Clinical and Molecular Characteristics of DDX41-Mutated Patients in a Large Cohort of Sporadic MDS/AML

Abstract Background: MDS and AML are mostly found in elderly patients. However, even in this population there is increasing evidence of predisposing genetic conditions, which have been underdiagnosed so far. Identifying inherited predisposition to myeloid disorders can be crucial especially in the context of hematopoietic stem cell transplantation (HSCT). Germline mutations in the DEAD/H-box helicase gene DDX41 have been identified in families with multiple cases of MDS or AML but also in sporadic cases. We aimed to analyze the prevalence and clinical features of DDX41-related myeloid malignancies within an unselected cohort of pts diagnosed with MDS or AML (MDS/AML). Methods Between March 2017 and June 2018, mutation screening was performed in 842 consecutive pts with a diagnosis of MDS/AML in a single center at Hôpital Saint-Louis, Paris. DNA was obtained from bone marrow or peripheral blood. Targeted sequencing of all exons of a panel of 80 genes recurrently mutated in myeloid malignancies was performed using custom capture-based library preparation (Agilent SureSelect) and Illumina sequencing. Sanger sequencing was performed on selected pts' cultured skin fibroblasts to check for the putative germline origin of the variants. Results We identified a DDX41 gene variant in 36 unrelated pts (4% of 842). We focused on the 32 pts having at least one DDX41 variant with a variant allele frequency (VAF) ranging from 40 to 60% highly suggestive of a germline origin, which was subsequently confirmed in all available cases (N=7). Sixteen variants were classified as pathogenic or likely pathogenic based on major predicted changes in protein sequence while the 16 others were missense variants of unknown significance (VUS), which scored deleterious in most algorithms (Figure 1A). An additional, likely somatic DDX41 mutation (VAF < 40%) was present in 18 of 32 pts (56%). Overall, 22 pts could be unambiguously considered as having a DDX41-related malignancy based on the presence of a major disturbing mutation and/or a second DDX41 mutation, while 10 pts had a single VUS. Twenty-six variants were newly described, including a recurrent one, G173R found in 5 pts, all having a second DDX41 mutation. Median age of the 32 patients was 70 years (35-88). Only 4 pts (12%) had a familial history of hematologic disorders. According to revised WHO classification, 4 (12.5%) had MDS-MLD, 8 MDS-EB (25%), 12 AML (37.5%), 6 MDS/MPN (18.7%), one 5q syndrome and one aplastic anemia. Strikingly, 15/32 (47%) pts had a history of cytopenia several years before blastic evolution and the 5 pts with G173R presented with hypoplastic MDS or initially isolated cytopenias, suggesting a specific functional effect of this mutation. Karyotype was normal in 16 pts (44%), complex in one, 12 pts had an isolated abnormality, and three had cytogenetic failure. Additional driver mutations were identified in most (27/32,84%) pts (Figure 1B), but we noticed that they were less frequent and at lower VAF in pts having both germline and somatic DDX41 mutations as compared to pts with a single variant (median 1.5 vs 3 mutations, median VAF 7% vs 29.5%, p<0.001). This suggests distinct oncogenic pathways, with DDX41 double-hit oncogenesis being relatively independent of other oncogenic drivers. Seven low-risk MDS pts were untreated, 7 received ESA and 5 (71%) responded. Ten high-risk MDS/AML pts received a hypomethylating agent and 8 (80%) achieved hematological response. Nine AML pts received intensive chemotherapy, with a complete response rate of 100% (7/7, 2 ongoing) and 5 of them had HSCT, all of them being alive with tolerable toxicity. Five pts died, median OS was 87 months, and 2-y OS was 89%. No difference on OS was observed between single and double-DDX41 mutated pts. Conclusions: DDX41 germline variant carriers represent a significant part of MDS/AML pts, the vast majority presenting without familial history. The predicted change in protein and/or the presence of a second somatic mutation strongly support the causality of the germline variant in most pts. By contrast with previous reports, pts frequently presented a phase of cytopenia before overt malignancy. Finally, outcome regarding response to treatment and OS in this DDX41-mutated cohort appeared relatively favorable. Figure 1. Figure 1. Disclosures Peffault De Latour: Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Fenaux:Otsuka: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Download Full-text

Efficacy and Safety of a FLAG-Sequential Regimen Followed By Hematopoietic Stem Cell Transplantation for Myelodysplasia or Acute Leukemia in Fanconi Anemia: A Franco-Brazilian Report

Blood ◽

10.1182/blood-2019-122007 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2498-2498

Author(s):

Pierre-Edouard Debureaux ◽

Flore Sicre de Fontbrune ◽

Carmem M. S. Bonfim ◽

Jean-Hugues Dalle ◽

Nimrod Buchbinder ◽

...

Keyword(s):

Cumulative Incidence ◽

Research Funding ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Infectious Complications ◽

Hematopoietic Stem ◽

Free Survival ◽

History Of ◽

Syncytial Virus

Background: Fanconi anemia (FA) is the most frequent genetic cause of bone marrow failure (BMF) due to a DNA repair mechanism defect. The natural history of FA is marked by progressive BMF during early childhood. Throughout life, the hematopoietic situation may change by clonal evolution toward myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure in FA patients. The role of HSCT for FA patients with AML or advanced MDS is less defined. Currently, HSCT first line result offers 50% Overall Survival (OS) for patients with cytogenetic abnormalities only and 30% OS for patients with advanced MDS or AML in FA (Ayas et al., JCO 2013; Mitchell et al., BJH 2014). We previously reported a FLAG-sequential approach in 6 patients with FA (5 AML and 1 advanced MDS), all alive at a median follow-up of 28 months (Talbot et al., Hematologica 2014). We update here those patients and report 12 more patients treated by FLAG-sequential since then. Materials & Methods: This retrospective study (2006-2019) was conducted in 7 centers in France and Brazil on behalf of the French Reference Center for Aplastic Anemia to evaluate FLAG-sequential in FA patients with morphological clonal evolution (no patients with cytogenetic abnormalities only). The study was conducted in accordance with the Declaration of Helsinki. Anonymous data collection was declared to the appropriate authorities. The FLAG-sequential treatment consisted of FLAG, Fludarabine 30 mg/m²/d for five days and Cytarabine 1 g/m²x2/d with G-CSF for five days, which was followed three weeks later by Cyclophosphamide 10 mg/kg/d for four days, Fludarabine 30 mg/m²/d for four days and TBI 2 Gy (Fig 1A). In a haploidentical setting, Cyclophosphamide at 30 mg/kg/d was performed only in post-transplantation, at Days +4 and +5 (Fig 1B). Results: Eighteen patients were included with 14 AML, 1 acute lymphoblastic leukemia (ALL), and 3 RAEB-2 (Table 1). The median age at the time of HSCT was 22 years (4-37 years). Fifteen patients (83%) were older than 10 years at the time of HSCT. The median follow-up was 31 months (3- 153 months). Eight patients (44%) had complex karyotype. None of the included patients had a history of solid malignancies before HSCT. All patients engrafted. The cumulative incidence of neutrophil engraftment at Day 60 was 94% (95% CI 63-100%) with a median of 18 days (12-343 days). The cumulative incidence of platelet engraftment at Day 60 was 83% (95% CI 50%-96%) with a median of 25 days (17-245 days). The donor chimerism was complete at Day +100 for 15 patients. The three patients without full donor chimerism at Day +100 either had a relapse (n=1) and 2 early deaths before Day+100 from steroid-refractory aGVHD (n=1) or septic shock (n=1). None of the patients received a second HSCT. Non-relapse mortality (NRM) at 3 years was 32% (95% CI 6-58%) (Fig 2). Cumulative incidence of grades II to IV aGVHD was 56% (35% grades III to IV). Cumulative incidence of extensive cGVHD was 16%. Infectious complications during HSCT include the following: CMV (n=8), EBV (n=2), adenovirus (n=4), BK virus (n=7), respiratory syncytial virus (n=1), candidaemias (n=2) and invasive aspergillosis (n=3). Progression free survival (PFS) and OS at 3 years were 53% (95%CI 32-89%) and 53% (95%CI 32-89%), respectively (Fig 2). Cumulative incidence of relapse at 3 years was 13% (95%CI 0-31%) (Fig 2). Seven patients died during the study. Causes of death were relapse (n=2), aGVHD (n=2), cGVHD (n=1), septic shock (n=1), and respiratory syncytial virus associated with invasive aspergillosis (n=1). GVHD-relapse free survival (GRFS) at 3 years was 48% (95%CI 29-78%). One patient had anal epidermoid carcinoma at 4 years after HSCT, which required multiple surgical ablations. Conclusion: With almost 3 years follow-up, which is long enough for our results to be considered robust, we report an OS and PFS of 53%, which compares favorably to historical controls since all of our 18 patients were treated with florid disease at time of HSCT (and not with cytogenetic abnormality only, known to be associated with a better prognosis). Toxicity is still a concern in this particular population of FA patients with notably a high rate of infectious complications. Future well designed prospective clinical trials will refine this sequential strategy, which appears promising in this particular difficult clinical situation. Disclosures Socie: Alexion: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Download Full-text

Intravesicular Cidofovir in the Treatment of BK Virus-Associated Hemorrhagic Cystitis (BKV-HC) Following Allogeneic Hematopoietic Stem Cell Transplant (HSCT)

Blood ◽

10.1182/blood-2018-99-110887 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2085-2085

Author(s):

Graham Tooker ◽

Ashraf Z. Badros ◽

Jennifer Nishioka ◽

David Riedel

Keyword(s):

Viral Load ◽

Median Time ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Bk Virus ◽

Response To Treatment ◽

Cell Transplant ◽

Initial Report ◽

Hematopoietic Stem ◽

Common Diagnosis

Abstract Background: BKV HC is a well-known complication following allo-SCT. Supportive care with bladder irrigation and blood transfusions were the only available treatment. Since our initial report (Bridges B et al. Am J Hematol 2006;81:535), several studies confirmed that intravesicular cidofovir is a potential effective treatment for BKV HC. In this study, we report a large series of consecutive patients who developed BKV HC following allo SCT and received intravesicular cidofovir. Methods: We conducted a retrospective review of allo SCT patients who developed BKV HC and were prescribed intravesicular cidofovir from 2012 to 2017. Results: 33 patients were diagnosed with BKV HC. The median age was 50 years (range=23-73), and 18 (55%) were male. Acute myeloid leukemia (n=12, 35%) was the most common diagnosis followed by non-Hodgkin lymphoma (n=7, 21%) and B cell acute lymphoblastic leukemia (n=4, 12%). Conditioning regimens were myeloablative (n=19, 58%) or reduced-intensity (n=14, 42%); 15 (45%) patients received cyclophosphamide, and 22 (67%) received total body irradiation. The median time to onset of HC symptoms following SCT was 37 days (range: 8-178); 17 (52%) patients had acute graft vs. host disease. HC symptom severity ranged from grade 0-4 (median=2). The median BK urine viral load pre-treatment was 100,000,000 IU/ml. Patients received a median of 2 intravesicular treatments (range=1-7) at a dose of 5 mg/kg. Four patients (12%) were also treated concurrently with intravenous cidofovir. 19 (59%) patients demonstrated complete clinical resolution of symptoms, 9 (28%) demonstrated partial response to treatment, and 4 (13%) had no change in symptoms following treatment. These improvements in clinical status were independent of viral load, though most had reductions in the viral load. The median time to symptom resolution was 17 days (range=7-53; n=28). 82% of patients had no recurrent symptoms of HC. The main side effect of intra-vesicular instillation was increased discomfort and bladder spasms; severe in 3 patients (9%). No patient had impaired renal function directly attributable to intra-vesicular cidofovir. At 12 months after BKV HC diagnosis, 26 (79%) patients were alive. Conclusions: To our knowledge this is the largest study of intravesicular cidofovir for BKV HC reported to date; 77% of patients with BVK HC achieved clinical improvement of symptoms with minimal side effects. Clinical trials of intravesicular cidofovir could provide further evidence for adding intravesicular cidofovir as a standard tool for the treatment of BKV HC. Disclosures Badros: Celgene: Consultancy, Research Funding; Karyopharm: Research Funding; GSK: Research Funding.

Download Full-text

Profiling the Genomic Landscape at the Early Stages of CLL: Low Genomic Complexity and Paucity of Driver Mutations in MBL and Indolent CLL

Blood ◽

10.1182/blood.v128.22.3214.3214 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3214-3214 ◽

Cited By ~ 1

Author(s):

Andreas Agathangelidis ◽

Viktor Ljungström ◽

Lydia Scarfò ◽

Claudia Fazi ◽

Maria Gounari ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Driver Mutations ◽

Whole Genome ◽

Sequencing Analysis ◽

Driver Genes ◽

Hematopoietic Stem ◽

Genomic Complexity ◽

Targeted Deep Sequencing

Abstract Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for >10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF <10% in POT1 (n=2), TP53 (n=1), and SF3B1 (n=1) in 4 HC-MBL samples. In conclusion, LC-MBL and CLL with stable lymphocytosis for >10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

Download Full-text

Clonal Hematopoiesis Is Driven By Aberrant Activation of TCL1A

Blood ◽

10.1182/blood-2021-153242 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 597-597

Author(s):

Jk Gopakumar ◽

Joshua Weinstock ◽

Bala B Burugula ◽

Nikolaus Jahn ◽

Charles Kooperberg ◽

...

Keyword(s):

Research Funding ◽

Clonal Expansion ◽

Expansion Rate ◽

Driver Mutations ◽

Birth Date ◽

Mutant Clone ◽

Driver Genes ◽

Myeloid Malignancies ◽

Clonal Hematopoiesis ◽

Passenger Mutations

Abstract Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) may occur when a hematopoietic stem cell (HSC) acquires a fitness-increasing mutation resulting in its clonal expansion. A diverse set of driver genes, such as regulators of DNA methylation, splicing, and chromatin remodeling, have been associated with CHIP, but it remains largely unknown why HSCs bearing these mutations are positively selected. It has been challenging to identify the genetic and environmental factors mediating clonal expansion in humans, partially due to a lack of large cohorts with longitudinal blood sampling of participants. To circumvent this limitation, we developed a method to infer clonal expansion rate from single timepoint data called PACER (passenger-approximated clonal expansion rate). Methods: PACER is based on the principle that genomic passenger mutations can be used to infer the birth date of pre-malignant clones because these mutations accumulate fairly linearly over time. Thus, an individual with CHIP with a greater number of passenger mutations in the mutant clone is expected to have acquired the clone at a later age than someone with fewer passenger mutations. For two individuals of the same age and with clones of the same size, we expect the clone with more passengers to be more fit, as it expanded to the same size in less time. Typically, one would need to isolate single-cell colonies derived from HSCs in order to calculate the total passenger mutation burden. However, we hypothesized that this measure could also be approximated from whole genome sequencing of blood cell DNA, such as that used in large biobank projects. The expansion rate (PACER) is then estimated by adjusting the total passenger count for age and variant allele fraction in each individual. The ability of passengers to predict future clonal expansion was validated using longitudinal blood samples from 51 CHIP carriers in the Women's Health Initiative taken ~10 years apart (Figure 1). It also accurately predicted the known fitness effects due to different driver mutations in 5,551 CHIP carriers from the Trans-Omics for Precision Medicine (TOPMed) program (Figure 2). Results: Having validated the approach, we next hypothesized that we could identify germline variants influencing PACER, thus revealing genes and pathways mediating clonal expansion. The lead hit in a genome-wide association study (GWAS) of PACER was a common single nucleotide polymorphism (SNP) in the TCL1A promoter that was associated with slower clonal expansion in CHIP overall (Figure 3). TCL1A is an oncogene that is activated via translocation in T-cell prolymphocytic leukemia, but has no known role in CHIP or myeloid malignancies. A gene-level analysis indicated that the TCL1A SNP was associated with slower growth of clones bearing TET2 mutations, but had no effect on DNMT3A-mutant clone growth. We further found that those carrying two copies of the protective SNP had 40-80% reduced odds of having clones with driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A. A concomitant decrease in incident myeloid malignancies was also seen in carriers of this protective SNP. Next, we interrogated how the protective SNP influenced TCL1A activity in HSCs. Normal human HSCs lacked open chromatin at the TCL1A promoter and TCL1A expression, but inducing frameshift mutations in TET2 via CRISPR editing led to accessibility of the promoter and gene/protein expression in HSCs (Figure 4). This effect was abrogated in HSCs from donors of the protective TCL1A SNP in a dose-dependent manner. Finally, we found that HSCs from donors homozygous for the protective SNP had markedly less expansion of phenotypic stem and progenitor cells in vitro after the introduction of TET2 mutations than TET2-edited HSCs from donors with two copies of the reference allele. Conclusions: In summary, we developed a novel method to infer the expansion rate of pre-malignant clones and performed the first ever GWAS for this trait. Our results indicate that the fitness advantage of several common driver genes in CHIP and hematological cancers is mediated through TCL1A activation, which may be a therapeutic target to treat these conditions. PACER is an approach that can be widely adopted to uncover genetic and environmental determinants of pre-malignant clonal expansion in blood and other tissues. Figure 1 Figure 1. Disclosures Desai: Bristol Myers Squibb: Consultancy; Kura Oncology: Consultancy; Agios: Consultancy; Astex: Research Funding; Takeda: Consultancy; Janssen R&D: Research Funding. Natarajan: Blackstone Life Sciences: Consultancy; Boston Scientific: Research Funding; Novartis: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Apple: Consultancy, Research Funding; Amgen: Research Funding; Genentech: Consultancy; Foresite Labs: Consultancy. Jaiswal: Novartis: Consultancy, Honoraria; AVRO Bio: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Foresite Labs: Consultancy; Caylo: Current holder of stock options in a privately-held company.

Download Full-text

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Blood ◽

10.1182/blood-2021-151691 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3308-3308

Author(s):

Bianca L Rivera ◽

Shanisha Gordon ◽

Srinivas Aluri ◽

Yang Shi ◽

Samarpana Chakraborty ◽

...

Keyword(s):

Stem Cells ◽

Transgenic Mice ◽

Rna Sequencing ◽

Murine Model ◽

Research Funding ◽

Publicly Traded ◽

Gfp Expression ◽

Hematopoietic Stem ◽

Myeloid Malignancies ◽

Stop Cassette

Abstract Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. Figure 1 Figure 1. Disclosures Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

Download Full-text

KIT D816 Mutated / CBF-Negative Acute Myeloid Leukemia (AML): A New Poor-Risk Subtype Associated with Systemic Mastocytosis (SM-AML)

Blood ◽

10.1182/blood-2018-99-115895 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1535-1535

Author(s):

Mohamad Jawhar ◽

Konstanze Döhner ◽

Sebastian Kreil ◽

Juliana Schwaab ◽

Khalid Shoumariyeh ◽

...

Keyword(s):

Research Funding ◽

De Novo ◽

Systemic Mastocytosis ◽

Response To Treatment ◽

Treatment Modalities ◽

Molecular Characteristics ◽

Molecular Analyses ◽

Poor Risk ◽

Myeloid Neoplasm ◽

De Novo Aml

Abstract Systemic mastocytosis with an associated hematologic neoplasm (SM-AHN) is the most common subtype of advanced SM (advSM), diagnosed in up to 80% of patients. The AHN is most frequently diagnosed as a myeloid neoplasm, e.g., SM-MDS/MPNu or SM-CMML. Acquired mutations in KIT (usually KIT D816, KIT D816mut) are detectable in >90% of patients. The basis for the SM-AHN phenotype is usually the multi-lineage involvement, e.g. monocytes, eosinophils and other non-mast cell lineages, of KIT mutations. Core binding factor (CBF) positive AML (CBFpos AML) represents a distinct subtype and is identified in 5-8% of all AMLs. KIT mutations, most frequently KITD816mut, are detectable in up to 45% of CBFpos AML patients and are associated with an adverse prognosis. There is, however, only little information on KIT D816mut/CBFneg AML. We therefore evaluated a) clinical and molecular characteristics, b) response to treatment and, c) survival and prognostic factors in 40 KIT D816mut/CBFneg patients with histologically proven SM and associated AML (SM-AML), collected at 4 centers of the European Competence Network on Mastocytosis (ECNM). Molecular analyses (n=32) revealed at least one additional somatic mutation (median, n=3) apart from KIT D816, most frequently SRSF2 (n=12, 38%), RUNX1 (n=11, 34%), TET2 (n=11, 34%), ASXL1 (n=10, 31%), or NPM1 (n=7, 22%). At least one mutation in SRSF2, ASXL1 or, RUNX1 (S/A/Rpos) was identified in 21/32 (66%) patients. At diagnosis of SM-AML 21/40 (52%) patients had an aberrant karyotype. Secondary AML evolved in 29/40 (73%) patients from SM ± associated myeloid neoplasm and longitudinal molecular analyses revealed acquisition of new somatic mutations (TP53, n=2; NPM1, n=1; RUNX1, n=1, ASXL1, n=1; BCOR, n=1; IDH1/2, n=1) and/or karyotype evolution in 15/16 (94%) patients at the time of SM-AML. Thirty-one of 40 (78%) patients were treated with intensive chemotherapy (ICT) with a complete response (CR) rate of 40%. Allogeneic stem cell transplantation (SCT) was performed in 12/40 (30%) patients with durable CR in 6/12 (50%) patients. S/A/Rpos and/or the presence of a poor-risk karyotype were adverse predictive markers for response to treatment. To further investigate whether KITD816mut/CBFneg AMLdefines a distinct AML subtype associated with SM, two independent AML databases (AMLdatabases) were retrospectively screened and 69 KIT D816mut/CBFneg AML patients identified. The comparison between KIT D816mut/CBFneg SM-AML from ECNM (n=40) centers with KIT D816mut/CBFneg AMLdatabases(n=69) revealed remarkable similarities: a) a high KIT D816 variant allele frequency (VAF) (median 34% vs. 29%), b) with the exception of SRSF2 (38 vs. 18%), a highly similar mutation landscape, rather comparable to that of advSM (Jawhar et al., Blood 2017) than to that of de novo AML, c) in contrast to de novo AML, a low frequency of FLT3 mutations (3 vs. 7%), and d) a high frequency of an aberrant karyotype (52 vs. 42%). The median overall survival (OS) of 40 KIT D816mut/CBFneg SM-AML and 17 evaluable KIT D816mut/CBFneg AMLdatabases was 5.4 (95% confidence interval, CI [1.7-9.1]) and 26.4 (95% CI [0-61.0]) (P=0.015) months, respectively (Figure 1). However, if only the patients with ICT ± allogeneic SCT were compared, median OS between the two groupswas not different (16.7 vs. 26.4 months, P=0.4). In multivariate analyses, S/A/Rpos and a poor-risk karyotype remained the only independent poor-risk factors with regard to OS. These results were independent of treatment modalities. We conclude that KIT D816mut/CBFneg AML is a new poor-risk subtype associated with SM (SM-AML). The remarkable clinical, genetic and prognostic similarities between SM-AML and AMLdatabases suggest that a significant proportion of the AMLdatabases patients may in fact have SM-AML. We therefore strongly recommend to determine serum tryptase and KIT D816 mutation status in all AML patients, and to perform bone marrow histology in KIT D816mut patients. These simple diagnostic measures would allow reclassification to SM-AML and inclusion of KIT inhibitors in established treatment modalities of AML. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Döhner:Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Jazz: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Reiter:Incyte: Consultancy, Honoraria.

Download Full-text

A Prospective, Multicenter Surveillance Study for Adverse Events Associated with Hematopoietic Stem Cell Infusion: Analysis of Pediatric and Low Body-Weight Recipients

Blood ◽

10.1182/blood-2018-99-118328 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2105-2105

Author(s):

Kazuhiko Ikeda ◽

Hitoshi Ohto ◽

Yoshiki Okuyama ◽

Minami Yamada-Fujiwara ◽

Heiwa Kanamori ◽

...

Keyword(s):

Body Weight ◽

Multivariate Analysis ◽

Stem Cell ◽

Adverse Events ◽

Research Funding ◽

Allergic Reactions ◽

Hematopoietic Stem ◽

History Of ◽

Pediatric Recipients ◽

Transfusion Reactions

Abstract Adverse events (AEs) associated with blood component transfusion have been widely surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) infusion in HSC transplant (HSCT),including bone marrow transplant (BMT), peripheral blood stem cell transplant (PBSCT), and cord blood transplant (CBT),has been less rigorous, even though HSC products contain cells of diverse maturity and viability,plasma with various antigens, cytokines and antibodies, and dimethyl sulfoxide (DMSO) in the case of cryopreserved products. In fact, HSC infusion is associated with several AEs, e.g., allergic reactions, flushing, hypo- or hypertension, and respiratory distress, which have been attributed to toxicity of dead cells and DMSO (Otrock et al, Transfusion, 2017). However, our recent prospective surveillance revealed that HSC infusion-related AEs often occurred in each HSC type and the overall rates of AEs were greater in allo-BMT with no DMSO, compared with auto-PBSCT, allo-PBSCT, and allo-CBT typically cryopreserved with DMSO. Hypertension was the most common AE in each HSC source, with the highest rate in BMT, while allergic reactions were the most frequent in allo-PBSCT. A multivariate analysis identified a history of transfusion reactions as a risk factor of HSC infusion-related AEs (Ikeda et al, Transfus Med Rev, 2018). These findings suggest that some DMSO-independent factor(s), such as plasma components, may contribute to HSC infusion-related AEs. Thus, we asked if HSC volume and component effects were more substantial in small recipients, and if age-related factors alter susceptibility to HSC infusion-related AEs in pediatric patients. So far, data on HSC infusion-related AEs in pediatric and low body-weight recipients are lacking. Here, to address these issues, we investigated AEs due to HSC infusions in 219 recipients of <45 kg body weight, including 90 recipients 15 years old or younger, versus data from 1,125 recipients in general (general recipients)in the prospective study described above. The rates of overall HSC infusion-related AEs were quite similar among HSC sources among low body-weight/pediatric recipients (Table 1) andexclusivepediatric recipients (P= .158 in auto-/allo-HSCT and .867 in allo-HSCT), in contrast to general recipientswhose rate of AEs was highest in BMT. In addition, bradycardia was more often reported in CBT compared with PBSCT and BMT (Table 1), especially in pediatric recipients (30.8% in CBT and 0% in others, P< .001). On the other hand, there were some similar trends between low body-weight/pediatric recipients and general recipients: PBSCT and CBT recipients, but not BMT recipients, complained of malodor, whereas the rate of hypertension was highest in BMT (Table 1). Next, we compared HSC infusion-related AEs between auto- and allo-HSCT using cryopreserved products (Table 2). This comparison showed higher overall rates of AEs in allo-HSCT compared with auto-HSCT, especially in pediatric recipients, suggesting that plasma componentsrather than DMSO contribute to HSC infusion-related AEs in low body-weight/pediatric recipients as well as in general recipients. Of note, pediatric recipients showed a 10-foldhigher incidence of nausea/vomiting in allo-HSCT versus auto-HSCT, instead of allergic reactions, for which the incidence was significantly higher in allo-HSCT than auto-HSCT in general recipients. Subsequently, we sought factors that correlate with HSC infusion-related AEs in allo-HSCT using multivariate analysis with logistic regression, which identified lymphoid neoplasms over myeloid neoplasms as a factor for overall AEs (OR 3.013, P= .026), while history of transfusion reactions did not reach statistical significance (OR 2.368, P= .066). Notably, in a multivariate analysis for any grade ≥2 AEs, there were some factors that did not correlate with general recipients but did with low body-weight/pediatric recipients, including complications before HSCT (OR 5.764, P= .019), without plasma or red blood cell removal for ABO mismatch (OR 3.815, P= .032), and >10 ml/kg infusion volume (OR 5.306, P= .027). In conclusion, our data quantifysome specific symptoms associated with HSC infusionin low body-weight and pediatric recipients. We should be mindful ofinfusion volume and preexisting complications when small recipients receive HSC infusion. Disclosures Fujiwara: Astellas: Consultancy; Kyowa-Hakko: Consultancy; Kirin: Consultancy; Chugai: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Muroi:JCR: Speakers Bureau; Becton: Speakers Bureau; Dickinson and Company: Speakers Bureau; Japanese Red Cross Society: Speakers Bureau. Mori:Astella Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Japan Blood Products Organization: Honoraria; MSD: Research Funding; Ono: Honoraria; Novartis Pharma: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Asahi Kasei: Research Funding; Novartis Pharma: Research Funding; Celgene: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Shire Japan: Honoraria; Pfizer: Honoraria; CHUGAI: Honoraria; MSD: Honoraria; SHIONOGI: Honoraria. Nagai:Ono Pharmaceutical Co.Ltd.: Consultancy; Kaneka Corporation: Research Funding; Kawasumi Laboratories Inc.: Research Funding.

Download Full-text

Role of Surgical Resection in Allogeneic Hematopoietic Stem Cell Transplant Recipients with Prior Pulmonary Aspergillosis

Blood ◽

10.1182/blood.v124.21.5846.5846 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5846-5846

Author(s):

Ren Lin ◽

Li Gao ◽

Zhiping Fan ◽

Qianli Jiang ◽

Hongsheng Zhou ◽

...

Keyword(s):

Surgical Resection ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Antifungal Treatment ◽

Unrelated Donor ◽

Hematopoietic Stem ◽

Guangzhou City ◽

Acute Myelogenous ◽

History Of

Abstract Backgroud: Invasive fungal disease (IFD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients with a history of invasive pulmonary aspergillosis (IPA), especially those with residual pulmonary lesions at transplantation, are at high risk for IFD relapse after allo-HSCT. Since antifungal chemotherapy might be unable to eradicate IPA, surgical resection of pulmonary lesions is suggested to decrease the risk of IPA relapse. In this study, we retrospectively analyzed the outcome of patients who had persistent pulmonary cavity lesions or nodule after initial antifungal treatment. Methods: Between January 2007 and June 2014, a total of 129 patients with a history of IPA underwent allo-HSCT at Nanfang Hospital. Fourteen of these patients had persistent cavity lesions or nodule with more than 2cm in diameter in lung (cavity lesion in 10 and nodule in 4) after initial antifungal treatment (>1 month). Eight of the 14 patients underwent thoracescopic surgery before transplantation including 6 receiving wedge resections and 2 receiving lobectomies. The other 6 patients did not have surgery because of primary underlying diseases (non-complete remission) or multiple sites of lesions. The median duration of initial antifungal treatment pre-transplants were 179 days (range, 92-338 days) and 117.5 days (range, 60-132 days) in the patients with and without surgery, respectively. All the patients were given secondary antifungal prophylaxis (SAP) from the start of the conditioning until 90 days post-transplantation or until eradication of residual lesions. The SAP agents were chosen based on treatment response to initial antifungal therapy. Results: Of the 8 patients undergoing surgery, the underlying primary diseases included acute lymphoblastic leukemia in 5 and acute myelogenous leukemia in 3. The median time from diagnosis of IPA to surgery was 169.5 days (range, 73-330 days). Seven patients received related donor and 1 received unrelated donor transplantation. HLA-matched transplantation was conducted in 6 patients and HLA-mismatched transplantation was conducted in 2. Three patients received standard conditioning and 5 received intensified conditioning. In the 6 patients without surgery, the underlying primary diseases were acute myelogenous leukemia in 3, acute lymphoblastic leukemia in 2 and acute biphenotypic leukemia in 1. Five of these 6 patients underwent related donor transplantation and 1 underwent unrelated donor transplantation; HLA-matched transplants was conducted in 5 patients and HLA-mismatched transplants was conducted in 1. Standard conditioning were given in 3/6 patients and intensified conditioning in 3/6. After transplantation, none of the 8 patients with surgery experienced IFD relapse while 3 of the 6 patients without surgery experienced IFD progressing including 1 died of hemoptysis caused by IPA. The median duration of SAP after HSCT were 96 days (range, 73-119 days) in patients with surgery and 116.5 days (range, 101-183 days) in patients without surgery. The patients with surgery seemed to have the trend of low incidence of IPA relapse than those without surgery after allo-HSCT (P=0.055). Besides, the incidences f IPA relapse were comparative among the patients with surgery and those who achieved complete remission of IPA at transplantation (P=0.595). Conclusions: For patients with persistent cavity or nodules in lung, surgical resection followed by SAP might be effective to decrease the risk of IPA relapse after transplantation. Disclosures Liu: National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

Download Full-text

Single-Cell Multi-Omics in Human Clonal Hematopoiesis Reveals That DNMT3A R882 Mutations Perturb Early Progenitor States through Selective Hypomethylation

Blood ◽

10.1182/blood-2020-142574 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Anna S Nam ◽

Neville Dusaj ◽

Franco Izzo ◽

Rekha Murali ◽

Tarek H Mouhieddine ◽

...

Keyword(s):

Single Cell ◽

Research Funding ◽

Gene Set Enrichment Analysis ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Binding Motifs ◽

Mutation Status ◽

Clonal Hematopoiesis ◽

Cd34 Cells ◽

Myeloid Progenitors

Leukemia driver mutations have been identified in clonal hematopoiesis (CH; Jaiswal et al, NEJM, 2014). This provides a window of opportunity to interrogate the downstream impact of driver mutations in the earliest stages of neoplasia, before the accumulation of additional drivers that lead to frank malignancy. However, CH mutated cells are morphologically and phenotypically similar to normal cells. Thus, previous characterization in primary human samples have largely focused on genetic identification of these clonal outgrowths. To overcome this limitation and define the downstream effects of CH mutations, we leveraged multi-omic single-cell sequencing to profile the mutation status and whole transcriptome for the same cells at high-throughput (GoT; Nam et al, Nature, 2019). To identify CH samples, we screened 136 stem cell grafts collected for autologous transplant from multiple myeloma patients in remission (Mouhieddine et al, Nat Comm, 2020). We identified four individuals with DNMT3AR882 mutations with VAF > 0.05, allowing the profiling of 27,324 CD34+ cells (Fig. 1a-b). DNMT3A R882 mutations in these human CH samples did not result in a significant differentiation block in the hematopoietic stem cells, as assessed by pseudotime analysis (Fig. 1c). However, specific differentiation skews were apparent in multi-lineage progenitors, corresponding to the highest expression of DNMT3A in mutated immature myeloid progenitors (IMP, i.e. common myeloid progenitors/granulocyte-monocyte progenitors, Fig. 1d). We observed the expansion of mutated IMPs (Fig. 1e) that showed priming toward the megakaryocytic-erythroid (ME) lineage (Fig. 1f). DNMT3AR882 lympho-myeloid primed progenitors showed a myeloid bias and disfavored lymphoid development (Fig. 1g). In these CD34+ progenitors, we identified dysregulated expressions of megakaryocytic lineage markers, such as CD9 and PLEK, consistent with the ME-bias (Fig. 1h). We also observed downregulation of lymphoid differentiation gene ZBTB1, corresponding to the myeloid over lymphoid skewing (Fig. 1h). Finally, we identified dysregulated expression of genes involved in TNF-alpha signaling (e.g. TNFRSF4, TNFSF13B), suggesting a pro-inflammatory state. To identify activated pathways, we performed a gene set enrichment analysis that revealed activation of MYC and IL-6 signaling (FDR <0.05, Hallmark). A focused analysis of regulatory networks identified enhanced activity of key transcription factors (TFs) involved in ME differentiation such as GATA1 and FLI1, as well as those involved in inflammation such as NFKB/REL (FDR <0.05). We also identified increased expression of polycomb repression complex 2 (PRC2) target genes (FDR <0.05). To determine how aberrant DNA methylation may serve as a link between DNMT3A mutations and the observed transcriptional dysregulation, we performed single-cell multi-omics that simultaneously profiles the cells' methylome and transcriptome, linked with mutation status (Gaiti et al, Nature, 2019; Fig. 1i). By comparing the methylation rates in mutated vs. wildtype CD34+ cells within the same sample, we found that DNMT3AR882 result in selective hypomethylation that preferentially impacts CpG islands (Fig. 1j) and PRC2 targets (Fig. 1k), which may underlie the dysregulated expression of PRC2 targets in DNMT3AR882 progenitors. Notably, DNMT3AR882-induced hypomethylation was enriched in a specific sequence context in which the CpG is followed by a T nucleotide (Fig. 1l). This DNMT3AR882 motif was enriched in the DNA binding motifs of central hematopoietic TFs, including MYC/MAX (Fig. 1m), suggesting that DNMT3AR882-induced hypomethylation may serve as a mechanism to preferentially increase the activity of these TFs. Consistent with this hypothesis, joint single-cell methylome and transcriptome data revealed that the expression of MYC targets increased with hypomethylation of its binding motifs (Fig. 1n), providing a mechanism of enhanced MYC activity due to DNMT3AR882 mutations. Altogether, we report the direct examination of the consequences of DNMT3AR882 mutations in primary CD34+ cells in CH. We discovered that DNMT3A is most highly expressed in mutated multi-lineage progenitors, promoting their expansion and biasing their downstream differentiation. This is accompanied by a disruption to differentiation through PRC2 and MYC target reactivation via selective hypomethylation. Disclosures Abdel-Wahab: Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy. Ghobrial:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; Cellectar: Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy; Noxxon Pharma: Consultancy; Genentech: Consultancy; GlaxoSmithKline: Consultancy; GNS Healthcare: Consultancy; AbbVie: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

Download Full-text

Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Hematopoietic Stem Cells in JAK2-Mutant Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2020-141643 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-8

Author(s):

Debra Van Egeren ◽

Javier Escabi ◽

Maximilian Nguyen ◽

Shichen Liu ◽

Christopher R. Reilly ◽

...

Keyword(s):

Peripheral Blood ◽

Research Funding ◽

Jak2v617f Mutation ◽

Newly Diagnosed ◽

Advisory Committees ◽

Hematopoietic Stem ◽

History Of ◽

Mutant Cells ◽

Individual Human

*equal contribution, #co-corresponding authors Although the JAK2V617F mutation is the most common MPN phenotypic driver mutation, the precise consequences of the mutation on the behavior of individual human hematopoietic stem cells (HSCs) in vivo remains unknown. We used whole genome sequencing and single-cell profiling of hematopoietic stem and progenitor cells (HSPCs) to quantify the impact of JAK2V617F on the proliferation dynamics of HSCs and the differentiation trajectories of their progenies in individual newly diagnosed MPN patients. We reconstructed the lineage history of individual HSCs obtained from patients with newly diagnosed essential thrombocythemia (ET), using the pattern of spontaneous somatic mutations accrued in their genomes over decades (Figure 1). Intriguingly, our analysis indicates that the JAK2V617F mutation occurred in a single HSC many years before MPN diagnosis - at age 9±2 years in a 34 year-old patient, and at age 19±3 years in a 63 year-old patient. In each patient, we inferred the number of mutated HSCs over the years and computed their fitness. After escaping stochastic extinction, the population of mutated HSCs grew exponentially by 63±15% and 44±13% every year in the two patients respectively. To contrast the differentiation trajectories of the JAK2-mutant HSCs with those of healthy HSCs, we simultaneously measured the full transcriptome and somatic mutations in single HSPCs in the two ET patients in whom we had performed whole genome sequencing and in one additional ET patient (N=3 total) and also in patients with polycythemia vera (PV) (N=3). We observed, at the time of MPN diagnosis, a consistent lineage bias of JAK2-mutant HSPCs toward megakaryocyte-erythrocyte fate, across ET and PV patients. Exploiting our ability to discriminate JAK2-mutant cells from JAK2 wild-type cells within individual MPN patients, we identified genes involved in antigen presentation and inflammation as differentially up-regulated in JAK2-mutant HSPCs, in particular within the JAK2-mutant CD14+ monocytic cell population. Although we found a range of peripheral blood JAK2V617F variant allele fractions (VAFs) in newly diagnosed ET and PV patients, approximately 20% HSCs in these patients were JAK2-mutant suggesting that peripheral blood mutational burden does not accurately capture the composition and output of JAK2-mutant HSCs. There are several implications of these findings: first, our studies suggest that the JAK2V617F mutation alone is sufficient to initiate and engender MPN, which has important therapeutic implications. Second, our findings indicate that the JAK2-mutant clone may manifest as clonal hematopoiesis of indeterminate potential (CHIP) for a decade or more before presenting as an overt MPN. This is important not only because JAK2-mutant CHIP has clinical implications, but also because the long "pre-MPN phase" uncovered by our work suggests that MPN could potentially be prevented, by targeting the JAK2-mutant MPN disease-initiating clone prior to exponential expansion. Third, in addition to revealing the limitations of peripheral blood JAK2V617F allele burden measurements, we found that the fraction of JAK2-mutant cells varied significantly across different populations of progenitor cells within the same MPN patient (for example the erythroid progenitors versus the granulocyte-monocyte progenitors). Surprisingly, almost all of the red blood cells, even in ET patients, descended from JAK2-mutant HSCs. Given that the JAK2V617F mutation has been shown to have cell-intrinsic consequences not only in leukocytes, but also in erythroid cells and in platelets, this finding may help explain the development of thrombosis in patients with low peripheral blood JAK2V617F allele burdens. Together, our study provides the most detailed portrait to date of the functional and transcriptional consequences of the JAK2V617F mutation in individual human HSCs in vivo, and a comprehensive molecular history of MPN initiation and pathogenesis in individual patients. The technology platforms and computational frameworks developed here are broadly applicable to other types of myeloid malignancies and blood cancers. Disclosures DeAngelo: Jazz: Consultancy; Incyte Corporation: Consultancy; Forty-Seven: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding; Agios: Consultancy; Autolos: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Glycomimetics: Research Funding; Abbvie: Research Funding; Takeda: Consultancy; Shire: Consultancy. Stone:Syndax: Consultancy, Research Funding; Macrogenics: Consultancy; Syntrix: Other: DSMB; Takeda: Other: DSMB; Stemline: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Arog: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gemoab: Consultancy; Syros: Consultancy; Jazz: Consultancy; Astellas: Consultancy; Aztra-Zeneca: Consultancy; Daiichi-Sankyo: Consultancy; Janssen: Consultancy; Biolinerx: Consultancy; Celgene: Consultancy, Other; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy; Argenix: Other. Garcia:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Eli Lily: Research Funding. Hobbs:Jazz: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Constellation: Honoraria, Research Funding; Bayer: Research Funding; Incyte: Research Funding; Merck: Research Funding. Mullally:Janssen: Consultancy, Research Funding; Actuate Therapeutics: Research Funding.

Download Full-text