Leukemia-associated Mutations Research Articles

Exploring Aging Dependent Adaptations of Splicing Factor Mutations in Clonal Hematopoiesis

Introduction: It has been shown that leukemia-associated mutations can be detected in the blood of healthy individuals and their clonal expansion in hematopoietic stem and progenitor cell (HSPC) populations is referred to as Clonal Hematopoiesis (CH). In one study, a population analysis of a cohort of 17,182 patients performed by Bick et al. [PMID7944936] selected without prior screening for hematological malignancies, indicated that advancing age is directly associated with an increase in CH. When the mutant clone accounts for 4% of the nucleated blood cells, it is then referred to as Clonal Hematopoiesis of Indeterminant Potential (CHIP). CHIP has been classified by the World Health Organization as a premalignant state and risk criteria for neoplastic development. CHIP is highly associated with adverse outcomes including auto-immune or auto-inflammatory conditions, cardiovascular disease, cytopenia, cancers in general, and other hematological neoplasms. The associated hematopoietic neoplasms include myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute myeloid leukemia (AML). Population studies reveal splicing factor mutations to be among the most common mutations in CHIP and are highly correlated with age and show a high hazard ratio for risk of developing secondary neoplasms including AML. RNA splicing factors U2AF1, SRSF2, and SF3B1 make up core components of the spliceosome and are critical to prevent aberrant splicing. Mutations in these factors have widespread effects implicating a multitude of cellular processes. There are several established mutant splicing factor mouse models that can be utilized; however, many employ non-specific or leaky Cre drivers. Methods: To avoid non-specific and leaky Cre activation during aging, we used a Cre/lox-mediated U2af1(Q157R) conditional knock-in mouse model that does not contain an internal Cre driver. This mouse has a floxed wild-type U2af1 cDNA insert that expresses wild-type U2AF1 under normal circumstances. However, upon the exogenous addition of recombinant Cre the U2af1 cDNA is excised and the U2af1(Q157R) splicing factor mutation in exon 6 is expressed instead. This mouse model also possesses lox-stop-lox-YFP, leading to expression of YFP along with mutant U2AF1(Q157R) when Cre is introduced. We then isolated bone marrow from 6 week (young) and 6 months (aged) floxed Q157R/YFP mice and treated lineage-depleted bone marrow with recombinant Cre protein for two hours, followed by transplantation into mildly conditioned 6 week (young) and 18 month (old) wild-type mice. The percentage of YFP in the peripheral blood is quantified every 3 weeks. Full analyses, including RNA-seq, of the bone marrow progenitor populations after 3 months is in progress. Results and Discussion: Using the previously mentioned bone marrow transplant model our work shows that when a U2AF1 mutation is introduced into young and old recipient mice, there is significant preferential outgrowth when the donor mice are aged (Figure 1). This is indicated by an increase in the ratio of YFP, which represents the U2AF1(Q157R) mutant, to CD45.1 events, which represent an internal engraftment control. This would suggest that the expansion of U2AF1(Q157R) splicing mutant cells seen in clinical data may be the result of intrinsic factors from the aging cell population. Comprehensive analysis of the effects of alternative splicing in this model is ongoing, however, these data provide support for further investigation of the intrinsic effects of splicing factor mutations in an aged context.

Molecular Measurable Residual Disease in Patients with Newly Diagnosed m IDH1 Acute Myeloid Leukemia Treated with Ivosidenib + Azacitidine

Background: Ivosidenib (IVO) + azacitidine (AZA) improved complete remission (CR) rates and overall survival (OS) relative to placebo (PBO) + AZA in patients (pts) with newly diagnosed mutant isocitrate dehydrogenase 1 (m IDH1) acute myeloid leukemia (AML) in the pivotal AGILE study (Montesinos et al. NEJM 2022). Prior analyses showed clinical responses to IVO+AZA were associated with deep clearance of m IDH1 (limit of detection [LOD]: 0.02-0.04% variant allele frequency [VAF]), as well as clearance of baseline co-mutations below the threshold of conventional next-generation sequencing (NGS) (LOD: 2.0% VAF) (Döhner et al. Blood 2022; Daigle et al. Blood 2021). Measurable residual disease (MRD), a negative prognostic marker, following chemotherapy is often detected via multiparametric flow cytometry or quantitative polymerase chain reaction although both techniques have their limitations. (Heuser et al. Blood 2021). NGS can detect a wide range of AML mutations and studies show that NGS-assessed MRD is highly predictive of both relapse and survival (Thol et al. Blood 2018; Tsai et al. Blood Adv 2021). However, a limitation of NGS is difficulty in distinguishing between somatic leukemia-associated mutations and clonal hematopoiesis of indeterminate potential (CHIP) (Bacher et al. Blood Cancer J 2018). We aimed to perform an NGS analysis of MRD responses in the AGILE study. Methods: All AGILE pts who had a best overall response (BOR) of CR, CR with incomplete count recovery, or CR with incomplete platelet recovery, and had ≥1 on-treatment bone marrow mononuclear cell (BMMC) sample available were included. Suggested timepoints for MRD testing included day 1 of treatment cycles 3, 5, 7, 9, 11, 14, 20, 26, and 32. Baseline and on-treatment BMMC DNA samples were analyzed using a diagnostic 51-gene myeloid NGS panel (LOD: 3% VAF) and a 26-gene AML MRD panel, respectively. For variants detected at baseline, the LOD was 0.1% VAF; for variants not detected at baseline (either emerging mutations or if baseline samples were unavailable), the LOD was 0.5% VAF. All variants above the applicable LOD with known or potential clinical significance were considered evidence of positive MRD, aside from DNMT3A, TET2 and ASXL1 (“DTA”) mutations to reduce the risk of false-positive MRD due to CHIP. Results: The analysis set comprised 173 samples from 33 IVO+AZA-treated pts and 10 PBO+AZA-treated pts (Fig. 1). Clinical response data were based on a March 18, 2021 primary analysis data cut; OS data were based on an updated June 30, 2022 data cut. The median number of baseline mutations was 4 (range: 1-10) per patient, median number of MRD assessments was 3 (range: 1-9) and median follow-up period was 189 days (range: 49-875). Ten (30.3%) IVO+AZA-treated pts became MRD-negative (MRD neg), compared with 2 (20.0%) PBO+AZA-treated pts. All pts attaining an MRD neg response had a BOR of CR (10 [34.5%] of 29 IVO+AZA-treated pts in CR; 2 [22.2%] of 9 PBO+AZA-treated pts in CR). Seven (70.0%) of the 10 MRD neg IVO+AZA-treated responding pts converted to an MRD neg response by day 1 of cycle 7 (C7D1). Fewer pts with an MRD neg response had an Adverse risk at baseline according to European LeukemiaNet 2022 genetic risk classification versus those pts with an MRD-positive (MRD pos) response (26.3% vs 73.7%) (Table 1). In the IVO+AZA arm, rates of event-free survival at 12 months and OS at 24 months were numerically higher in MRD neg vs MRD pos responders (80.0% vs 60.6% and 88.9% vs 72.3%, respectively) but did not reach statistical significance. All 5 (15.2%) IVO+AZA-treated pts with confirmed relapses had detectable MRD at the last completed assessment prior to relapse. One pt converted from an MRD neg to an MRD pos response (“MRD relapse”) 169 days prior to overt clinical relapse. Among the 23 IVO+AZA-treated pts without an MRD neg response, 16 were alive as of the updated data cut; OS duration ranged from 20.3-48.9 months. Conclusions: IVO+AZA induced molecular MRD negativity in approximately one third of responding pts with newly diagnosed m IDH1 AML; MRD neg responses were most often observed on or before C7D1. Interestingly, several pts had durable clinical responses and OS despite ongoing MRD positivity, potentially due to persistent CHIP rather than true MRD, illustrating the complexity of interpreting NGS MRD data. Additional analyses, including associations between specific molecular alterations, MRD response, and OS are ongoing and will be presented.

DNMT3A-Mutated Stem and Progenitor Cells Contribute to Altered T Cell Activation in Clonal Hematopoiesis

Clonal hematopoiesis is characterized by an expansion of blood cells derived from a single mutated hematopoietic stem cell (HSC). When this expansion is driven by a leukemia-associated mutation that exceeds a variant allele frequency (VAF) of 2%, it is termed clonal hematopoiesis of indeterminate potential (CHIP). Approximately half of CHIP cases are driven by mutations in the DNA methyltransferase gene DNMT3A. In patients with CHIP, greater clonal expansion is associated with an increased risk of cardiovascular diseases and hematologic malignancies. While DNMT3A impairment has been shown to alter inflammatory signaling in HSCs and myeloid cells, less is known about the impact of DNMT3A-mutated CHIP on T cell biology. Here, we leverage a cohort of human CHIP samples to assess how the expansion of DNMT3A-mutated cells affects gene expression across blood cell types and generate a new mouse model to assess antigen-specific immune cell interactions. We find evidence that the accumulation of DNMT3A-mutated stem and progenitor cells can cause T cell dysfunction. We first identified CHIP in a cohort of 191 sternum bone marrow samples collected during heart surgery. We detected CHIP in 45 samples (23.6%), including 22 samples with mutated DNMT3A (48.9% of CHIP samples). To mitigate confounding factors that might be introduced by comparing CHIP to healthy individuals, we used ten DNMT3A-mutated CHIP samples for this study, splitting them into a low-VAF (<10%, three samples) and high-VAF (>10%, seven samples) cohorts. We performed single-cell RNA sequencing on these 10 samples, generating high-quality data for 95,245 cells. We clustered and annotated 33 cell types according to the expression of canonical marker genes, representing the full spectrum of hematopoietic progenitors to myeloid, erythroid and lymphoid lineages. To evaluate how greater DNMT3A clonal expansion affects immune cell states, we performed differential gene expression analysis in the high-VAF compared to the low-VAF cohort with DESeq2 using a pseudobulk approach followed by gene set enrichment analysis using Hallmark pathways. We found that TGF-beta signaling, known for its immunosuppressive role, is upregulated in hematopoietic stem and progenitor cells (HSPCs), myeloid and T cell populations in the high-VAF compared to the low-VAF cohort. Both interferon alpha and gamma signaling pathways are consistently downregulated in HSPCs and multiple myeloid cell types as well as T cells. TNF-alpha signaling is upregulated in HSPCs and myeloid cells, consistent with recent observations linking TNF-alpha to the survival and proliferation of HSPCs and myeloid cells. Overall, greater expansion of DNMT3A-mutated cells is associated with variable gene expression changes in progenitor and myeloid cell types, whereas gene expression changes in T cells are consistent with diminished activation (Figure 1). To evaluate mechanisms contributing to altered T cell activation in DNMT3A-mutated CHIP, we combined our Dnmt3a-fl-R878H/+ inducible mouse model with the ovalbumin (OVA) trackable antigen system. Endogenously expressed OVA is processed into a peptide that is loaded onto MHCI and recognized by transgenic CD8+ T cells from OT-I mice. We crossed the Dnmt3a-fl-R878H/+ mouse with a mouse expressing OVA under the actin promoter, resulting in Dnmt3a-mutated mice with constitutive OVA expression. To interrogate antigen-specific T cell responses, we co-cultured Dnmt3a-mutated OVA+ lineage negative (Lin-) stem and progenitor cells with CD8+ T cells from OT-I mice. By measuring T cell proliferation using CFSE dilution, we observed an increase in T cell proliferation when CD8+ T cells were co-cultured with Dnmt3a-mutated OVA+ Lin- cells compared to wildtype OVA+ Lin- cells (Figure 2). These results suggest that Lin- Dnmt3a-mutated cells have an increased capacity for antigen-specific T cell activation. In conclusion, our studies demonstrate that greater expansion of DNMT3A-mutated CHIP is associated with reduced expression of activation signatures in T cells. In apparent contrast, Dnmt3a-mutated mouse cells have an increased capacity for antigen-specific T cell stimulation. Since chronic antigenic stimulation causes T cell exhaustion, our findings are consistent with a model in which the expansion of DNMT3A-mutated progenitor and myeloid cells causes chronic stimulation and dysfunction of T cells.

Abstract A28: Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones

Abstract Hematopoietic stem cells (HSC) frequently acquire gene mutations with age. An expansion of blood cells derived from a single HSC is termed clonal hematopoiesis (CH), a condition that often precedes acute myeloid leukemia (AML). CH is common in elderly individuals: by age 60, approximately 10% of individuals have a clonally expanded blood cell population with a leukemia-associated mutation. The most common mutations in CH (50% of all cases) inactivate the DNA methyltransferase DNMT3A, which regulates gene expression via methylation of CpG rich regions. These mutations provide a selective advantage over healthy HSCs during aging and inflammation. Our goal is to discover mechanisms that allow for clonal expansion of DNMT3A-driven CH. A better understanding of these mechanisms will inspire new therapeutic strategies to limit clonal expansion and increase survival. To evaluate the transcriptional consequences of DNMT3A mutations in myeloid cells, we analyzed our previously published single-cell RNA-sequencing data of DNMT3A-mutated AML patient samples. We found a significant enrichment of antigen presentation signatures in DNMT3A mutated cells along the myeloid differentiation trajectory. CD8+ T cells from these same patients had significantly increased signatures of T cell activation and T cell exhaustion. T cell exhaustion occurs after an activated T cell has been continuously exposed to antigens. Exhausted T cells lack most effector functions and the ability to properly surveil (i.e. recognize mutant peptides presented on the surface of mutated cells and kill the target upon recognition) and restrain mutant cells from expanding. We hypothesize that DNMT3A-driven CH leads to escape of immune surveillance via chronic antigen stimulation and eventual promotion of T cell exhaustion. This would allow for further expansion of the mutated cells and the transformation of CH into AML. To functionally test this hypothesis, we evaluated whether a mouse model of Dnmt3a-CH recapitulated our human data. Indeed, scRNA-seq data of mouse Dnmt3a-CH cells showed significantly increased antigen presentation signatures across the myeloid differentiation trajectory. Additionally, CD8+ T cells had increased signatures of activation. We next functionally tested whether Dnmt3a-mutated CH cells had enhanced antigen presentation by pulsing bone marrow cells derived from Dnmt3a-CH mice with DQ-Ovalbumin, a self-quenched exogenous protein that becomes fluorescent when endocytosed and digested in the phagolysosome. Dnmt3a-mutated HSCs and LSKs had significantly increased DQ-OVA uptake and processing. In conclusion, we found that DNMT3A-CH cells have increased antigen presentation and processing which corresponds with enhanced immunogenicity. These findings support a model in which T cells constrain the expansion of DNMT3A-mutated HSCs, until T cell exhaustion occurs, leading to further expansion of the mutated clone and an increased chance of transformation into AML. Citation Format: Kyle A Romine, Daniel Ssozi, Tina Mujica, Yoke Lee, Jennifer Trowbridge, Peter van Galen. Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A28.

Clonal Hematopoiesis of Indeterminate Potential Predicts Adverse Outcomes in Patients With Atherosclerotic Cardiovascular Disease

BackgroundClonal hematopoiesis of indeterminate potential (CHIP)—the age-related clonal expansion of blood stem cells with leukemia-associated mutations—is a novel cardiovascular risk factor. Whether CHIP remains prognostic in individuals with established atherosclerotic cardiovascular disease (ASCVD) is less clear. ObjectivesThis study tested whether CHIP predicts adverse outcomes in individuals with established ASCVD. MethodsIndividuals aged 40 to 70 years from the UK Biobank with established ASCVD and available whole-exome sequences were analyzed. The primary outcome was a composite of ASCVD events and all-cause mortality. Associations of any CHIP (variant allele fraction ≥2%), large CHIP clones (variant allele fraction ≥10%), and the most commonly mutated driver genes (DNMT3A, TET2, ASXL1, JAK2, PPM1D/TP53 [DNA damage repair genes], and SF3B1/SRSF2/U2AF1 [spliceosome genes]) with incident outcomes were compared using unadjusted and multivariable-adjusted Cox regression. ResultsOf 13,129 individuals (median age: 63 years) included, 665 (5.1%) had CHIP. Over a median follow-up of 10.8 years, any CHIP and large CHIP at baseline were associated with adjusted HRs of 1.23 (95% CI: 1.10-1.38; P < 0.001) and 1.34 (95% CI: 1.17-1.53; P < 0.001), respectively, for the primary outcome. TET2 and spliceosome CHIP, especially large clones, were most strongly associated with adverse outcomes (large TET2 CHIP: HR: 1.89; 95% CI: 1.40-2.55; P <0.001; large spliceosome CHIP: HR: 3.02; 95% CI: 1.95-4.70; P < 0.001). ConclusionsCHIP is independently associated with adverse outcomes in individuals with established ASCVD, with especially high risks observed in TET2 and SF3B1/SRSF2/U2AF1 CHIP.

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Accumulation of Specific Somatic Leukemia-Associated Mutations in Congenital Neutropenia Precedes Malignant Transformation - New Preconditions for Treatment Decisions

Severe congenital neutropenia (CN) and Shwachman-Diamond Syndrome (SBDS) are pre-leukemia bone marrow failure syndromes with a high cumulative incidence of developing MDS or AML. Clonal hematopoiesis (CH) due to nonsense mutations in the intracellular part of CSF3R or missense TP53 mutations has been reported previously in CN or SBDS patients, respectively. So far, studies of CH in CN were conducted by sequencing CSF3R only (Germeshausen et al., 2007, Klimiankou et al., 2019) or in ELANE-CN patients (Jun Xia et al., 2018). Precise characterization of the timeline and occurrence of somatic mutations found at the neutropenia, pre-leukemia or leukemia stage in CN is essential to make a timely and correct decision on the requirement of bone marrow transplantation prior to transformation to overt leukemia. We therefore aimed to assess the acquisition of somatic leukemia-associated mutations in a large number of genes in a worldwide largest and most representative cohort of CN patients. We performed error-corrected sequencing with fixed single-stranded unique molecular indexes (UMIs) on panel of 31 genes and "hotspot" regions in 340 genes reported to be mutated in de novo MDS/AML. We analyzed 146 CN patients representing most of genetic subtypes of CN, 21 SBDS patients, 29 CN patients who progressed to MDS or AML, and 18 healthy individuals. The median age of CN and SBDS patients was 13,73 and 10,5 years, respectively. For 53 CN patients, we collected sequential samples with a median observation time of 1,1 years. We found that the prevalence of CH with at least 1 somatic DNA lesions in CN group was 49,5 % which is much higher than in healthy individuals of the same age. The prevalence of CH with CSF3R mutations varied between 20 and 38 % in patients with genetic defects in ELANE (n = 75), HAX1 (n = 26), SRP54 (n = 8), JAGN1 (n = 6), and G6PC3 (n = 5). The percentage of CH positive cases without CSF3R mutations ranged between 12 % and 50 %. CN patients acquired somatic coding mutations most frequently in CSF3R (49 %) following by TP53 (4,1 %), ASXL1 (4,1 %), DNMT3A (2,1 %), TET2 (2,1 %), JAK2 (1,37 %), SETBP1 (1,37 %), ARID1B (1,37 %), GNAS (1,37 %), and ZRSR2 (1,37 %). Interestingly, in the SRP54-CN group, we found 1 patient with CSF3R mutation, 1 patient with TP53 mutations, and 1 patient with both mutations. Out of 3 CLPB-CN patients, 1 patient has CSF3R and 1 patient acquired TP53 mutations. High prevalence of CSF3R (67 %), RUNX1 (44 %), SETBP1 (22 %), ASXL1 (17 %), ZRSR2 (11 %), NPM1 (11 %) and PTPN11 (11 %) mutations was detected in CN patients with overt MDS/AML, arguing for a specific pathway which initiates and drives leukemic transformation in CN. Comparison of CN patients who later developed MDS/AML with MDS/AML-free CN cohort revealed enrichment of CSF3R (Fisher's exact test; OR 3,42; 95% CI, 1,27, - 9,36, p = 0,01), and ASXL1 (Fisher's exact test; OR 7,4; 95% CI, 0,93 - 59,16, p = 0,03) mutations. In contrast, RUNX1 and SETBP1 mutations were associated with overt MDS/AML in CN. These data argue that the acquisition of CSF3R and ASXL1 mutations are early events in CN leukemogenesis, whereas RUNX1 and SETBP1 mutations may have stronger leukemogenic potential and therefore are rarely found at the neutropenia stage. Moreover, mutation burden at the neutropenia stage in CN patients who later progressed to MDS/AML was significantly higher (p = 0.016, by Fisher's exact test), as compared to CN group who haven't developed leukemia so far. The number of mutations in samples prior AML/MDS was dependent on duration to overt AML/MDS with gradual accumulation of genetic lesions over time. CH was detected in 66,7 % of SBDS patients with a high prevalence of TP53 mutations (9 out of 14 patients with CH) followed by missense mutations in IDH1 (n = 1), FOXP1 (n = 1), and PTPN11 (n = 1). Our results are in line with previous publications about dominant role of TP53 mutation in SBDS patients. Prevalence of CH didn't differ between CN patients depending on G-CSF dose (median = 4,06 μg/kg/d) in two biggest CN cohorts harboring ELANE or HAX1 mutations. Taken together, CN per se contributes to high level of CH already in the young age, as compared to healthy population. Acquisition of multiple genetic lesions, especially RUNX1, SETBP1, ASXL1, TP53, PTPN11 in association with CSF3R mutation might be a strong indicator of the advanced pre-leukemia stage in CN patients at neutropenia stage and requires closer patient follow up.

Clonal Hematopoiesis Evolves into Dendritic Cell Neoplasm through the Acquisition of UV Mutations in the Skin

Background Expansion of blood cells derived from a single mutated blood stem cell - clonal hematopoiesis - often precedes leukemic transformation. However, the circumstances that promote or prevent transformation are poorly understood. Here, we investigate the evolution of clonal hematopoiesis into blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive blood cancer that often presents with malignant cells isolated to the skin (referred to as skin-only disease). We gain a better understanding of the influence of site-specific selective pressures and the role of TET2 mutations, which may inform interventions to prevent disease progression. Methods We analyzed bone marrow and skin from 13 BPDCN patients using targeted, exome, and whole-genome sequencing, with germline controls. We performed multimodal single-cell sequencing (10x 3' v3.1 single-cell RNA-seq with genotyping of expressed variants) on bone marrow from 6 healthy donors (20,411 cells), 5 skin-only BPDCN patients with malignant cells in the skin but not conventionally detectable in marrow (36,018 cells), and 6 BPDCN patients with overt bone marrow involvement (30,582 cells). For functional studies, we targeted Tet2 using CRISPR/Cas9 in mouse blood progenitor cells immortalized by an estrogen receptor hormone binding domain fused to HoxB8, which undergo synchronized differentiation to myeloid and dendritic cells upon estrogen withdrawal. We used CellRanger, Seurat, RandomForest machine learning, and the R tidyverse package collection for bioinformatics. Results Genetics allowed us to reconstruct the evolution of BPDCN across bone marrow and skin (representative patient, panel a). Invariably, we detected founder mutations in marrow, including TET2 inactivation in 9/13 patients (two mutations, likely bi-allelic, in 5/9). Single-cell sequencing of marrow from 5 skin-only patients showed that pre-malignant mutant stem cells contributed to multiple lineages, and that TET2 mutated clones were myeloerythroid-biased. We generated a gene expression signature to identify rare malignant BPDCN cells in marrow of skin-only patients. In 3/5 patients, we identified rare cells (0.03-0.19%) with the transcriptional profile of fully transformed cells, and concurrent single-cell genetic analysis suggested that these cells originated from the skin tumor (panel b). To assess whether disseminated skin tumor cells establish bone marrow disease, we quantified mutational signatures: skin tumors and malignant BPDCN marrow cells in 5/5 patients harbored UV-induced mutations, likely acquired in a single skin-resident plasmacytoid dendritic cell (pDC) during malignant transformation. Finally, we edited Tet2 in mouse myelo-dendritic progenitors and tested the response to UV irradiation in vitro. In addition to observing that Tet2 inactivation increases dendritic cell commitment, the more striking result was a survival advantage of Tet2-mutated cells in the presence of UV (n = 6, fold change 1.803, P = 0.0012). These results highlight an unexpected function of TET2 that may predispose mutated pDCs to survive UV-induced DNA damage in the skin, leading to disease progression. Conclusion The complex evolution of BPDCN traverses multiple anatomic sites prior to full transformation. This allows for accurate sequencing of oncogenic events. Disease evolution frequently starts with acquisition of a leukemia-associated mutation in a multipotent blood stem cell, leading to clonal expansion. In the most frequently observed scenario, bi-allelic TET2 inactivation promotes myeloerythroid bias, increases dendritic cell commitment, and confers resistance to UV-mediated cell death. Subsequent evolution of skin-resident mutant pDCs leads to a high burden of UV-induced mutations and ultimate transformation. Dissemination of malignant cells, which can be detected by integrating single-cell gene expression and mutation profiles, initiates "retrograde" bone marrow disease. Our discovery that multiple anatomic sites - and local selective pressures - play a role in disease progression provides a template to study multi-site evolution of clonal hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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Clonal hematopoiesis are not associated with an increased systemic inflammation, ATHerosclerosis nor incidence of atherothrombosis: Results from the 3-city study (CHIP-3C)

Background and Aims : Clonal hematopoiesis of indeterminate Potential (CHIP) is defined by the detection of leukemia-associated mutations in the absence of hematological malignancy. This condition is associated with an increased mortality mainly driven by athero-thrombotic complications. Previous studies in mouse models demonstrated that CHIP increase atherosclerosis development. However the association between CHIP, atherosclerosis and athero-thrombosis remains poorly evaluated in patients.Methods: The 3-city study is a population-based longitudinal study that enrolled individuals aged ≥65 years. In this cohort, we selected 322 persons who had no cardiovascular event before inclusion. Eighty five of them suffered from a myocardial infarction or stroke during the 12-year follow-up. We searched for CHIP by a targeted NGS strategy on DNA collected at inclusion. Anthropomorphic, cardiovascular (risk factors, diet, atherosclerosis) and biological (CRP level) data at inclusion as well as incidence of athero-thrombotic events were compared between patients with or without CHIP.Results: A CHIP was detected in 41% of patients. As described, most patients presented mutations in DNMT3A (46%) and TET2 (30%). Patients with CHIP were slightly older than patients without CHIP (74.2 years VS 73 years, p=0.03). Neither the cardiovascular risk profile, nor the CRP levels (1.66 VS 1.75), nor the number of atheromatous plaques nor the intima-media thickness (0.67 VS 0.68) were different between patients with and without CHIP. The incidence of athero-thrombotic complications (myocardial infarction or stroke) was similar between patients with a CHIP and patients without.Conclusions: In conclusion, CHIP, in particular involving DNMT3A mutations, are not strongly associated with systemic inflammation, atherosclerosis or athero-thrombotic events. Background and Aims : Clonal hematopoiesis of indeterminate Potential (CHIP) is defined by the detection of leukemia-associated mutations in the absence of hematological malignancy. This condition is associated with an increased mortality mainly driven by athero-thrombotic complications. Previous studies in mouse models demonstrated that CHIP increase atherosclerosis development. However the association between CHIP, atherosclerosis and athero-thrombosis remains poorly evaluated in patients. Methods: The 3-city study is a population-based longitudinal study that enrolled individuals aged ≥65 years. In this cohort, we selected 322 persons who had no cardiovascular event before inclusion. Eighty five of them suffered from a myocardial infarction or stroke during the 12-year follow-up. We searched for CHIP by a targeted NGS strategy on DNA collected at inclusion. Anthropomorphic, cardiovascular (risk factors, diet, atherosclerosis) and biological (CRP level) data at inclusion as well as incidence of athero-thrombotic events were compared between patients with or without CHIP. Results: A CHIP was detected in 41% of patients. As described, most patients presented mutations in DNMT3A (46%) and TET2 (30%). Patients with CHIP were slightly older than patients without CHIP (74.2 years VS 73 years, p=0.03). Neither the cardiovascular risk profile, nor the CRP levels (1.66 VS 1.75), nor the number of atheromatous plaques nor the intima-media thickness (0.67 VS 0.68) were different between patients with and without CHIP. The incidence of athero-thrombotic complications (myocardial infarction or stroke) was similar between patients with a CHIP and patients without. Conclusions: In conclusion, CHIP, in particular involving DNMT3A mutations, are not strongly associated with systemic inflammation, atherosclerosis or athero-thrombotic events.

Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation.

Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221.

Dysregulated minor intron splicing in cancer.

Pre‐mRNA splicing is now widely recognized as a cotranscriptional and post‐transcriptional mechanism essential for regulating gene expression and modifying gene product function. Mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are now considered among the most recurrent genetic abnormalities in patients with cancer, particularly hematologic malignancies. These include mutations in the major (U2‐type) and minor (U12‐type) spliceosomes, which remove >99% and ~0.35% of introns, respectively. Growing evidence indicates that aberrant splicing of evolutionarily conserved U12‐type minor introns plays a crucial role in cancer as the minor spliceosome component, ZRSR2, is subject to recurrent, leukemia‐associated mutations, and intronic mutations have been shown to disrupt the splicing of minor introns. Here, we review the importance of minor intron regulation, the molecular effects of the minor (U12‐type) spliceosomal mutations and cis‐regulatory regions, and the development of minor intron studies for better understanding of cancer biology.

Phase II trial assessing safety and preliminary efficacy of high-dose intravenous ascorbic acid in patients with TET2-mutant clonal cytopenias of undetermined significance.

TPS7076 Background: Clonal cytopenias of undetermined significance (CCUS) is operationally defined as unexplained persistent cytopenias arising in the context of leukemia-associated somatic driver mutations in hematopoietic stem and progenitor cells, without morphologic evidence for an underlying hematological neoplasm. Patients with TET2 mutant ( TET2MT) CCUS have a high probability of progression to myeloid neoplasms, with approximated 10-year cumulative rates of progression being 90%. To date, no FDA-approved treatment for CCUS has been established, and we have shown that these patients can have transfusion burdens akin to patients with myelodysplastic syndrome (MDS) (Li M et al. Blood Adv 2020). Thus there is an urgent and unmet need for therapies for these patients. Intravenous (IV) ascorbic acid (AA) dosed at pharmacologic concentrations could have anti-cancer activity via two mechanisms: (1) hydrogen peroxide-induced oxidative stress and (2) DNA demethylation mediated by TET activation (cofactor for TET1/2/3). TET2, the primary enzyme for catalyzing oxidative reactions, converts 5-methylcytosine to 5-hydroxymethylcytosine, a crucial step leading to iterative DNA demethylation. Our study hypothesis is that IV AA in TET2MT CCUS will restore hematopoiesis and limit clonal progression by stimulating TET2 (the unmutated allele) and TET3 activities. Methods: LS1781 (NCT03418038) is an investigator-initiated, prospective, single-arm, single-institution, phase II trial assessing the safety and preliminary efficacy of high dose of IV AA for patients with TET2MT CCUS (harboring ≥1 TET2MT or TET2MT combined with concurrent spliceosome MTs). IV AA (1g/kg, maximum 100g) is given 3 times weekly for 12 weeks through a central line with assessments being carried out before each cycle (4 weeks), at 12 weeks, and one year. The primary endpoint is the hematologic response rates determined by MDS IWG 2018 criteria. Secondary endpoints include safety and side effects. Exploratory endpoints include the impact of AA on TET2MT variant allele fraction, plasma cytokine levels, DNA methylation, and hydroxymethylation (as measured by Infinium MethylationEPIC array), and TET activity. Patients 18 years or older with TET2MT CCUS, and with any of the following laboratory criteria: (1) hemoglobin ≤10g/dL, (2) absolute neutrophil count (ANC) ≤1000/mm3, (3) platelet count ≤100,000/mm3 are eligible. Fifteen patients will be enrolled at Mayo Clinic Rochester and the Mayo Clinic Health System. Dose modifications have been established and will be considered based on adverse events. Treatment will be discontinued for disease progression or in the event of ≥Grade 3 adverse events that don’t resolve promptly. Enrollment to the trial began in June 2021, with 7 patients enrolled at abstract submission. Clinical trial information: NCT03418038.

Immunogenetic Drivers in Post-Transplant Acute Myeloid Leukemia Recurrence: Genomic Dysfunction of Human Leukocyte Antigen Diversity

Structural and functional variability of human leukocyte antigen (HLA) is the foundation for competent anti-tumor and infectious adaptive immune responses. HLA genomic heterogeneity enables the presentation of a broad immune-peptidome, sustaining an efficient diversification of T cell receptor repertoires (TCR). 1,2,3 Any perturbation impacting this diversity may be at the basis of pathological processes, hampering antigen presentation capabilities and T-cell reactivity. In allogeneic hematopoietic cell transplantation (allo-HCT) setting, the graft versus leukemia (GvL) effect should ensure disease control allowing the recognition of recipient neoantigen burden by donor T-cell effectors. However, the molecular dissection of graft versus host responses (GvH) remains elusive. Herein, by means of a broad immunogenetic study of a cohort of patients with myeloid malignancies who received a donor matched allo-HCT, we investigated how dysfunction of HLA variability could have an impact on alloreactive responses, ultimately hindering disease control. To that end, we combined NGS-based HLA genotyping and TCR-beta sequencing to molecularly characterize the HLA region in terms of locus-specific divergence and somatic mutational profile, and dissect features and clonotypic spectra of TCR repertoires.We first hypothesized that more diverse HLA genotypes could better present leukemic neoantigen burden than less diverse complexes, enhancing the GvL effect. Hence, we performed a matched-pair analysis between allo-HCT recipients relapsing after 3mo (median 6.2 mo. [IQR=4.6-12]), N=75) compared to patients without recurrence (N=193, matched for ethnicity, age, disease, graft source and conditioning regimens) and characterized the patterns of HLA evolutionary divergence (HED), 1 a metric recently conceived to quantitate the pair-wise distance (based on physiochemical composition) between the amino acids located within the peptide-binding groove of two homologous HLA alleles. Overall, the relapsed group was characterized by a lower global (class I/II) mean HED (p=.0029) compared to non-relapsed patients, with major differences seen for C (p=.0041), DQB1 (p=.0291), and DPB1 (p=.0396) loci. When studying the landscape of post-transplant TCR reconstitution (+3 months) in a subset of 25 patients, we observed an inverse correlation between TCR clonal expansion and global HED (AdjR 2=0.04, p=<2e-16), contributing to decrease the diversity of TCR repertoires in patients with lower HED. Although not different in number, the expansion of clonotypes with known anti-cancer specificity was higher in non-relapsing group (p=6.3e-08), possibly underlying a better tumor-surveillance. Next, we sought to investigate the patterns of somatic HLA dysfunction in relapsing patients (intended as allelic loss or mutations). Indeed, through a recently implemented HLA mutational calling algorithm, we observed somatic events encompassing both class I and II alleles in 23% (N=8/34 profiled patients). Interestingly, when analyzing patients with relapse who received a donor lymphocyte infusion-based treatment (DLI), none of the cases harboring mutational events (N=4/4) responded to this salvage strategy. It is noteworthy that in this last group, one patient relapsed with an extramedullary localization along with the acquisition of HLA mutations. HLA mutated group had a higher (although not significant) leukemia mutational burden compared to non-mutated group (mean number of leukemia-associated mutations: 3.6 vs 1.9/patient), underscoring the need for further driver mutational events compensating the possible lower immunogenic potential of HLA mutant clones. Despite a mild increase in mutational burden, driver hits (such as IDH1/2, FLT3, TP53, NPM1) were never present in patients carrying HLA aberrations, who instead harbored in a few cases mainly lesions in epigenetic regulators and chromatin modifiers (TET2, EP300, DNMT3A, EZH2).Altogether these findings pinpoint the role of the dysfunction of the structural variability of HLA complexes within both germline (HED) and somatic (HLA loss/mutations) scenarios as mechanisms hampering a successful neoantigen presentation and TCR recovery processes, possibly conveying a higher risk of disease relapse or treatment-resistance. DisclosuresBalasubramanian: Servier Pharmaceuticals: Research Funding. Carraway: Takeda: Other: Independent review committee; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee. Hamilton: Syndax: Membership on an entity's Board of Directors or advisory committees; Equilium: Membership on an entity's Board of Directors or advisory committees. Majhail: Anthem, Inc: Consultancy; Incyte Corporation: Consultancy. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.

Prevalence and Dynamics of Clonal Hematopoiesis Caused By Leukemia Associated Mutations in Elderly Individuals without Hematologic Disorders

Clonal hematopoiesis is frequently observed in elderly people and may represent a premalignant condition. Evidence suggests that genetic alterations found in patients with myeloid malignancies may also be causative for induction of clonal hematopoiesis in healthy elderly. Discrimination between age-associated and leukemia-associated mutations is of crucial clinical importance. We sought to investigate the prevalence and dynamics of genetic alterations among elderly individuals without hematologic/oncologic disorders by genotyping a cohort of 50 elderly individuals (29 women; median age 84 years; range 80-90 years) for a panel of 30 commonly mutated leukemia-associated genes by targeted deep next-generation sequencing (NGS). Buccal cells were analyzed to confirm the somatic origin of mutations. A total of 16 somatic mutations in leukemia-associated genes were identified in 13 of 50 (26%) elderly individuals. One subject presented with two, another subject with three different mutations. Ten of 16 mutations (63%) affected epigenetic modifier genes (DNMT3A, n=8; TET2, n=1; IDH2, n=1). Four somatic mutations affected genes involved in the RNA splicing machinery (SRSF2, n=2; SF3B1, n=1; U2AF1, n=1). Mutations in TP53 and NRAS were identified in two individuals. All but one mutation were missense mutations with cytosine to thymine transitions being the most common base pair change (n=7). Mutations occurred at low variant allele frequencies (VAF) with a median of 11.7% (range, 1.0 to 30.7%) indicating that mutations were presented in only a subset of nucleated blood cells. Mutation kinetics remained virtually stable over a follow-up observation of three years as measured by targeted NGS (median VAF 13.1%; range, 1.0 to 35.3%). Two subjects with mutations in splicing factor genes died, one with an SF3B1 mutation developed pancreatic cancer, the other harbored a U2AF1 mutation and died from a stroke. All other individuals with mutations are alive without any evidence for cancer or cardiovascular disorders. To study the prevalence of DNMT3A mutations in more detail, a DNMT3A specific high-sensitive (sensitivity 1-3%) NGS assay was established for investigation of an additional cohort of 163 healthy individuals including younger people (84 women; median age 39 years; range 1-79 years). No DNMT3A mutation was detected in 40 children and adolescents (1-19 years). Somatic DNMT3A mutations were found in 2/43 (4.6%) individuals with an age of 20-39 years, 1/40 subjects (2.5%) between 40-59 years and in 3/40 subjects (7.5%) between 60-79 years. Again, mutations occurred at low VAF with a median of 8.2% (range, 1.5 to 37.3%). Two DNMT3A variants were detected both in blood and buccal cells indicating germline variants or rare polymorphisms (a 34-year old female and a 74-year old male, respectively). These findings indicate that the appearance of low-level clones characterized by mutations in epigenetic regulator genes and the RNA splicing machinery is a common age-associated phenomenon which may represent a premalignant condition in the development of cancers and may also predispose to other aging associated disorders. DNMT3A mutations were the most common mutations identified in elderly individuals but were also apparent in younger healthy people. The question why subclones are initially selected but apparently enter a stage of clonal size stability without further selection in healthy individuals in contrast to patients with myeloid disorders is of particular interest and needs further investigation. DisclosuresHochhaus:Incyte: Research Funding; ARIAD: Research Funding; BMS: Research Funding; MSD: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

AML-Associated Somatic Mutations Can be Detected in Stem and Progenitor Populations in Patients with Antecedent Multiple Myeloma Years before Onset of Therapy-Related Transformation

Introduction: Clonal hematopoiesis is an increasingly well-established risk factor for developing therapy-related acute myeloid leukemia and myelodysplastic syndrome (t-AML/t-MDS), particularly in patients receiving chemotherapy. In some cases, leukemia-associated mutations can be detected at low frequencies in prior samples, and the same mutation is expanded significantly in t-AML/t-MDS samples. However, most of these studies have been conducted using whole mononuclear cells populations and it is unclear whether these mutations reside in the stem cell compartment. Here, we present DNA sequencing results from sorted mobilized stem cells from patients with multiple myeloma (MM) treated with autologous hematopoietic stem cell transplant (AHSCT) who subsequently developed t-AML/t-MDS.Methods: Patients with MM treated with AHSCT at the University of Arkansas for Medical Sciences Hospital who subsequently developed t-AML/t-MDS were identified. Clinical data, including the results of commercially available targeted gene sequencing, was recorded and unused stem cell collection specimens were retrieved. Specimens were sorted by flow cytometry into subpopulations, including normal hematopoietic stem cells (nlHSCs, Lin-/CD34+/CD38-), aberrant hematopoietic stem cells (Ab-HSC, defined as phenotypic HSCs that are additionally either CD99+ or CD123+ or IL1RAP+), megakaryocyte-erythroid progenitors (MEPs), granulocyte-macrophage progenitors (GMPs), granulocytes (Grans), T-cells, and B-cells. DNA was extracted and targeted deep sequencing was performed by NGS (Genoptix). The low frequency variants (<1%) are presently being validated by independent high sensitivity assays. If available, DNA from CD138+ myeloma cells collected at the time of initial diagnosis was sequenced. The libraries were then queried for mutations identified by commercially available targeted sequencing performed on t-AML/t-MDS samples, after the exclusion of germline mutations. The protocol was approved by the IRBs at the participating institutions.Results: Six patients were identified. All had unused stem cell collection samples and commercially available DNA sequencing performed at the time of t-AML/t-MDS diagnosis. Two had DNA from CD138+ MM cells available for sequencing. Clinical characteristics of these patients are shown in Table 1. The results of mutational frequencies among the subpopulations are show in in Table 2. In all patients, mutations subsequently found in t-AML/t-MDS samples were found among at least one stem or progenitor subpopulation. Variant allele frequency ranged from 0.02%- 55.1%. The low frequency variants are presently being validated by independent assays. Mutations were particularly enriched in nlHSCs or Ab-HSCs in 4 out of 6 patients and were enriched in phenotypic GMP or MEP progenitors in the other two cases. In particular, Patient 3 was found to have high frequencies of RUNX1, IKZF1, and BCOR mutations in nlHSC, Ab-HSC, and GMP populations, with much lower frequencies in the mature populations, and with none in the MEP population. In both patients with evaluable CD138+ MM samples, the same TP53 mutations present in the MM were also present in the HSCs as well as mature B lymphocytes, demonstrating persistence during plasma cell differentiation. .Conclusions: Our results show for the first time the presence and distribution of t-AML/t-MDS-associated mutations in patients at the time of MM treatment, many years before eventual transformation. While mutations to tumor suppressor genes such as TP53 have previously been detected at low frequency in unsorted antecedent samples, we show that these mutations preferentially reside in nlHSCs, Ab-HSCs, and immature progenitor populations which enrich over time, leading to high frequencies at the time of t-AML/t-MDS diagnosis. Finally, in 2 of 2 evaluable patients, we demonstrate the presence of the same tumor initiating mutation in both CD138+ MM and t-MDS samples, suggesting that common ancestral tumor initiating cells harboring these mutations may have given rise to both the MM and t-MDS. [Display omitted] DisclosuresBarlogie:Millenium Pharmaceuticals: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers: Consultancy, Honoraria. Steidl:Bayer Healthcare: Consultancy; Novartis: Research Funding; Celgene: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Consultancy, Research Funding.

Mutational Burden before and after Allogeneic Hematopoietic Cell Transplantation (HCT) in Patients with Acute Myeloid Leukemia (AML)

BackgroundAllogeneic HCT remains the only curative option for patients (pts) with high-risk or relapsed AML. Genome sequencing technologies such as next generation targeted deep sequencing (NGS) or whole exome sequencing have identified several recurrent somatic mutations in AML that may be associated with HCT outcomes. Genomic abnormalities may persist in pts who achieve complete remission (CR) after induction chemotherapy; however, the impact of HCT on the mutational burden before and after transplant in such pts has not been described. We explore the dynamic changes of the clonal architecture in serial samples obtained from AML pts before and after HCT.MethodsNGS data on bone marrow and peripheral blood samples obtained at diagnosis, prior to transplant, and at day 30 and day 100 after transplant from pts with AML (according to 2016 WHO criteria) who were treated at our institution between 11/2015 - 1/2017 were analyzed. A total of 170 somatic and germline gene mutations that have been described in hematologic malignancies were sequenced at each time point. The mean depth of the targeted sequencing was 300 fold for the entire cohort. Clonal architecture was defined by using variant allelic frequency (VAF) adjusted for zygosity at each time point.ResultsA total of 28 pts with serial samples were included. The median age at transplant was 57 years (range, 25-74). Cytogenetic analysis at diagnosis included normal karyotype in 15 pts (54%), 9 (32%) with unfavorable risk per Alliance criteria (including 5 pts with complex karyotype), 1 (3%) with t(8,21), and 3 pts (11%) with other (intermediate risk) abnormalities. All pts received induction chemotherapy at diagnosis with cytarabine and anthracycline (7+3) +/- combination except for one pt who received azacitidine. All pts achieved complete remission (CR/CRi) per 2003 IWG criteria prior to transplant. Conditioning regimens were myeloablative (N= 17, 61%) and reduced-intensity (N=11, 39%). Graft sources were bone marrow (N = 17, 61%), and peripheral blood (N =11, 39%). Donor source was matched sibling (N= 9, 32 %), matched unrelated (N= 14, 50 %) or haplo-identical (N= 5, 18%). Among pts with samples available at diagnosis, the most frequently mutated genes were: DNMT3A (46%), NPM1 (21%), FLT3 (ITD/TKD, 20%), TET2 (13%), and WT1 (13%). All pts with abnormal cytogenetics (13 pts) achieved complete cytogenetic remission prior to transplant. Common leukemia-related mutations such as DNMT3A, ASXL1, and TET2 persisted among others prior to transplant even in pts with CR and complete cytogenetic response. Among pts who achieved full engraftment at day 30 and/ or day 100 post-transplant, persistent leukemia-related mutations were still identified. In 2 pts with heterozygous BRCA2 and 1 pt with a heterozygous BRCA1 mutation at diagnosis, mutations persisted at CR, prior to transplant and at day 30 and 100 post-transplant, with a VAF of 50%, suggesting that these likely represented germline mutations. None of these pts had a history of breast or gynecological cancers. The median follow up after transplant was 9.97 months (range, 6.07- 20.17). Among pts with persistent mutations at day 100, 37% relapsed and the rest continued to be in remission until their last follow up.ConclusionGenomic analysis showed persistent leukemia-associated mutations in pts with AML in morphological and cytogenetic CR prior to HCT. Regardless of the conditioning regimen, a significant proportion of pts had persistent mutations at day100 after transplant even in pts with full engraftment. Persistent mutations predicted relapse though not all pts relapsed during this study period. These data suggest that deep genome sequencing may help in identifying higher risk pts of relapse post HCT, though longer follow up is needed to monitor the persistent mutations in pts who did not relapse during study time. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Majhail:Sanofi: Honoraria; Anthem, Inc.: Consultancy.

Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns1–4. Although hypothesized to serve important regulatory functions5, physiologic roles for the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations6–9, yet functional connections between minor introns, hematopoiesis, and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNAs converged on LZTR1, a regulator of Ras-related GTPases10–12. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing, and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers, and links between ZRSR2 mutations, LZTR1 regulation, and leukemias.