EZH2 Mutations Research Articles

EXPLORING DIFFERENTIAL MUTATIONAL DISTRIBUTION BY TARGETED SEQUENCING IN A R‐CHOP TREATED COHORT OF DIFFUSE LARGE B‐CELL LYMPHOMA PATIENTS

Background: Diffuse large B-cell lymphoma (DLBCL) harbors striking clinical and molecular heterogeneity, which continues to encumber risk stratification with a consequently high rate of refractory/relapsed patients (pts). Though the genomic landscape of DLBCL has been comprehensively uncovered, the search for eligible molecular biomarkers continues. Exploratively, we set out to retrospectively evaluate differential mutational distributions in a DLBCL cohort using targeted sequencing. Patients and method: A cohort of 105 de novo DLBCL pts, uniformly treated with first-line R-CHOP, were studied. Thirty pts relapsed, of which 70% had early relapses (<2 years after completing therapy), while 30% had late relapses (≥2 years). Targeted next-generation sequencing (90 gene panel) was applied on archival paraffin-embedded diagnostic samples and, if available, first relapse samples. Results: In total, 118 samples were sequenced, each displaying a median of 12 (range 1–46) putative somatic protein-coding mutations. Germinal center B-cell (GCB) DLBCL (n = 60) displayed a differential somatic signature with enrichment of EZH2, TNFRSF14, BCL2, ACTB, SOCS1, and FAS mutations (Fisher's Exact, p < 10-4), whereas mutations in CDKN2A, PRDM1, MYD88, and CD79B were significantly enriched in non-GCB pts (p < 10-4, n = 44). Survival analysis confirmed inferior overall survival (OS) in non-GCB DLBCL (5-year OS, log-rank (Mantel-Cox), p = 0.01, hazard ratio (HR) 1.7). Pts with double-hit biology (DHB), defined as either MYC and BCL2 and/or BCL6 fluorescent in situ hybridized positive and/or with MYC and BCL2 immunohistochemical double expression (n = 32), did not display a significant mutational signature or differential OS. However, a subgroup of DHB pts characterized by mutations in MYC, PRDM1, or IRF4 (n = 15) had markedly inferior OS (5-year OS, log-rank, p = 0.0003, HR 3.34) compared to negative non-DHB pts (n = 55) or negative non-DHB/DHB pts (p = 0.0017, HR 2.7, n = 72). Noticeably, ACTB mutations were only present in non-DHB pts. The mutational distribution at diagnosis did not differ between pts with relapse and in sustained remission. However, in the relapse group, GNA13 mutation at diagnosis was more frequent in early relapses than late relapses (43% vs. 0%, Fisher’s Exact without correction, p = 0.03). The presence of GNA13 in the entire cohort showed a trend towards inferior survival. Conclusion: Differential distribution with significant mutational signatures were expectedly found in GCB versus non-GCB, but not in subgroups with DHB or relapse. Interestingly, mutations in MYC, PRDM1 or IRF4 identified a distinct subset of DHB pts with inferior survival. Furthermore, GNA13 mutations were significantly enriched in early relapse pts. These preliminary findings call for validation of GNA13 as a potential marker of early relapse as well as the prognostic value of MYC, PRDM1 and IRF4 mutations in DHB DLBCL. The research was funded by: The Danish Cancer Society and The Vissing Foundation Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, tumor biology and heterogeneity Conflicts of interests pertinent to the abstract T. S. Larsen Consultant or advisory role Roche, Gilead, Novartis, Celgene/BMS Research funding: Genentech

Tazemetostat for tumors harboring SMARCB1/SMARCA4 or EZH2 alterations: results from NCI-COG pediatric MATCH APEC1621C.

National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice assigns patients aged 1-21 years with refractory solid tumors, brain tumors, lymphomas, and histiocytic disorders to phase II trials of molecularly targeted therapies based on detection of predefined genetic alterations. Patients whose tumors harbored EZH2 mutations or loss of SMARCB1 or SMARCA4 by immunohistochemistry were treated with EZH2 inhibitor tazemetostat. Patients received tazemetostat for 28-day cycles until disease progression or intolerable toxicity (max 26 cycles). The primary endpoint was objective response rate; secondary endpoints included progression-free survival and tolerability of tazemetostat. Twenty patients (median age = 5 years) enrolled, all evaluable for response and toxicities. The most frequent diagnoses were atypical teratoid rhabdoid tumor (n = 8) and malignant rhabdoid tumor (n = 4). Actionable alterations consisted of SMARCB1 loss (n = 16), EZH2 mutation (n = 3), and SMARCA4 loss (n = 1). One objective response was observed in a patient with non-Langerhans cell histiocytosis with SMARCA4 loss (26 cycles, 1200 mg/m2/dose twice daily). Four patients with SMARCB1 loss had a best response of stable disease: epithelioid sarcoma (n = 2), atypical teratoid rhabdoid tumor (n = 1), and renal medullary carcinoma (n = 1). Six-month progression-free survival was 35% (95% confidence interval [CI] = 15.7% to 55.2%) and 6-month overall survival was 45% (95% CI = 23.1% to 64.7%). Treatment-related adverse events were consistent with prior tazemetostat reports. Although tazemetostat did not meet its primary efficacy endpoint in this population of refractory pediatric tumors (objective response rate = 5%, 90% CI = 1% to 20%), 25% of patients with multiple histologic diagnoses experienced prolonged stable disease of 6 months and over (range = 9-26 cycles), suggesting a potential effect of tazemetostat on disease stabilization.

Abstract CT082: Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints

Abstract Introduction: The PI3K inhibitor copanlisib (C) plus the anti-CD20 antibody rituximab (R) was superior to R plus placebo (P) in patients (pts) with relapsed iNHL (Matasar et al. Lancet Oncol 2021). We previously reported that C+R improved median progression-free survival (PFS) in the iNHL, follicular lymphoma (FL), and non-FL groups, with a statistically significant improvement in the PTEN-positive population (Shalini et al., AACR 2022). Here we conducted NGS analysis from pts treated with either C+R or P+R for possible impact on PFS outcomes. We also determined if there was an association between plasma cytokine levels at baseline with survival. Methods: Adult pts with relapsed B-cell iNHL were randomly assigned 2:1 to either C+R or P+R; standard dosing on a 28-day cycle applied. Either fresh or archival tumor tissues were collected for central pathology review and biomarker analysis. NGS was conducted using the TSO500 platform for 476 genes. Analyses included EZH2 and BCL2 mutation status in FL subset, and other genes for FL subsets and overall iNHL, as prognostic and predictive markers. The effect of mutations on PFS and overall survival (OS) outcomes was assessed. Cytokines from baseline plasma were assessed using the multiplex MSD platform (71-plex). Association between cytokine levels and PFS and OS were explored. Results: Baseline tumor samples were evaluable by NGS from 200 pts; 127 C+R and 73 P+R. Variant alleles with frequencies &amp;gt;10% were analyzed. One quarter (25.7%) of FL pts were determined to have EZH2 mutations. C+R treatment significantly improved PFS for FL pts with in mutant EZH2 (p=0.0066) compared to P+R. FL pts with mutant BCL2 had a statistically improved PFS compared to WT BCL2 when receiving C+R treatment. Plasma samples were available for 71-plex assessment in 373 pts; 246 C+R and 127 P+R. Pts were assigned as high or low baseline cytokine groups based on the median values. For majority of cytokines, the low cytokine group correlated with better PFS for all pts, FL and Other iNHL pts in the C+R arm. For OS, there were too few events at the time of primary data cutoff to make any associations. However, with longer follow up (2 years from primary completion) there was a significant improvement in OS in iNHL pts with low IL2 values at baseline receiving C+R compared to pts receiving P+R; HR 0.495 (95%CI: 0.270,0.908), p=0.0230. This association was only seen for pts with low (or undetected) levels of IL2 at baseline. Conclusions: Mutation status was associated with improvements in PFS in FL pts with mutant BCL2 or mutant EZH2 treated with C+R. iNHL pts with low plasma levels of IL2 levels treated with C+R showed a significant improvement in OS. Citation Format: Shalini Chaturvedi, Anke Weispfenning, David Bauer, Rong Du, Tine Descamps, Sara Bellinvia, Teresa Lunt, Lidia Mongay Soler, Barrett H. Childs, Pier Luigi Zinzani. Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT082.

Abstract 4784: Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer

Abstract Early breast carcinogenesis is characterized by an intermittently hypoxic, acidic and nutrient-deprived microenvironment, eventually selecting for the invasive, aerobically glycolytic phenotype known as Warburg Effect (WE), a universal cancer hallmark. We have reproduced the emergence of WE in vitro, by subjecting the nontumorigenic breast epithelial cell line MCF10A to multiple intermittent cycles of media starvation followed by recovery in growth media. Clonal expansion of surviving cells demonstrated increased lactate production in aerobic conditions (WE). A comprehensive molecular characterization, through WES and single cell multiomics, was performed to study the relationship between mutations, CNA, cytogenetic abnormalities, transcriptional dysregulation, and epigenetic modifications. Mutational analysis of 6 WE clones identified 249 shared mutations, including HRAS(G12V), BRAF(P523L) and ALK(Q180X). COSMIC mutational signature 18 (ROS-induced) was identified as the main DNA lesion across all WE clones, suggesting oxidative stress as a main source of mutagenesis associated with WE development. Non-synonymous mutations in FUS, HDAC3, EZH2, MAZ, KMTD2, MET, and other oncogenes were found in the clones, consisting in secondary events in the WE transformation. Geneset enrichment analysis identified over-expression of MYC/E2F targets, MTORC1 signaling, glycolysis and cell cycle pathways, as well as under-expression of EMT/cell adhesion pathways, in WE clones. CNA, based on a consensus from WES, scRNAseq (inferCNV) and scATACseq (epiAneufinder), confirmed amplification of MYC, E2F1 and MYBL2, as well as Gain(19q) and Gain(20) in all WE clones, as well as loss of TP63 in 5 out of 6 WE clones. Integrative single cell transcriptome/chromatin accessibility analysis (ArchR) identified decreased accessibility and expression of EMT-related genes, as a consequence of differential expression of pioneer transcription factors (i.e. reduced expression of TP63, due to deletion, led to silencing of TP53/63/73 targeted genes), as well as well-established EMT regulators (ZEB1, SNAI1/2, NFKB1), among others. We propose that the WE phenotype emerges after intermittent nutrient-derived oxidative stress induces increased expression of HRAS-regulated genes, acutely increasing mutation rate and transcriptionally inducing an EMT-like state. Once HRAS(G12V), MYC/E2F1 amplification and TP53 deletions are fixed in the population, the clones will maintain the ROS production and EMT-like state, even in optimum growth media conditions, completing the transition from an acute response to a chronic-inheritable phenotype known as WE. These findings reinforce the importance of microenvironmental stress driving early carcinogenesis, bridges the gap between environment and genomic-driven causes of malignant transformation and provides testable hypotheses of causal events in connection with WE. Citation Format: Rafael Renatino Canevarolo, Praneeth Reddy Sudalagunta, Joon-Hyun Song, Erez Persi, Mehdi Damaghi, Ariosto Siqueira Silva. Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4784.

Abstract 6271: Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor

Abstract Polycomb repressive complex 2 (PRC2) catalyze the writing of H3K27me3 epigenetic marker and suppresses the expression of genes including tumor suppressors. Activation mutation of EZH2, the catalytic subunit of the PRC2 complex, drives the progression of tumors such as DLBCL. The embryonic ectoderm development (EED) protein acts as the scaffolding and H3K27me3-binding subunit of PRC2 and is essential for its activity. We have developed potent, selective, and orally bioavailable EED inhibitor BR1733. BR1733 blocks the interaction between EED and H3K27me3, and inhibits catalytic activity of PRC2. BR1733 showed superior PK profile across species and exhibited tumor growth inhibition against several B cell lymphoma CDX models. Indication expansion studies demonstrated that BR1733 could inhibit solid tumors with SWI/SNF loss-of-function mutation only when both CDKN2A and TET1 are expressed. BR1733 also inhibited tumor angiogenesis and showed tumor growth inhibition in several CDX models of highly vascularized solid tumors such as HCC and RCC. BR1733 is currently in Phase I clinical trials. Citation Format: Xinghao Wang, Shichao Ma, Min (Bella) Xie, Lele Wang, Yajun Wang, Song Zhang, Sheng Chen, Lynette Zhang, Yongqi Zhou, Mengxi Zhao, Dandan Li, Guobin Li, Xiao Fan, Hailong Zhang. Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6271.

Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells.

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.

Pembrolizumab for the treatment of disease relapse after allogeneic hematopoietic stem cell transplantation.

A failed graft-versus-tumor (GVT) effect is a common mechanism of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Although targeting the PD-1/PD-L1 axis may restore GVT effects, PD-1 blockade exacerbates graft-versus-host disease (GVHD) in murine models, and severe GVHD can occur in patients treated with anti-PD-1 therapy after alloHCT. Therefore, we developed a prospective study to assess the safety and efficacy of pembrolizumab in patients relapsing after alloHCT. Eligible patients received pembrolizumab (200 mg every 3 weeks) for up to 2 years. Twelve patients were enrolled (8patients with acute myeloid leukemia, 1 patient with myelodysplastic syndrome, 1 patient with classical Hodgkin lymphoma, and 2 patients with diffuse large B-cell lymphoma [DLBCL]). All participants received reduced-intensity preparative regimens with invivo T-cell depletion. The median time from alloHCT to enrollment was 587 days (range, 101-4211). Three participants (25%) experienced grade 3 to 4 immune-related adverse events (irAE) (pneumonitis, 2 patients; hyperthyroidism, 1 patient), all occurring after 1 to 2 cycles, and resolving after pembrolizumab discontinuation and corticosteroid treatment. irAEs of any grade occurred in 5 patients (42%). No treatment-emergent GVHD was observed. Overall and complete response (CR) rates were 22% (2/9). Both patients achieving CRs hadPD-L1 gene-amplified lymphomas and diffuse PD-L1 expression on pretreatment biopsies. An acquired EZH2 mutation was identified at relapse in a patient with DLBCL who achieved an initial CR to pembrolizumab, which was associated with downregulated HLAexpression on malignant B cells, implicating EZH2 mutations as a potential immune escape mechanism after PD-1-blockade therapy. In conclusion, after alloHCT, treatment with pembrolizumab is feasible and associated with objective responses in relapsed lymphoid malignancies but can induce severe irAEs, requiring vigilant monitoring. Thistrial was registered at www.clinicaltrials.gov as #NCT02981914.

EZH2 W113C is a gain-of-function mutation in B-cell lymphoma enabling both PRC2 methyltransferase activation and tazemetostat resistance

Polycomb Repressive Complex2 (PRC2) suppresses gene transcription by methylating lysine 27 of histone H3 (H3K27) and plays critical roles in embryonic development. Among the core PRC2 subunits, EZH2 is the catalytic subunit and EED allosterically activates EZH2 upon binding trimethylated H3K27 (H3K27me3). Activating mutations on Y641, A677, and A687 within the enzymatic SET domain of EZH2 have been associated with enhanced H3K27me3 and tumorigenicity of many cancers including B-cell lymphoma and melanoma. To tackle the critical residues outside the EZH2 SET domain, we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel gain-of-function (GoF) mutation W113C, which increases H3K27me3 in vitro and in vivo and promotes CDKN2A silencing to a similar level as the EZH2 Y641F. Different from other GoF mutations, this mutation is located in the SAL motif at the EZH2 N-terminus, which stabilizes the SET domain and facilitates substrate binding. This may explain how the W113C mutation increases PRC2 activity. Tazemetostat is an FDA-approved EZH2 binding inhibitor for follicular lymphoma treatment. Intriguingly, the W113C mutation leads to tazemetostat-resistance in cell and in vivo in both H3K27 methylation and tumor proliferation. Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance, effectively decreases H3K27me3, and blocks tumor proliferation in cells expressing EZH2 W113C. As this mutation is originally identified from lymphoma samples, our results demonstrated its activating characteristic and the deleterious consequence, provide insights on PRC2 regulation, and support the continued exploration of treatment optimization for lymphoma patients.

Prevalence of IRF4 rearrangement in large B-cell lymphomas of the Waldeyer’s ring in adults

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (B-NHL) in adults. These lymphomas are classified according to gene expression profiling (GEP) into germinal center B-cell (GCB) and activated B-cell type (ABC). Recent studies have suggested new subtypes of large B-cell lymphoma, based on genetic and molecular alterations, among them is large B-cell lymphoma with IRF4-rearrangement (LBCL-IRF4). We used fluorescence in situ hybridization (FISH), GEP (using the DLBCL COO assay by HTG Molecular Inc), and next generation sequencing (NGS) to comprehensively characterize 30 cases of LBCLs located in Waldeyer’s ring in adult patients and to identify LBCL-IRF4. FISH revealed breaks of IRF4 in 2/30 cases (6.7%), BCL2 breaks in 6/30 cases (20.0%), and IGH breaks in 13/29 cases (44.8%). GEP classified 14 cases each as GCB or ABC subtype, and 2 cases remained unclassified; this was concordant with the immunohistochemistry (IHC) in 25/30 cases (83.3%). A subgrouping, based on GEP, was performed: group 1 included 14 GCB cases with the most frequent mutations in BCL2 and EZH2 in 6/14 cases (42.8%). The two cases with IRF4 rearrangement were assigned to this group by GEP and showed IRF4 mutations, supporting the diagnosis of LBCL-IRF4. Group 2 included 14 ABC cases; the most frequent mutations were CD79B and MYD88 identified in 5/14 patients (35.7%). Group 3 included 2 unclassifiable cases in which no molecular patterns were detected. Overall, LBCLs of Waldeyer’s ring in adult patients are a heterogeneous group, including LBCL-IRF4, which shares several features with cases in the pediatric population.

Predicting chemoevasion potential of cancer cells from statistical chromatin packing behavior.

In malignancy, genetic, metabolic, or environmental insults may remodel chromatin structure, potentially increasing the plasticity of aberrant cell state transitions. Observed alterations to the epigenetic landscape of chromatin include gain-of-function mutations of Polycomb repressor EZH2 that block differentiation. Nucleosome remodeling and higher-order chromatin organization may also increase malignancy, with abnormalities ranging from prevalent mutations of ATP-dependent chromatin remodelers to mixing between transcriptionally active and inactive compartments. However, a universal relationship between chromatin structure and the disease state does not yet exist. Here, using the first principles of physics, we demonstrate that chromatin packing, or the statistical distribution of chromatin density in 4D, is a genome-wide regulator of phenotypic plasticity. The nucleus is a highly crowded, heterogeneous environment where DNA, RNA, proteins, and other macromolecules exert excluded volume effects, altering the kinetics and efficiency of transcription reactions. As chromatin is the major crowder in the nucleus, characterizing the chromatin mass density distribution facilitates a more complete understanding of transcription processes in a realistic nuclear environment. Combining high-resolution chromatin scanning transmission electron microscopy (ChromSTEM) and live-cell, label-free Partial Wave Spectroscopy (PWS) along with polymer physics-based analysis, we identified the existence of spatially separable packing domains, around 80 nm in radius. We then employed our understanding of fundamental chromatin packing to build a chromatin-mediated model of transcription that incorporates two pertinent aspects of phenotypic plasticity: transcriptional malleability, the ability of a cell to upregulate certain genes within a critical time frame, and transcriptional heterogeneity, a cell population's range of functional states. Finally, we extended our transcription model to predict how the average chromatin packing behavior of a cell population contributes directly to cancer cell survival upon chemotherapy treatment. Experimental observations confirmed model predictions, underlying the importance of chromatin packing in chemoevasion processes.

EZH inhibitors in lymphoma therapy

Enhancer of zeste homolog (EZH), a subunit of polycomb repressive complex 2 (PRC2), suppresses gene expression by methylation of H3K27. EZH is closely associated with B-cell development and pathogenesis of certain malignant lymphomas. In follicular lymphoma (FL), gain-of-function mutation and upregulation of EZH2 are observed in approximately 30% and 15% of cases, respectively. Moreover, one-third of diffuse large B-cell lymphomas carry an EZH2 mutation, mostly co-existing with translocation involving Bcl-2. Genome-wide trimethylation of H3K27 is a unique characteristic induced by upregulation of both EZH2 and EZH1, and is responsible for more than half of the gene suppression that occurs in adult T-cell leukemia/lymphoma (ATL). Inhibition of EZH can reduce H3K27 methylation and subsequently restore epigenetically suppressed genes. Currently, an EZH2 inhibitor and dual EZH1/2 inhibitor have been clinically used to treat relapsed/refractory FL and ATL, respectively. EZH-targeted treatment for lymphoma has only just begun, and further development of these drugs for various other malignancies, both alone and in combination with other therapeutics, is ongoing.

ETNK1 mutation occurs in a wide spectrum of myeloid neoplasms and is not specific for atypical chronic myeloid leukemia.

ETNK1 mutation has been suggested as a useful tool to support the diagnosis of atypical chronic myeloid leukemia. ETNK1 mutations, however, occur in other myeloid neoplasms. The authors assessed the clinicopathologic and molecular genetic features of 80 ETNK1-mutated myeloid neoplasms. Thirty-seven neoplasms (46%) were classified as myelodysplastic syndrome, 17 (21%) were classified as myelodysplastic/myeloproliferative neoplasm, 14 (18%) were classified as acute myeloid leukemia, and 12 (15%) were classified as myeloproliferative neoplasm. ETNK1 mutations were detected at the first test in 96% of patients, suggesting that ETNK1 mutation is an early event in pathogenesis. ETNK1 mutations represented the dominant clone in 63% of patients and was persistently dominant in 93%. The variant allele frequencies were usually higher in acute myeloid leukemia and increased upon leukemic transformation. ETNK1 mutation was accompanied by coexisting mutations in all patients, with ASXL1 (50%), TET2 (25%), EZH2 (24%), RUNX1 (24%), and SRSF2 (24%) mutations being the most common. Neoplasms with ETNK1 mutations were associated with morphologic dysplasia, increased blasts, myelofibrosis, and noncomplex karyotypes. With a median follow-up of 16.5months, 30 patients died, 44 had persistent disease, and four achieved complete remission after stem cell transplantation. ETNK1 mutation is present in various myeloid neoplasms, often as an early event and a dominant clone and always with concurrent mutations. It may play an important role in the pathogenesis and progression of myeloid neoplasms by causing DNA damage and inducing other mutations and genomic instability, and it may serve as a potential therapeutic target. ETNK1 mutation is not disease-specific and should be interpreted with caution to classify myeloid neoplasms.

Clinical characteristics and outcomes of EZH2-mutant myelodysplastic syndrome: A large single institution analysis of 1774 patients

EZH2 mutations in myeloid neoplasms are loss of function type, and have been linked to poor overall survival (OS) in patients with myelodysplastic syndrome (MDS). However, the specific determinants of outcomes in EZH2-mutant (mut) MDS are not well characterized. In this single-center retrospective study, clinical and genomic data were collected on 1774 patients with MDS treated at Moffitt Cancer Center. In our cohort, 83 (4.7%) patients had a pathogenic EZH2 mutation. Patients with EZH2mut MDS were older than EZH2-wild type (wt) group (median age- 72 vs. 69 years, p = 0.010). The most common co-occurring mutation in EZH2mut MDS was ASXL1, with a significantly higher frequency than EZH2wt (54% vs. 19%, p < 0.001). Patients with EZH2mut MDS had lower response rates to hypomethylating agents compared to EZH2wt MDS (26% vs. 39%; p = 0.050). Median OS of patients with EZH2mut MDS was 30.8 months, with a significantly worse OS than EZH2wt group (35.5 vs. 61.2 months, p = 0.003) in the lower-risk IPSS-R categories. Among patients with EZH2mut MDS, co-presence of ASXL1 or RUNX1 mutations was associated with inferior median OS compared to their wt counterparts (26.8 vs. 48.7 months, p = 0.031). Concurrent chromosome 7 abnormalities (12%) were also associated with significantly worse OS (median OS- 20.8 vs. 35.5 months, p = 0.002) in EZH2mut MDS. Future clinical trials should explore the potential role of novel targeted therapies in improving outcomes in patients with EZH2mut MDS.

Abstract B013: Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition

Abstract Polycomb repressive complex 2 (PRC2) drives the expansion of H3K27me3 epigenetic marker and suppress the expression of target genes including tumor suppressors. Activation mutations of EZH2, the catalytic subunit of the PRC2 complex, are thought to be the driver for certain malignancies such as diffused large B cell lymphoma (DLBCL). The SWI/SNF complex remodels nucleosomes to modulate transcription, they are generally considered as tumor suppressors and loss-of-function (LOF) mutations are frequently found across a wide variety of cancers. Many studies suggest that PRC2 is important for the proliferation of tumor cells with SWI/SNF LOF mutations, indeed, EZH2 inhibitor tazemetostat was approved by FDA for epithelioid sarcoma with SWI/SNF subunit SMARCB1 deletion. While not all SWI/SNF LOF tumors respond to PRC2 inhibition, we would like to know what subset of these tumors are sensitive to PRC2 inhibition. In this study, we have developed a potent and selective PRC2 inhibitor BR1763 which blocks the interaction of EED with tri-methylated histone H3 at Lys 27 (H3K27me3) and inhibits cellular H3K27me3 levels. By analyzing the gene expression patterns of BR1763 treated cells, we found that up-regulation of CDKN2A/p16 and down-regulation of E2F downstream gene are essential for cellular response to PRC2 inhibition. Further studies also confirmed that PRC2 inhibition caused P16 up-regulation and induced cell senescence for the sensitive cells. Importantly, suppression of TET1 expression contributes to the resistance to PRC2 inhibition in SWI/SNF LOF tumors, which may be caused by PcG hypermethylation on p16 promoters. TET1 expression, together with SWI/SNF LOF mutation status, can largely predict the sensitivity of tumor cells to PRC2 inhibition. Citation Format: Hailong Zhang. Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B013.

Significance of cardiovascular comorbidity in patients with chronic myelomonocytic leukemia.

In aretrospective study, we analyzed the prevalence of common cardiovascular comorbidities in 310patients with chronic myelomonocytic leukemia (CMML), their potential prognostic impact, and potential correlations with laboratory and molecular features. 115 (36%) patients had adocumented cardiovascular comorbidity. In these patients, coronary heart disease 41/115 (36%), atrial fibrillation 34/115 (29%), and hypertension 75/115 (64%) were documented. None of these conditions had asignificant impact on survival. Unexpectedly, patients with cardiovascular comorbidity had alower number of circulating blasts and alower prevalence of EZH2 mutations. Moreover, time to transformation was significantly longer in these patients. Cardiovascular comorbidity does not seem to have amajor impact on prognosis in CMML patients. The unexpected lower transformation rate in these patients needs to be further investigated.

EZH2 Inhibition Mediates Proptosis Via DHX9 and S100A9 in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) is considered as an archetypal epigenetic disorder due to the aberrant epigenome, frequent epigenetic regulator mutations and the most responsive disease to DNA methylation inhibitors. Among numerous epigenetic regulators, Enhancer of zeste 2 homologue 2 (EZH2) occupies an important position owing to its involvement in multiple epigenetic modifications. In MDS, EZH2 mutation frequency is 5%-10%. Given that the EZH2 gene is located at 7q36.1, abnormalities of chromosome 7q disturb EZH2 as well. To investigate the expression and effects of EZH2 in MDS patients without EZH2 mutation and chromosome 7 abnormalities, qPCR was performed. We found that EZH2 transcript level was significantly higher in MDS patients and its expression was highest in very high risk group, followed by the high risk group. The survival time of EZH2 high expression was shorter than the low expression group. Multivariable Cox regression showed that EZH2 expression was an independent prognostic factor. Next, we treated MDS cell lines MDS-L and SKM-1 with EZH2 inhibitor DZNep and found that DZNep inhibited cell proliferation in a dose- and time-dependent manner. To determine which pattern of cell death played the most important role, we pretreated different cell death inhibitors for 1 hour before adding DZNep. Compared with the group without cell death inhibitors, pyroptosis inhibitor (VX765) was the most effective in restoring cell viability. Besides, both DZNep and knockdown of EZH2 could trigger pyroptosis. To elucidate the possible mechanism of pyroptosis induced by EZH2 inhibition, we searched for RNA-seq data from tumor cells treated with EZH2 inhibitors by using the GEO database. We overlapped 53 genes by analyzing GSE151576, GSE80239 and GSE119088, of which DHX9 was the highest expressed in BM and attracted attention.Meanwhile, down-regulation of DHX9 was observed after pharmacological inhibition or EZH2 knockdown. There was a positive correlation between EZH2 and DHX9 in MDS patients. Furthermore, DHX9 expression was increased in MDS patients in contrast to control, and its overexpression was connected with shorter OS. Knockdown of DHX9 with two shRNAs induced pyroptosis. CUT&RUN and qPCR assay results validated that EZH2 and H3K4me3 occupancy at DHX9 promoter. Moreover, with RNA sequencing we found DHX9 can regulate S100A9 and confirmed by western blot. Overexpression of S100A9 could cause pyroptosis of MDS cell lines, which could be reversed by a S100A9 inhibitor. DHX9 and S100A9 are negatively correlated in MDS patients. Finally, we established xenograft mouse models by intravenously injecting luciferase-labeled SKM-1 cells. Intraperitoneal injection of 2mg/kg DZNep daily could significantly reduce the tumor burden and prolong the survival time of mice. We further confirmed that inhibition of EZH2 regulated DHX9 and S100A9 and induced pyroptosis in mouse bone marrow cells. In conclusion, we found that EZH2 expression in de novo MDS patients without EZH2 mutation and chromosome 7 abnormality was higher than that of the control group, and its high expression was correlated with inferior prognosis. EZH2 suppression inhibited tumor growth and induced pyroptosis both in vitro and in vivo. Inhibition of EZH2 triggered pyroptosis by reducing its binding in the DHX9 promoter and upregulating S100A9. In this present study, we reveal a novel molecular mechanism by which EZH2 inhibition induced pyroptosis and provide a basis for the treatment of MDS with EZH2 inhibitor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutations Associated with Progression in Follicular Lymphoma Predict Inferior Outcomes in Newly Diagnosed Patients (Alliance 151303)

Follicular lymphoma (FL) is a clinically heterogeneous B cell non-Hodgkin lymphoma (B-NHL) subtype. While some patients (pts) tolerate watch-and-wait for years, others require prompt treatment. Further, a subset of pts suffers progression of disease within 24 months (mo) of treatment initiation (POD24) and/or aggressive histologic transformation (tFL). No predictive model guides FL treatment, but the FLIPI stratifies pt outcomes using clinical parameters. The m7-FLIPI integrates FLIPI, performance status (PS), and mutations in EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11; however, it is not routinely used to guide treatment due to unproven generalizability and actionability to date. We conducted a clinicogenomic analysis of 343 pts with FL or tFL [enrolled on CALGB/Alliance trials 50402, 50701, or 50803 (n = 58); or real-world cohorts from WUSTL (n = 191), Cleveland Clinic (n = 77), and U Miami (n = 17)], applying a custom 2,611-gene sequencing panel to B-NHL specimens. We explored patterns of mutated genes in newly diagnosed FL (n = 231), relapsed (rel) FL (n = 65), and tFL (n = 47). We also asked whether specific genomic features were associated with clinical parameters and assessed the association of different mutations with pt outcomes. Median age was 60 years (range: 22-89); stage distribution was 23% early vs 77% advanced; FLIPI distribution was 34% low vs 34% intermediate vs 32% high. Progression-free and overall survival (PFS/OS) of pts in our cohort exhibited anticipated relationships with key parameters (e.g., histology: 48-mo OS 96% vs 69% for FL vs tFL). We applied the m7-FLIPI to our 231 newly diagnosed FL samples, observing weak prognostic impact [low vs high risk; HR for PFS 1.37 (95% CI 0.43-1.23), p = 0.2]. Given heterogeneous treatment histories of our pts compared to uniform pt treatment in the initial m7-FLIPI cohort, we tested for an interaction between m7-FLIPI and frontline treatment. We observed a strong interaction between m7-FLIPI risk stratification and receipt of cytotoxic chemotherapy (p < 0.01; Fig 1A). 48-mo PFS was 80% for pts with low risk m7-FLIPI receiving chemo-based frontline treatment, vs 50-55% for all three other pt subsets (high risk m7-FLIPI pts irrespective of chemo-status, and low risk m7-FLIPI pts receiving chemo-free treatment). This suggests that the prognostic value of the m7-FLIPI mutational pattern is impacted by pt treatment. To probe the links between the B-NHL genomic landscape and pathologic or clinical features, we compared the mutation burden (median number of mutations) across a range of B-NHL subsets. FL cohorts based on grade 1-2 vs 3A, early vs advanced stage, FLIPI, initial treatment vs observation, and POD24 vs no POD24 did not differ by mutation burden. However, mutation burden was significantly higher (p < 0.01) in tFL than rel FL, and in rel FL than newly diagnosed FL. Using a Cox proportional hazard penalized splines model, we did not find a significant relationship between mutation burden at FL diagnosis and PFS. CREBBP was the only gene in which mutations were significantly more common in FL than tFL (BH FDR q < 0.1), yet mutated CREBBP was associated with shorter frontline PFS in pts with FL (p < 0.05). Conversely, mutations in 23 other genes were more common in either rel FL or tFL compared to newly diagnosed FL (BH FDR q < 0.1), including 6 significantly mutated genes (SMGs; by MutSigCV): STAT6, TP53, IGLL5, B2M, SOCS1, and MYD88. We hypothesized that pts with newly diagnosed FL may be at high risk of poor outcomes if their pre-treatment tumors harbor mutations more common in progressive/transformed disease. We defined a 'mutations associated with progression' (MAP) signature as 2 or more mutations in these 7 genes (6 SMGs enriched in progressive/transformed disease and CREBBP). Applying univariate and multivariate Cox proportional hazard modeling (adjusting for FLIPI and PS), pts with newly diagnosed FL possessing a MAP signature had significantly shorter frontline PFS [multivariate HR for PFS 1.9 (95% CI 1.12-3.2), p < 0.05; median PFS 39 vs 89 mo, p < 0.05; Fig 1B]. In summary, our analyses highlight a set of MAPs that may offer unique insight into progression risk in FL, which may be more generalizable than the m7-FLIPI. Future studies are warranted to validate the poor prognosis associated with the presence of MAPs in pts with untreated FL, which could facilitate prospective studies of novel therapies in this subset of high risk pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omics Profiling of Longitudinal Samples Reveals Early Genomic Changes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: To identify early genetic events during FL pathogenesis and molecular events underlying progression and transformation, we performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients; 22 who experienced transformation (tFL) and 22 with progression of FL (nFL). Nineteen patients(both groups) experienced POD24. SNP6.0 data was available for 93 sequenced tumors and was used to infer allele-specific copy number alterations. Bulk RNA sequencing data was available for 64 sequenced tumors. Several computational tools were applied to identify potential cancer driver genes (IntOGen, MutSig2CV, 2020plus), mutational signatures, and to study clonal evolution (PyClone, ClonEvol). CIBERSORTx was used to infer B-cell specific transcriptomes from bulk RNAseq data. Results: Deep whole exome sequencing confirmed recurrent mutations in epigenetic regulators (CREBBP, KMT2D, EZH2, and EP300) and known copy number alterations. The mutational landscape was similar in nFL and tFL patients, except for higher recurrence of EZH2 mutations in tFL. We used CIBERSORTx to impute B-cell transcriptomes from bulk RNA-sequencing data to identify differentially expressed genes associated with common mutations. We discovered that numerous histone genes were downregulated in biopsies with EZH2 mutations, and in pretreatment samples from tFL patients, potentially contributing to epigenetic deregulation. Based on the mutational pattern in each biopsy, we calculated the genomic distances between longitudinal samples. This revealed great variability in nFL and tFL patients, compatible with complex evolutionary patterns independent of transformation. Tumor clonal evolution analysis identified CREBBP and KMT2D mutations as early genetic events in FL pathogenesis, likely occurring prior to the early CNAs that remained stable throughout disease progression such as gain in 12p and 18p and loss in 6p. Conclusions: Identification of such early and potentially essential genetic events may therefore provide opportunities for early disease detection and monitoring of progression.

Transient Monosomy 7 Is a Rare Event in Young Children with SAMD9L Syndrome

Monosomy 7 and deletion 7q are among the most common chromosomal aberrations in pediatric myeloid malignancies and generally associated with an increased risk of rapid progression to advanced neoplasia, warranting allogeneic hematopoietic stem cell transplantation (HSCT). However, several cases of transient monosomy 7 and spontaneous hematologic recovery upon loss of monosomy 7 in young children have been described in the literature. In several cases, germline mutations in sterile alpha-motif domain-containing protein 9 (SAMD9) and SAMD9-like (SAMD9L), both located on chromosome 7q, have been identified. Gain of function (GOF) mutations in SAMD9 and SAMD9L cause phenotypically overlapping syndromes that predispose to pediatric myelodysplastic syndrome (MDS) and are associated with non-hematological anomalies and variable immunodeficiency. GOF SAMD9/9L mutations inhibit cell proliferation and reduce cell survival of hematopoietic stem and progenitor cells (HSPCs). This may exert strong selection pressure on HSPCs to acquire somatic aberrations that improve their competitive fitness. To no surprise, a distinctive feature of SAMD9/9L syndromes is their propensity for somatic genetic reversion, including somatic loss of function mutations in SAMD9/9L in cis, uniparental isodisomy 7q, and non-random loss of chromosome 7 or 7q of the mutant allele. Multiple hematopoietic subclones with clone-defining somatic mutations can exist in the bone marrow and their composition may change over time due to the exceptionally high plasticity of hematopoiesis in young children. Consequently, monosomy 7 may spontaneously disappear in young patients with SAMD9/9L syndromes. Earlier observations had suggested that somatic compensatory mutations may be more frequent in SAMD9L than in SAMD9 mutated patients. Here, we investigate the natural history of young children with SAMD9L syndrome and monosomy 7. We report on seven patients with SAMD9L syndrome and monosomy 7 presenting before the age of 5 years in whom an initial surveillance strategy was electively chosen rather than timely allogeneic HSCT. All patients presented with refractory cytopenia of childhood with monosomy 7 at a median age of 1.0 years (range: 0.4-3.1 years). Six patients had an additional non-hematologic phenotype, including prematurity (P2, P5), small for gestational age (P1, P2, P7), cerebellar atrophy and developmental delay (P1), bilateral cleft lip and palate (P2), macrocephaly (P3, P6), shorter thumbs (P6), and big eye bulbs and dystrophy (P7). Immunodeficiency was present in five patients ranging from hypogammaglobulinemia (P1, P2, P5, P6, P7) to B and NK cell deficiency patients (P2, P5, P6, P7). At a median follow-up of 35 months (range: 10-51 months), allogeneic HSCT had been employed in four patients for severe neutropenia and/or bacterial infection (P1, P7), persistence of monosomy 7 beyond the age of 5 years (P5) or disease progression to MDS with excess blasts (P2). One patient (P2) suffered transplant-related mortality, while the remaining patients were alive at last follow-up. Three patients did not require definitive treatment. Two of the three patients had complete loss of the monosomy 7 clone with concomitant recovery of blood counts (P4, P6), a third patient had normalized blood counts, but cytogenetic and molecular studies at follow-up were missing (P3). Secondary somatic mutations were detected in six patients. Five patients carried somatic SAMD9L mutations in cis (P2, P3, P4, P6, P7). Two patients with spontaneous loss of monosomy 7 carried large somatic SAMD9L subclones that were stable over time and presumably maintained hematopoiesis (P4, P6). In addition, two patients carried somatic mutations in oncogenic driver genes (P2, P5). P2, who developed MDS-EB 14 months after diagnosis, had two RUNX1 mutations at the time of progression. P5 had one RUNX1 and one EZH2 mutation, but without evidence of disease progression. Our results suggest that loss of monosomy 7 can occur in young patients with SAMD9L syndrome but is a rare event. If a watch and wait strategy is chosen, close monitoring with frequent bone marrow examinations including SAMD9L sequencing, cytogenetics and search for somatic oncogenic events is advised. Importantly, presence of severe infections, severe neutropenia, increase in blast percentage, and persistence of monosomy 7 beyond the age of 5 years should prompt timely allogeneic HSCT.

Molecular Response Analysis By High Throughput Sequencing in Higher Risk Myelodysplastic Syndrome (HR-MDS) Treated Intensively with CPX-351

Context and objective Intensive chemotherapy (IC) remains an important therapeutic option in HR-MDS, but in-depth evaluation of response to IC remains ill studied. We previously reported results of CPX-351 in 31 untreated higher risk MDS patients (pts) (GFM-CPX-MDS study, NCT04273802, ASH 2021 Abst #243). Responses were associated with a significant decrease of CD34/CD117 progenitors and restoration of normal hematopoiesis patterns in bone marrow (BM) samples in multiparameter flow cytometry. In this ancillary study, the aim was to evaluate molecular responses by high-throughput sequencing (HTS) centrally. This involved description of the pts molecular landscape and an assessment of variant allele frequency (VAF) variations after induction treatment with CPX-351. Methods The mutational landscape was explored in BM nuclear cells by capture HTS of 42 genes (SureSelect XTHS2 Agilent) with unique molecular identifiers (UMI), unique dual index (UDI) and Illumina paired-end sequencing (Miseq and Nextseq). Somatic mutations (SM) were considered significant for VAFs >2%. Molecular response was evaluated by comparing baseline and post-induction. Molecular clearance was defined according to (i) clearance (<2%) of all SM excluding the "pre-leukemic” SM of DNMTA, TET2, ASXL1 (DTA) (ii) clearance of all non-DTA SM < HTS detection threshold (iii) normalized VAF decrease of the major non-DTA baseline SM (nmVAF). Results Median age of the 31 pts included was 62 years (range 31-69). There were 26 MDS-EB2 and 5 CMML-2. IPSS was int-2 in 26 pts and high in 5. Cytogenetic IPSS-R was very good (6.5%), good (71%, including normal in 22 pts), int (9.5%), poor (6.5%) or very poor (6.5%). After induction with CPX-351 (3 days cycle and a possible 2-day second induction cycle for non-responders), 16 pts (52%) reached CR, 4 (13%) CRi, 7 (22%) MLFS while 4 (13%) were in stable disease according to ELN 2017 criteria (which appeared more adequate than MDS IWG 2006, as reported at ASH 2021, Abst #243). Median follow-up was 511 days (179-710), 29/31 patients (94%) received allogeneic hematopoietic stem cell transplantation (Allo-HSCT) after, no, 1, 2, 3 or 4 CPX-351 consolidation cycles (that included only 1 day of CPX-351) in 10, 16, 7, 3 and 1 patients respectively. So far, 4 relapses have occurred, including 3 in transplanted pts. At inclusion, all pts had at least one mutation (median 3, [1-14]) with a mean VAF of 30% (2-89), implicating at least one DTA (71%; 22/31) or one non-DTA genes (93%; 29/31) (fig). Very high or high risk was seen in 19/31 (61%) pts according to the new IPSSm classification. Post induction molecular response was evaluated at a median of 53 days from CPX-351 onset (28-112) in 26 of the 27 who reached at least MLFS, and had a post-induction sample. Molecular clearance was observed (i) at the 2% threshold of non-DTA genes in 17/26 (65%) pts (ii) at the HTS detection threshold (0.1% i.e. minimum considered 3X with median coverage > 3000X) in 11/26 pts (42%) (iii) by nmVAF decrease in 25/26 pts (96%) with a median of -62% (-2 to -100%) (fig) while the remaining pt had an increase of SRSF2 (+67%) and RUNX1 (+46%) SMs but disappearance of three STAG2 SMs and was still in CR after allogeneic SCT. Of note, DTA SMs remained detectable in all pts with a median nmVAF modulation of -86% (+83% to -99.6%, fig) from baseline to post-induction. Of the 4 non-responders to CPX-351, 2 had high risk anomalies (inv(3) and EZH2 / RUNX1 SMs). The 3 relapses observed after alloHSCT, at 7, 8 and 15 months had nmVAFs decrease from baseline to post-induction of -95%, -98% and -50% respectively. Among them, one had a very complex karyotype and a homozygous TP53 mutation, the two others had a high mutational load and EZH2 mutations and one of the latest was transplanted in refractory state. Clonal evolution was observed in 5/26 pts with significant emergence of new mutations involving PPM1D, ASXL1 or JAK2 and in one pt the emergence of EZH2, RUNX1 and TET2 SMs. Four of these pts remained in morphological CR while one progressed to AML. Conclusion In 25/26 CPX-351 responders, a significant decrease of nmVAF was observed, confirming the interest of CPX-351 treatment in higher risk MDS, especially as a bridge to transplant. Follow up is ongoing and few relapses were seen to evaluate the predictive role of molecular analysis. An update will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal