with MDS-IB2 and MDS-biTP53 was poor, with mOS of 1.5 years and 0.8 years, respectively (Table 1). Conclusion: Our data demonstrated the clinical and molecular characteristics of MDS pts evaluated within the WHO 2022 schema and showed that differing mutational features were prominently associated within the morphologically as well as the genetically defined subgroups. Further, OS differed substantially among the defined subgroups. Our incorporation of the IPSS-M and IPSS-R provides additive prognostic and survival components into the WHO 2022 classification, which together enhance this framework's utility for studying MDS and its natural history. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recombinant Human Erythropoietin Combined with All-Trans Retinoic Acid and Testosterone Undecanoate in the Treatment of Anemia in Patients with Lower-Risk Myelodysplastic Syndromes: A Multicenter, Open-Label, Prospective Trial

[Background] More than 90% of low-risk MDS face refractory anemia and blood transfusion dependence for a long time. At present, recombinant human erythropoietin (EPO) is the main treatment, but the effective rate of single agent is low, which seriously affects the quality of life. The anemia treatment in low-risk MDS remains a high unmet clinical need, and new treatment strategies are urgently needed. [Methods] This study is a multicenter, single-arm clinical study of EPO combined with all-trans retinoic acid (ATRA) and testosterone undecanoate in the treatment of anemia in patients with low-risk MDS [Results] As of June 2022, a total of 26 patients were enrolled, with a median age of 67 years, 15 males and 11 females, with median hemoglobin of 63 g/L, EPO concentration > 500 IU/L in 9 patients, and the overall hemoglobin response rate of 65.4% (17/26). The hemoglobin response rates were 55.6% (5/9) in patients with EPO concentrations greater than > 500 IU/L and 70.6% (12/17) in patients with EPO concentrations ≤ 500, which were not statistically different. Among them, 112 known or putative mutational gene targets in hematologic malignancy were examined for mutations in 16 patients using a custom targeted next-generation sequencing (NGS) gene panel. The frequency of SF3B1 mutation was 43.8% (7/16). The hemoglobin response rate was 85.7% (6/7) and 55.6% (5/9) in patients with or without SF3B1 mutation. [Conclusion]: This study showed that EPO combined with tretinoin and testosterone undecanoate regimen can effectively improve the efficacy of patients with lower risk MDS, and is not affected by EPO concentration. It has the better efficacy in patients with SF3B1 mutation and is worthy of further randomized controlled studies to verify the efficacy.

Luspatercept for Treatment of Lower Risk Myelodysplastic Syndromes: Real World Data Replicates Medalist Study Results and Confirms Activity Among Hypomethylating Agents and Lenalidomide Treated Patients

Introduction: Luspatercept, TGF-B fusion trap protein, is a first in class erythroid maturating agent approved by FDA for treatment of red blood cell transfusion dependent (RBC-TD) lower risk myelodysplastic syndromes (LR-MDS) with ring sideroblasts (RS) based on the MEDALIST clinical trial. Red blood cell transfusion independence (RBC-TI) was observed in 38% of 153 patients (pts) treated with luspatercept compared to 13% of 76 pts in the placebo group. We present to our knowledge first real-world data (RWD) in a large cohort of pts treated at our center with luspatercept for LR-MDS. More importantly, we address impact of prior therapies on response namely hypomethylating agents (HMA) as the original clinical trials excluded HMA treated pts. Methods We identified all pts treated at Moffitt Cancer Center with luspatercept since the FDA approval. Baseline RBC transfusion burden (TB) was defined as: non- transfusion dependent (NTD) (0 units in 8 weeks prior luspatercept), low TB (LTB) (1-5 units /8weeks) and high TB (HTB) (≥ 6 units /8 weeks). A hematological response (HI) was defined as an (i) objective Hgb increase of > 1.5 g/dl in NTD, and (ii) RBC-TI with Hgb increase of 1.5 g/dl, or RBC-TI without Hgb 1.5 g/dl increase, or >50% reduction in RBC transfusion burden among RBC-TD. Results Between February 2020 and December 2021, 114 pts were treated with luspatercept. Table-1 summarizes baseline characteristics in comparison to Medalist trial, the median age was 70 years. Seventy-three pts (67%) had MDS-RS subtype, 91% were intermediate or lower risk MDS by R-IPSS. SF3B1 mutation (MT) was detected in 71% (80/112). The mean Hgb level was 7.90 g/dl and 47% were RBC HTB transfusion dependent. The median serum erythropoietin (epo) level at time of referral was 122 U/L (19% > 500 U/L) (n=77). Majority had prior erythroid stimulating agents (ESA) (89%) with 29% hematological response to prior ESA, 59 pts (53%) had prior HMA therapy, and 42 patients (47%) had prior lenalidomide (len). The overall HI rate to luspatercept was 39.5% (45/114). Table-2 summarizes detailed responses. HI correlated with baseline RBC-TB where 69% (9/13) NTD, 47% (21/47) LTB and 25% HTB (13/53) achieved HI respectively, p=.004. Almost 55% of pts needed dose escalation, 62 pts received 1.33 mg/kg and 63 pts received 1.75 mg among whom 40% (25 pts) demonstrated response to higher doses. Based on RBC-TB, 75% (6/8) NTD patients achieved HI with highest dose escalation, 40% (10/25) LTB pts achieved HI with dose escalation and 30% (9/30) HTB pts achieved response with dose escalation. Among responders, the median duration of response was 15.6 months (2.6-27.3). As off last follow up, 56 pts (49%) discontinued treatment. Reason of discontinuation was lack of response in 47 pts, loss of response in 4 patients and adverse events in 5 pts (58 pts are still on therapy). The most common captured adverse events were fatigue (n=8), GI (n=4), shortness of breath and arthralgia (1 each). The response rates were higher among SF3B1 MT pts compared to wild type, 48% (34/80) compared to 16% (5/32), p=.002. The response rate among pts with RS subtypes versus non-RS were 49% versus 28% respectively, p=.05. Among 14 pts with MDS/MPN-RS-T the HI rate was 79%. HI for pts with serum epo < 500 U/L, was 48% (30/62) compared to 13% 2/15) for those with serum epo > 500, p=.01. Majority of pts with high serum epo level were RBC HTB at baseline 67% (10/15). The HI rate was 30% (18/60) after HMA failure compared to 50% (27/54) in HMA naïve pts, (p=.03). More pts post HMA failure were RBC-HTB 62% compared to 30% in HMA naïve. The luspatercept HI among RBC-LTB HMA failure pts was 39% compared to 54% in HMA naïve LTB, however, it was 24% among HTB HMA failure compared to 25% among HMA naïve HTB. The HI rate was 33% (15/46) for len failure pts compared to 43% (29/67) naïve patients, (p=.3). In multivariable analysis both SF3B1 mutation status and baseline RBC TB independently correlated with luspatercept response but not HMA failure. Conclusions Our RWD confirms luspatercept clinical benefit and safety profile observed in the MEDALIST trial. Responses are dose dependent with 1/3 of patients who were dose escalated responding. Low baseline RBC-TB dependency and SF3B1 MT correlated with higher response rates. Luspatercept retained activity after HMA or lenalidomide failure however a trend of lower responses observed correlated with RBC-HTB among those patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic Modulation of the Marrow Microenvironment in MDS: A Phase I Trial of Abaloparatide and Bevacizumab

Background: The bone marrow microenvironment (BMME), including mesenchymal stromal cells (MSCs) and osteolineage cells, vascular endothelial growth factor (VEGF) signaling, and marrow angiogenesis, is altered in myelodysplastic syndromes (MDS). BMME manipulation represents a novel therapeutic strategy for MDS. Preclinical studies have demonstrated that parathyroid hormone (PTH) increases MSC and osteoblast support of hematopoietic stem cells and remodels marrow vascularity. This effect can be augmented with the addition of bevacizumab (BEV), which also decreases angiogenesis. Here we report the results of a phase 1 study of abaloparatide (ABL), a PTH-related protein analog, plus BEV in patients with MDS. Methods: Eligible patients were ≥ age 18 with MDS or non-proliferative chronic myelomonocytic leukemia and at least one cytopenia. Those categorized as very-low to intermediate risk per the Revised International Prognostic Scoring System (IPSS-R) who were treatment-naive or had received only prior growth factors were eligible. Patients of all risk categories who had not responded to disease modifying agents were also eligible. Prior therapies were stopped at least 14-days before study start. Patients received one 28-day cycle of ABL (80 mcg/day self-injected) followed by three 28-day cycles of ABL + BEV (5 mg/kg on days 1 and 15). Bone marrow samples were collected at baseline, after cycles 1 and 4, and 3 months after treatment completion. The primary endpoint was the rate of treatment-limiting toxicities (TLTs). Response rates, impact of therapy on BMME cell populations, and effect on health-related quality of life (HRQoL) and geriatric assessment were additional endpoints. Results: Nineteen patients were enrolled and treated. The majority (n=13) received prior therapy, most commonly with an erythropoietin stimulating agent (ESA; 77%). Typical molecular heterogeneity was present and included mutations in SF3B1 (44%), TET2 (33%), ASXL1 (22%), and DNMT3A (22%). No TP53 mutations were noted. Additional baseline characteristics are outlined in Table 1. Three patients (16%) experienced a TLT: a grade 3 infusion-related reaction and grade 2 fatigue with orthostatic hypotension attributed to ABL, and a grade 3 colonic perforation possibly attributed to BEV. There were no other grade 3 events related to treatment (TRAEs). Frequent (≥15%) TRAEs were: back pain (n=6), chills (n=3), and epistaxis (n=3). Analysis of bone marrow samples revealed a significant increase in mean MSC colony forming units (CFU-Fs) after cycle 1 of ABL compared to baseline (20.7 ± 5.8 vs 36.5 ± 6.4 CFU-F per 1x106 cells, p=0.004). This increase was not maintained with the addition of BEV. Of the 17 patients evaluable after at least 2 cycles on study, 15 (88%) had stable disease and 2 (12%) had progression. Five of 6 patients (83%) with MDS with excess blasts 2, prior hypomethylating agents, and/or high to very-high IPSS-R maintained stable disease over 7 months of follow up. The sixth patient achieved a neutrophil response by 2006 International Working Group criteria after 8 weeks of therapy. In the subset of patients with baseline neutropenia (n=4) there was a significant increase in absolute neutrophil count (ANC) after 8 weeks on study (0.89 ± 0.26 vs 1.28 ± 0.32 103 cells/µL, p=0.04). All patients with baseline neutropenia or thrombocytopenia that had improved ANC (n=4) or platelet counts (n=3), respectively, over the first 8 weeks of treatment, had increased CFU-Fs after cycle 1. Three patients (18%) had decreased transfusion needs over the first 8 weeks despite discontinuation of prior ESAs; all 3 also demonstrated increases in CFU-Fs after cycle 1. HRQoL and geriatric assessments were stable throughout the study. Conclusions: ABL + BEV therapy is safe and well-tolerated in MDS. A significant increase in MSCs was noted following single-agent ABL for 4 weeks, suggesting BMME remodeling. This corresponded with significant early improvements in ANC values and transfusion requirements during the first 8 weeks of therapy in some patients. Prolonged stable disease was maintained in a majority of patients, including several with high-risk clinical features. These data suggest successful manipulation of the BMME with effects on hematopoiesis, and warrant further investigation of longer ABL monotherapy as a new treatment paradigm for patients with MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Trajectories and Therapeutic Targeting of High-Risk SF3B1-Mutant Myelodysplastic Syndromes

Myeloid neoplasms are driven by acquisition of clonal genetic lesions, yet it remains unclear how these lesions cooperate to transform hematopoietic stem and progenitor cells (HSPCs) programs and modify drug responses. The integration of genomic profiling with clinical and cytogenetic data in the recent IPSS-M has significantly improved risk stratification of patients with myelodysplastic syndromes (MDS). Spliceosome mutations in SF3B1 are common initiating events in MDS and confer favorable prognosis, while secondary mutations in RUNX1 and STAG2 transform this low-risk condition into high-risk MDS or secondary acute myeloid leukemia. To understand how these events cooperate with SF3B1 mutations, we modeled these clonal trajectories in primary human HSPCs. SF3B1 K700E mutation was introduced in CD34+ cells isolated from cord blood or peripheral blood of healthy donors, using CRISPR/Cas9 editing and AAV6-mediated homology directed repair. By introducing an intronic fluorescent reporter, this system allows for the tracking and isolation of SF3B1-mutant cells for molecular and functional analysis. Edited cells were heterozygous for the K700E mutation and recapitulated alternative 3' splice site selection of genes previously identified in SF3B1-mutant patients. To model disease progression, we introduced secondary RUNX1 (S-R), STAG2 (S-S), or control AAVS1 (S-A) mutations into SF3B1 K700E knock-in CD34+ HSPCs, leading to a reduction of RUNX1 or STAG2 expression in SF3B1-mutant cells (50% and 90%, respectively). The S-R and S-S trajectories induced divergent alterations in lineage specification of SF3B1-mutant HSPCs. S-R promoted myeloid skewing at the expense of the erythroid lineage, while S-S induced a block in differentiation, impairing both myeloid and erythroid differentiation. Consistently, introduction of secondary RUNX1 or STAG2 mutations in an induced pluripotent stem cell (iPSC-HPCs) model of SF3B1-mutant MDS recapitulated S-R myeloid expansion and S-S maturation arrest identified in primary HSPCs. S-R and S-S genotypes resulted in divergent transcriptional programs including inflammation, immune response, and myeloid differentiation. However, both high-risk clonal trajectories expanded the immature CD34+CD38- HSCs/multipotent progenitors (MPPs). S-R selectively expanded CD34+CD38-CD133+ HSCs, whereas S-S expanded the more mature CD34+CD38-CD133- population. These data suggest that, despite divergent molecular pathways, high-risk mutations converge on expansion of stem cell potential of SF3B1-mutant HSPCs. To understand how genetic (co-mutation patterns) and epigenetic (HSC state) heterogeneity can influence drug responses, we profiled a panel of spliceosome inhibitors and 166 FDA approved and investigational compounds. Both S-R and S-S genotypes conferred decreased response to single and combinatorial agents conventionally used for the treatment of myeloid neoplasms. High-risk genotypes maintained elevated sensitivity to SF3B inhibition, but conferred differential response to novel classes of spliceosome modulators, with STAG2 but not RUNX1 loss selectively promoting response to type I PRMTs inhibitor MS023. By contrast, CHK1 inhibitor Prexasertib was highly selective for SF3B1-mutant cells irrespective of co-mutations, inhibiting growth and cell cycle progression. Taken together these data indicate that the presence of SF3B1 mutation offers therapeutic vulnerabilities that can be leveraged to target high-risk genotypes, and different SF3B1 co-mutated genes can further modulate drug response of SF3B1-mutant cells. In conclusion, progression from low-risk SF3B1-mutant MDS to high-risk disease is mediated by molecularly distinct trajectories driven by RUNX1 and STAG2 mutations that converge on expansion of the HSC compartment. Moreover, clonal progression is associated with genotype-specific drug responses and increased resistance to standard agents, and ongoing studies are elucidating how genetic and epigenetic states affect therapeutic responses.

FISH Analysis of CDKN2A/B Deletion in Chronic Lymphocytic Leukemia

Introduction: The course of chronic lymphocytic leukemia (CLL) ranges from indolent to rapid disease progression, including transformation to an aggressive lymphoma (Richter transformation, RT). A subset of CLL pts have been shown to acquire deletions of the tumor suppressor CDKN2A/B. RT has been associated with deletion of CDKN2A/B, however, prior studies’ sample populations were limited or not primarily focused on those with this deletion. Due to the rarity of this genetic abnormality in CLL and severity of RT, additional studies concerning this subgroup should be investigated. Here, we examine a larger cohort of CLL pts with CDKN2A/B deletion by fluorescence in situ hybridization (FISH) to provide further clarity on this genetic lesion. Methods: We conducted a retrospective case study for all pts for whom cytogenetic clinical samples were submitted for extended CLL FISH panel analysis over a 3.5 year (yrs) period, between 2015 and 2018 at The Ohio State University. Pts were screened for CDKN2A/B deletion with a new or follow up diagnosis of CLL or small lymphocytic lymphoma (SLL). Cytogenetic testing was performed on cells stimulated with pokeweed mitogen, phorbol myristate acetate (PMA), and CpG oligonucleotides. FISH for D13S319, D12Z3, ATM, TP53, BCL6, MYC, REL, SEC63, MYB, and CDKN2A/B was performed via manufacturer's recommendations. IGHV mutational status was determined using PCR. A 50-gene hematologic sequencing panel was performed via Ion Torrent. Overall survival (OS) from first detection of abnormal CDKN2A/B results were examined. Cox regression model was used to evaluate OS post CDKN2A/B deletion. The Fine and Gray Model was used to examine correlation of variables with regard to RT, treating death without RT as a competing risk. Results: 636 pts had sample(s) submitted for extended CLL FISH panel analysis. Of these, 43 (6.8%) pts with confirmed CLL or SLL were found to have loss of CDKN2A/B by FISH, including 6 pts within one year of diagnosis. Median age at diagnosis was 54.9 yrs (range 41.9-77.7). Loss of CDKN2A/B was detected at a median of 7.8 yrs (range diagnosis-26.1) from diagnosis. Of those with known IGHV status (n=33), 91% were unmutated. A complex karyotype (>3 clonal chromosomal abnormalities) was found in 90% percent of pts. In regard to CDKN2A/B deletion, 77%, 16%, and 7% of pts had heterozygous, homozygous, or subclonal populations of heterozygous as well as homozygous loss, respectively. Additional FISH testing showed 23% had gain of REL, 16% had BCL6 gain/rearrangement, 16% had deletion 6q, 37% had gain/rearrangement of MYC, 16% had loss of ATM, 14% had trisomy 12, and 58% had deletion 13q. Deletion of TP53 was seen in 63%. Sequencing data was available for 15 pts (35%), of note, 10 pts had mutations in TP53, with 4 also having mutations in SF3B1. In terms of OS, there were 30 deaths in the cohort, with median OS of 10.8 yrs from diagnosis (95% CI 8.0-14.4). With a median follow-up of 4.9 yrs among survivors, the median OS from time of detection of CDKN2A/B deletion was 1.7 yrs (95% CI 0.3-4.7). Multivariable analysis of OS post CDKN2A/B detection was significant for increasing beta-2-microglobulin measured at first visit at our institution (p=0.0044, hazard ratio (HR) 1.32 (95% CI 1.09-1.60)) and CDKN2A/B subclonal heterozygous and homozygous loss (p=0.0024, HR 9.81 (95% CI 2.25-42.73)). Of the 43 pts with loss of CDKN2A/B, 21 (49%) progressed via RT (n=20) or prolymphocytic leukemia (PLL) (n=1). The Cumulative Incidence Rate of RT development 12-months post CDKN2A/B deletion was 23.5% (95% CI 10.9-38.9), at 24-months, it was 29.4% (15.1-45.3). Deletion of TP53 was seen in 85.7%. Albeit there was limited pts with detection of CDKN2A/B deletion prior to RT, the multivariable model for RT found loss of TP53 (p=0.02, HR 4.53 (95% CI 1.33-15.43)) and homozygous and heterozygous loss of CDKN2A/B (p=0.0008, HR 6.08 (95% CI 2.13-17.39)) were independently significant. Conclusion: Here we show that deletion of CDKN2A/B in CLL is a very rare occurrence and was seen predominantly in pts with highly aggressive disease characteristics. Close to half of our cohort progressed with RT or PLL. Additional investigation of serial samples is needed to establish when CDKN2A/B deletion occurs relative to RT. Our results indicate that prospective FISH analysis for CDKN2A/B deletion, in conjunction with other high-risk genetic alterations, to establish RT prognostication in high-risk CLL is warranted.

First Single-Center Experience with Luspatercept Therapy in Low-Risk Myelodysplastic Syndrome (LR-MDS) Patients with Transfusion Dependence Refractory to Erythropoietin Therapy

Introduction: In the treatment of low-risk myelodysplastic syndromes (MDS) patients, anemia is the predominant problem. A recent breakthrough in the therapy of anemia in low-risk MDS patients are drugs involved in TGFβ pathway blockade. Luspatercept is a recombinant fusion protein derived from human activin receptor type IIB. It binds TGFβ superfamily ligands and inhibits the SMAD2/3 pathway leading to potentiation of red cell line differentiation and proliferation. Luspatercept was approved last year by the FDA and EMEA in the treatment of anemic low-risk MDS patients with ring sideroblasts (MDS-RS-MLD, MDS-RS-SLD) and/or SF3B1 mutated patients who are not eligible for erythropoiesis-stimulating agents (ESA) or for patients who failed on this therapy. Luspatercept showed high clinical activity for the treatment of anemia in the phase 2 trial (PACE-MDS) and subsequently in the phase 3 trial (MEDALIST). It has led to transfusion independence (TI) in about 40 % of patients. Methods: From January 2021 to July 2022, 27 patients were treated with luspatercept at the hematology department of Charles University Hospital in Prague, Czech Republic. The median age of the patients was 77 years (range 57-89) with a male to female ratio 17:10. WHO 2016 diagnosis were: MDS-RS-MLD (N = 23) and MDS/MPN-RS-T (N = 4). Median of IPSS-R was 2.5 (range 1-4). Median of IPSS-R cytogenetic score scaled from 0-2 with the median of 1. Median follow-up was 7.8 months (range 1-38). Luspatercept was administered in subcutaneous injections once every 3 weeks with an initial dose of 1 mg/kg. Dose escalation was done according to the level of response to 1.33 and 1.75 mg/kg. The median number of cycles administered was 7 (2-17). Transfusion dependency before luspatercept initiation ranged from 2 transfusion units (TU)/8 weeks to 12 TU/8 weeks, 11 patients had low transfusion burden (LTB), 16 high transfusion burden (HTB). Baseline erythropoietin (EPO) level was 374 IU/l in average (range 150-770) and ferritin level was 1307 μg/l in average (range 234-3832). 9 patients received chelation therapy. 22 patients were tested for SF3B1 mutation with 21 positive results (95.5 %) using Sanger sequencing (N = 3) or NGS approach (N = 18). Isolated mutation of SF3B1 was present in 8 patients (44 %). The most frequent mutations besides SF3B1 were TET2 (N = 8; 44 %), ASXL1 (N = 3; 17 %), DNMT3A and RUNX1 (N = 2; 11 %). Average mutations count per one patient was 1.7 (range 0-5). We did not observe any TP53 mutation. Results: There were 24 evaluable patients in terms of response. Thus far 11 (45 %) patients have responded by achieving TI, 5 (20 %) patients have had a reduction in transfusion dependence (HI, according to IWG criteria 2006), 8 (33 %) patients have not responded. One patient progressed to the higher risk MDS, and one transformed to acute myeloid leukemia. Median duration of response was 7.4 months (range: 0.7-31.8). Median number of cycles to achieve response was 4. Eleven patients responded to the basic dose 1 mg/kg, 2 additional patients on the dose 1.33 mg/kg and 2 more on the dose 1.75 mg/kg. There were differences in response according to transfusion burden, in LTB group achieved TI 8 patients and HI 1 patient, in HTB group we observed TI in 3 patients and HI in 4 patients. In 9 patients, we added ESA to luspatercept therapy, as they act both at different levels of erythropoiesis, which led to the improvement of response in 5 cases. In general, patients with lower initial baseline EPO level were better responders. We did not observe decrease in ferritin level during therapy. There was no significant effect of mutation burden on patient's OS, although patients with single mutated SF3B1 achieved better ORR (75 %), than patients bearing mutations in multiple genes (ORR = 40 %). There were no adverse effects of Grade II or more. Conclusion: In conclusion, luspatercept is certainly the next major step in the treatment of anemia in MDS patients. The advantage of the drug is very good tolerance and easy administration. We observed better responses in patients bearing single mutation in SF3B1, patients with LTB and lower initial baseline EPO levels. The higher response rate in our follow-up may be influenced by the combination with ESA and rapid dose escalation. This work was supported by the following grants: grant AZV 2021-2024 NU21-08-00312, Grant AZV 2019-2023 NV19-08-00144, UNCE/MED/016

Long-Term Evolution of Somatic Mutations in Patients with Del(5q) MDS Early Treated with Lenalidomide in the Sintra-Rev Clinical Trial: Safe and Effecitive Approach?

INTRODUCTION Lenalidomide (LEN) is the election treatment in del(5q) MDS, approved only in case of RBC transfusion dependency (TD). Only scarce retrospective data exploring LEN in non-TD setting are available, suggesting a benefit. SintraRev is the first randomized clinical trial evaluating LEN in del(5q) MDS with anemia but without TD. While efficacy has been confirmed in this trial, safety regarding clonal evolution and disease progression has not been addressed in this early treatment context. METHODOLOGY SintraRev is a phase III multicenter clinical trial in low-risk del(5q) MDS patients with anemia without TD. Patients were randomized (2:1) in a double-blind design to LEN (5mg/day continuously) vs placebo (PCB) for 2 years of treatment and 2 years of follow-up (FU). In addition to clinical results, we aimed to study the role of molecular profiling in this early therapeutic approach (LEN use prior to TD) in both arms during FU (baseline, week 12 and the last available FU). Next-generation sequencing (NGS) was performed in available samples. Sequential samples were collected before treatment initiation and then every 6 months until last FU. A capture-based NGS panel targeting 65 myeloid genes was used. Libraries were prepared following manufacturer's instructions (Twist BioScience) and run on Illumina sequencers. Mutations were considered at coverage of 2000x and >5% variant allele frequency (VAF). RESULTS Sixty-one patients were included in SintraRev with a median FU of 60.6 (32.3-73.9) months. A total of 47 patients (29 LEN, 18 PCB) had NGS data. At baseline, 32%, 36% and 32% of patients had 0, 1 and ≥2 mutations, respectively. More frequently mutated genes were SF3B1 (25.5% of patients) and TP53 (21%). No differences were observed regarding SF3B1 (p=0.74), TP53 (p=0.48) and DAT (DNMT3A, ASXL1 and TET2, p=1.0) mutations between groups. Furthermore, no differences in neither median number of mutations per patient (1: 0-4) nor VAF were found between LEN and PCB initially (p>0.05). At week 12, median VAF of mutations was significantly lower in the LEN group as compared to baseline (1.84% vs 9%, respectively p<0.01, n=21), but remaining stable in the PCB arm (18% w12 vs 17% baseline, p=0.594). Interestingly, in the long-term FU (median 22.4 months - 3.03 to 68.77) an increase in median VAF (from 17% to 29%, p<0.001, n=13) was observed in PCB arm (Fig 1). Clonal evolution among LEN & PCB patients during treatment was different, with a reduction in predominating clones in LEN arm, while remaining stable in the PCB group (Fig 2). In the long-term FU, both PCB & LEN showed acquisition of additional mutations (20% PCB & 22.7% LEN, p=1.0). Nevertheless, all these new mutations in the LEN group were acquired several months after loss of response to treatment or LEN discontinuation. Interestingly, dynamics of mutations under LEN treatment did not affect DAT mutations, which remained stable when cytogenetic response (CyR) was achieved. In this way, 87,5% of DAT mutations showed an independent behavior from the clone with 5q deletion. However, most SF3B1 (62,5%) and TP53 mutations (100%) were affected by LEN treatment in parallel with the disappearance of the del(5q) clone at the time of CyR. In this regard, responses were not affected by the persistence of DAT mutations (>80% of erythroid response, ER, and CyR) and interestingly, SF3B1mut patients achieved 100% of ER and CyR. In contrast, only 20% of TP53mut patients achieved ER and 60% CyR. AML evolution occurred in 13.8% and 22.2% patients in LEN & PCB arms, respectively (p=0.43). In patients transforming to AML, 50% had TP53mut in LEN while no TP53 mutations were found in PCB AML progression. No differences were found regarding median time to AML between the two groups (25.87 months, p=0.2). CONCLUSIONS MDS patients showing 5q deletion and without TD early treated with LEN did not showed more clonal evolution than the same set of patients in PCB arm. In fact, the acquisition of additional mutations in LEN patients was observed after loss of response or LEN discontinuation. Interestingly, molecular responses were confirmed in LEN patients by reduction of mutation's median VAF at week 12. Dynamic evolution of DAT alterations was not affected by LEN treatment, suggesting a role as CHIP-associated mutations. In contrast, most SF3B1 and TP53 mutations were modified in parallel with the del(5q) clone at the time of CyR. The maintenance of CHIP-related mutations did not affect either ER or CyR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal