Clonal Hematopoiesis in AML Patients in Hematological CR Is Present in Many Patients with Intermediate Risk AML and Is Associated with a High Prevalence of DNMT3A gene Mutations

Abstract Background De novo acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells usually characterized by a rapid clinical onset. Despite this clinical manifestation, recent evidence suggests that premalignant stem cells might be present in patients with AML, which can persist after chemotherapy in some patients and induce clonal hematopoiesis. Little is known about the prevalence of clonal persistence and about the molecular basis. In order to study the prevalence of this phenomenon and to better understand the underlying molecular mechanisms, we investigated AML patients in remission for clonal persistence of cells after chemotherapy. Patients and methods: All patients included in this analysis were treated within a prospective treatment protocol of the Study Alliance Leukemia (SAL). The primary study cohort consisted of 61 female patients with intermediate risk cytogenetics achieving hematologic complete remission (CR), whose DNA material was available at CR. Clonality analysis was based on X-chromosome inactivation testing using the HUMARA assay. DNA from diagnostic samples of patients presenting with evidence for X-chromosome skewing in CR was analyzed using amplicon based resequencing on a MiSeq next generation sequencing (NGS)-system for DNMT3A, ASXL1, ASXL2, TET1, TET2, EZH1, EZH2, IDH1 and IDH2. Results: Of the 61 patients included, 52 were heterozygous for the STR in the human androgen receptor gene. In CR, 22 of these 52 patients (42%) showed evidence for a skewed X-chromosome representation, indicating persistence of clonal hematopoiesis in remission. The NGS-based analysis of genes involved in epigenetic regulation revealed mutations in 13/22 (59%) of the patients. DNMT3A was most frequently mutated (11/13 patients), either alone or in combination with other alterations (TET2, EZH2). Interestingly, two patients showed somatic alterations in the TET1 gene. In remission, clonal persistence of these alterations was detected in all 13 patients with mutations at diagnosis at levels between 0.8 and 50% as documented using ultradeep-NGS. To get an idea on the prevalence of clonal persistence in other cytogenetic groups, we analyzed 22 low risk (i.e. CBF-leukemias) as well as 18 poor risk (-7, complex karyotype) patients using the HUMARA assay. Here we observed similar results, with 13/19 informative patients showing clonal persistence in low-risk group (68%) compared to 7/14 patients (50%) in the poor risk population. Since all these analyses were confined to female patients and potentially limited by the sensitivity of the HUMARA method, we went on to look for persistence of clonal molecular markers using more sensitive ultra-deep NGS. Because DNMT3A exon 23 was the common alteration in this initial analysis, we screened a cohort of 48 patients with mutations in NPM1 and comutations in DNMT3A. In this separate cohort, persistence of the DNMT3A mutations at CR or during follow-up (FU) was detected in 42 patients (87.5%) at levels between 0.5 and 50% (median 11.1%). No difference was seen between male and female patients, the median age was 51 years, persistence was seen even in young patients at 26 years of age. During FU, the DNMT3A VAF level rose further in all patients analyzed, arguing for a clonal advantage of the mutant cells. All patients with relapse and available material showed high levels of DNMT3A at time of relapse. However, correlation of DNMT3A mutant allele levels at CR1 with the incidence of relapse showed no significant impact of the VAF for the development of relapse. Conclusions: Our data indicate that clonal persistence of premalignant cells carrying clonal alterations in epigenetic regulator genes is a common phenomenon in patients in continuous CR. DNMT3A is the most common lesion persisting, the majority of patients with this mutations retain it at CR and during FU. These data indicate that de novo AML develops from preleukemic stem and progenitor cells in many patients. Preliminary data indicate that this persistence per se is not associated with inferior outcome. Disclosures Schuster: AgenDix GmbH: Employment. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

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Long-Term Clinical and Molecular Follow-up of Large Animals Receiving Retrovirally Transduced Stem and Progenitor Cells: No Progression to Clonal Hematopoiesis or Leukemia

Molecular Therapy ◽

10.1016/j.ymthe.2003.12.006 ◽

2004 ◽

Vol 9 (3) ◽

pp. 389-395 ◽

Cited By ~ 77

Author(s):

Hans-Peter Kiem ◽

Stephanie Sellers ◽

Bobbie Thomasson ◽

Julia C Morris ◽

John F Tisdale ◽

...

Keyword(s):

Progenitor Cells ◽

Clonal Hematopoiesis ◽

Stem And Progenitor Cells ◽

Large Animals

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Prognostic Value of Molecular Markers for Guiding Post-Remission Therapy in Patients with De Novo Acute Myeloid Leukemia (AML) and Intermediate-Risk Cytogenetics

Blood ◽

10.1182/blood.v112.11.2522.2522 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2522-2522

Author(s):

Marta Pratcorona ◽

Mireia Camós ◽

Montserrat Torrebadell ◽

Maria Rozman ◽

Ana Carrió ◽

...

Keyword(s):

High Risk ◽

Prognostic Value ◽

De Novo ◽

Low Risk ◽

High Dose ◽

Intermediate Risk ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Molecular Features ◽

Mutational Status

Abstract The heterogeneous prognosis of patients with intermediate-risk cytogenetics AML (AML-IR) can be partially clarified by screening of NPM1 mutations (NPMmut) and internal tandem duplication of FLT3 (FLT3-ITD). Nonetheless, additional factors might influence the prognostic effect of these molecular lesions, such as the FLT3-ITD mutant level. Moreover, the optimal post-remission strategy might differ depending on the underlying molecular lesion. In this regard, we analyzed the outcome, according to NPM1 and FLT3 mutations and post-remission therapy given, of a series of patients diagnosed with de novo AML-IR in a single institution who received intensive chemotherapy. Patients were treated following 4 sequential protocols of CETLAM group during the period 1994–2006, consisting of 1 or 2 cycles of standard induction chemotherapy and 1 course of high-dose cytarabine-based consolidation therapy. Thereafter, patients underwent hematopoietic stem cell transplantation (HSCT) according to donor availability and presumed risk (protocols LAM 99 & 2003). NPM1 mutations and FLT3-ITD were screened in diagnostic DNA by PCR amplification followed by Genescan analysis. The ratio between FLT3-ITD and wildtype FLT3 alleles (ITD/wt ratio) was calculated using the area under the peak of corresponding alleles. Overall, 134 patients (51% male; median age, 53; range: 17–70) with AML-IR (normal karyotype, 66%) were studied. NPM1mut and FLT3-ITD were found in 45% and 37% of patients, respectively, with a median ITD/wt ratio of 0.59 (0.045–5.5). After induction regimen, 109 patients (81%) achieved complete response (CR). The only variables predictive of a favorable response were NPMmut (90% vs. 75%; p=0.01) and age <60 (85% vs. 72.5%; p=0.05). After a median follow-up of 69 months, relapse risk (RR) at 5 years was 54% (±5%). RR was higher in patients presenting with hyperleukocytosis (>50 × 109/L), NPMwt, and FLT3-ITD. Interestingly, the prognostic value of FLT3-ITD depended on the relative mutant level, and detection of FLT3-ITD with a low ITD/wt ratio (i.e.,<0.3) did not increase the risk conferred by underlying NPM1 mutational status. In accordance, a composite variable based on NPMmut and quantitative FLT3-ITD was created defining 2 prognostic categories: a low-risk group (LOWmol), constituted by patients with NPMmut and either absence of FLT3-ITD or low ITD/wt ratio, and a high-risk subset (HIGHmol), defined by the absence of NPMmut and/or high ITD/wt ratio. This molecular stratification showed independent prognostic value for RR (5-year RR: 24%±10% vs. 81%±7 in LOWmol vs. HIGHmol patients, respectively; p<0.001), and survival (OS; relative risk: 2.8, 95% CI:1.6-5, p<0.001; figure Moreover, the effect of post-remission therapy varied in both molecular-defined subgroups. Thus, among patients with an age ≤60, 5-year survival in LOWmol patients with a planned autologous HSCT (autoHSCT) was 83%±9%, not differing significantly from that of patients undergoing allogeneic HSCT (intention-to-treat analysis; figure On the other hand, 5-year OS of HIGHmol patients who underwent allogeneic HSCT in first CR was 73%±13, which compared favorably with other post-remission strategies (5-yr OS: 27%±7%; p=0.019). In conclusion, in patients with intermediate-risk AML, the combined assessment of NPM1 mutations and quantitative estimation of FLT3-ITD allows the distinction of 2 categories of patients with different prognosis. Thus, whereas autoHSCT arises as an effective postremission therapy in patients harboring low-risk molecular features, allogeneic HSCT in first CR seems to overcome adverse prognosis of patients with high-risk disease. Nonetheless, the validity of this molecularly-based therapeutic algorithm warrants confirmation in other studies. Figure Figure

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Allogeneic Transplantation in First Remission Improves Outcome in Secondary Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.281.281 ◽

2014 ◽

Vol 124 (21) ◽

pp. 281-281

Author(s):

Christer Nilsson ◽

Erik Hulegårdh ◽

Vladimir Lazarevic ◽

Hege Garelius ◽

Asa Rangert Derolf ◽

...

Keyword(s):

De Novo ◽

Risk Group ◽

Low Risk ◽

Intermediate Risk ◽

Exact Test ◽

Secondary Aml ◽

Fisher's Exact Test ◽

Fisher’S Exact Test ◽

First Remission ◽

De Novo Aml

Abstract Allogeneic stem cell transplantation (SCT) is widely used as post-remission treatment in younger patients with poor or intermediate risk AML. Transplant decisions are mainly based on cytogenetic and molecular risk group, age, comorbidity and on the availability of a suitable donor. Secondary AML, including therapy-related AML (t-AML) and AML after an antecedent hematological disorder (AHD-AML), constitutes more than one fourth of the AML cases and is a predictor of a poor outcome. However, the extent to which SCT improves outcome of this patient group is poorly studied. In this study, we set out to investigate the role of SCT for the survival of secondary AML patients within the population-based Swedish Adult Acute Leukemia Registry. In total, 5881 patients with AML diagnosed during the period 1997 – 2013 were included in the study. Of these, 4233 (72%) were de novo AML, 1098 (19%) AHD-AML and 550 (9%) t-AML. The median age at diagnosis was 70 in de novo AML, 73 in AHD-AML and 70 in t-AML. The gender distribution was equal in de novo AML (51% males). In AHD-AML, there was a male predominance of 57% whereas in t-AML, there was a female predominance of 56%. The proportion of patients who underwent SCT in first remission (CR1) was 10% in de novo AML, 5% in AHD-AML and 8% in t-AML (de novo vs AHD-AML p < 0.001, de novo vs t-AML p = 0.068, AHD-AML vs t-AML p = 0.081; Fisher's exact test). In patients aged 65 or below, the proportion of SCT in CR1 was 24%, 21% and 20%, respectively. The median age of SCT patients was 48 (range 17 – 71) in de novo AML, 57 (27 – 76) in AHD-AML and 49.5 (18 – 68) in t-AML. In de novo AML, the distribution of genetic risk groups among SCT patients was 3% low risk, 55% intermediate risk and 42% high risk. Corresponding figures for AHD-AML was 0%, 34% and 66% and for t-AML 5%, 45% and 50% respectively (de novo vs AHD-AML p = 0.004, de novo vs t-AML p = 0.299, AHD-AML vs t-AML p = 0.124; Fisher's exact test). The estimated median survival after the date of SCT in CR1 was 15 months in AHD-AML and 22 months in t-AML but not reached in de novo AML (95% lower confidence limit 107 months). Among patients <65 years who had been in CR for 3 months (genetic low risk excluded), those with secondary AML had a greater benefit from consolidation with SCT than those with de novo AML (Figure 1). The projected 7-year survival in de novo AML was 60% with SCT and 44% with conventional post remission therapy (CPRT) as compared to 46% and 21%, respectively, in secondary AML. The survival hazard with SCT was 0.45 in secondary AML (95% CI 0.28-0.72) as compared to 0.66 in de novo AML (CI 0.53 – 0.82), by multivariable Cox regression adjusting for type of secondary AML, age, sex, and cytogenetic risk group. To refine the analysis correcting for major confounding factors, a matched pair analysis was performed in patients with CR longer than 3 months. Matching criteria were type of secondary AML (AHD or t-AML), cytogenetic risk group and age (+/- 3 years). Remission of the patient with CPRT was at least as long as the time between CR1 and transplantation for the matched patient undergoing SCT. The projected 7-year survival rate was 43% in the SCT and 8% in the CPRT group (p = 0.01; log-rank test, Figure 2) further indicating a benefit for SCT as post remission therapy in secondary AML. We conclude that SCT improves survival in patients with secondary AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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PU.1-Dependent Enhancer Decommissioning Drives Transformation of Tet2 deficient Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-142070 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-40

Author(s):

Maria M. Aivalioti ◽

Tushar D Bhagat ◽

Aditi Paranjpe ◽

Boris Bartholdy ◽

Kith Pradhan ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Leukemia ◽

Mutant Mice ◽

Self Renewal ◽

Hematopoietic Stem ◽

Elderly Individuals ◽

Myeloid Malignancies ◽

Clonal Hematopoiesis ◽

Healthy Elderly ◽

Stem And Progenitor Cells

Acute myeloid leukemia (AML) is the most frequent leukemia in elderly individuals with a median age at diagnosis of 67 years (Juliusson et al., Blood 2009). It arises in a step-wise process and originates from hematopoietic stem cells (HSC) (Jan et al.,Sci Transl Med. 2012). Genetic and epigenetic alterations drive the formation of pre-leukemic HSC clones with altered function, which can gain dominance and eventually give rise to AML upon the acquisition of cooperating lesions (Jan et al.,Sci Transl Med. 2012). However, it is currently impossible to predict which healthy elderly individuals with clonal hematopoiesis will eventually develop myeloid malignancies, as the pathways to leukemia are unknown. Heterozygous inactivating mutations of the epigenetic regulator Ten-Eleven Translocation-2 (TET2) are commonly found in patients with AML, yet also in a remarkable fraction of healthy elderly individuals in whom it is associated with clonal hematopoiesis (Busque, et al Nat Genet. 2012). These observations and studies in Tet2-deficient mice strongly suggest that TET2 inactivation is an early event in the pathogenesis of myeloid malignancies, but is not sufficient to fully transform HSC (Moran-Crusio et al., Cancel Cell 2011). TET2 cooperates with several transcription factors to regulate hematopoiesis (Rasmussen et al., Genome Res 2019), one of which is PU.1 (de la Rica et al., Genome Biol. 2013), an essential transcription factor governing normal hematopoiesis (Iwasaki et al., Blood 2005). In humans, PU.1 activity or expression is only moderately impaired in the majority of AML patients, and remarkably, also in aged HSC (Will et al., Nat Med. 2015), underscoring the essentiality of PU.1. Importantly, PU.1 target genes are frequently found hypermethylated in AML (Sonnet et al., Genome Med. 2014, Kaasinen et al., Nat Commun. 2019), suggesting a profound epigenetic inactivation of the PU.1 network. We hypothesized that moderate impairment of PU.1 abundance, as found in AML, can cooperate with loss-of-function mutations of Tet2 to initiate malignancy. We developed a novel tissue-specific compound mutant mouse model carrying heterozygous deletion of an upstream regulatory element (URE) of Pu.1 along with Tet2 deletion (Vav-iCre+ PU.1URΕ∆/+Tet2+/flox; Vav-iCre+ PU.1URΕ∆/+Tet2flox/flox). While none of the single mutant mice developed AML, compound mutant mice developed aggressive myeloid leukemia whose penetrance and latency exhibited Tet2 dose dependency. The disease presented with leukocytosis, anemia and splenomegaly. By cell morphology analysis of the peripheral blood, bone marrow and spleen, the leukemic mice exhibited accumulation of differentiation-blocked myeloblasts, myelocytes and/or metamyelocytes, that was confirmed using detailed myeloid differentiation markers, distinguishing the disease in immature or mature AML. Furthermore, gold standard in vitro and in vivo assays, assessing both self-renewal and differentiation capacity of double mutant mice-derived cells, revealed that the expanded differentiation-blocked stem and progenitor cells bear aberrant self-renewal and disease-initiating capacities. Comprehensive molecular profiling by next generation sequencing of disease-initiating cells uncovered a substantial overlap with human AML, such as functional GF1b loss with concomitant overexpression of CD90/Thy1 (Thivakaran et al., Haematologica 2018). Importantly, our analyses also revealed transcriptional dysregulation, hypermethylation of PU.1 regulated enhancers with concomitant loss of enhancer activity and alterations in chromatin accessibility of particularly genes co-bound by PU.1 and TET2. Current efforts focus on identifying key effectors of the dysregulated PU.1/TET2 sub-network driving malignant transformation in clonal hematopoiesis. Our collected data provide proof of concept that moderate PU.1 dose impairment can functionally cooperate with the inactivation of Tet2 in the initiation of myeloid leukemia and uncovers a likely unifying AML pathomechansim. Disclosures Will: Novartis Pharmaceuticals: Other: Service on advisory boards, Research Funding.

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Deregulation of Splicing in Pediatric Acute Myeloid Stem and Progenitor Cells

Blood ◽

10.1182/blood-2021-154120 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2227-2227

Author(s):

Phoebe Mondala ◽

Inge Van Der Werf ◽

Larisa Balaian ◽

Kathleen Steel ◽

Luisa Ladel ◽

...

Keyword(s):

Progenitor Cells ◽

Research Funding ◽

De Novo ◽

Exon Skipping ◽

Rna Seq ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Pediatric Aml ◽

Dose Dependent

Abstract - Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study. Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation. After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal. Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence. Disclosures Cloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

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CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21124266 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4266

Author(s):

Evgenia Sarrou ◽

Laura Richmond ◽

Ruaidhrí J. Carmody ◽

Brenda Gibson ◽

Karen Keeshan

Keyword(s):

Progenitor Cells ◽

Gene Editing ◽

De Novo ◽

Chromosomal Rearrangements ◽

Chromosomal Translocation ◽

Fusion Product ◽

Acute Lymphocytic Leukaemia ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.

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Fatty Bone Marrow Positively Selects Pre-Leukemic HSPCs with a DNMT3A-mutation

Blood ◽

10.1182/blood-2021-149360 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 596-596

Author(s):

Naama Zioni ◽

Noa Chapal Ilani ◽

Ekaterina Petrovich-Kopitman ◽

Mehmet Saçma ◽

Hartmut Geiger ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Research Funding ◽

Selective Advantage ◽

Middle Aged ◽

Clonal Hematopoiesis ◽

Nsg Mice ◽

Age Related ◽

Stem And Progenitor Cells

Abstract With aging, humans accumulate preleukemic mutations (pLMs) in hematopoietic stem and progenitor cells (HSPCs) which was termed age-related clonal hematopoiesis (ARCH). To gain a better insight changes in the HSPCs-environment crosstalk upon aging that might contribute to ARCH, it is critical to develop a multilayer perspective that integrates information on mutations, epigenetics the cellular context and the bone marrow (BM) microenvironment, since all these layers are changing during ageing. Accordingly, a key question in the field is how the ageing BM microenvironment influences clonal expansion of HSPCs. Fatty bone marrow (FBM) is one of the environmental factors that may influence clonal hematopoiesis (CH) with age. As we age, our bone marrow shifts from red to adipocyte-enriched yellow BM. We hypothesize that age related BMF accumulation may provide a selective advantage to specific pre-leukemic stem and progenitor cells (preL-HSPCs) carrying pLM. To support this hypothesis, we established a FBM model in NSG mice to enable the study of both human and rodent preL-HSPCs. Transplantation of primary human preL-HSPCs from AML patients (DNMT3A, NPM1 mutations ) into FBM resulted in enhanced engraftment compared to control mice without FBM. We further demonstrate that DNMT3A-R882H+/- mice derived BM HSPCs, engrafted significantly higher in NSG mice with FBM compared to controls. Interestingly, when DNMT3A-R882H+/- derived BM cells from middle-aged mice (12-month old) were injected into FBM mice, engraftment increased tenfold. Secondary engraftment of aged DNMT3A-R882H +/-BM derived cells resulted in an increase in engraftment upon transplantation into to FBM, suggesting enhanced in vivo self-renewal capacity of HSPCs in FBM. To study the underlying molecular mechanisms provided by the FBM to preL-HSPCs carrying DNMT3A-R882H +/-, we used a multiplex cytokine assay. In this approach we analyzed 17 common cytokines in BM following transplantation of young, two-month old, or middle-aged, 12-month old, DNMT3A-R882H +/-or control - BM derived cells into FBM. Our results show that transplanting two months old, middle-aged DNMT3A-R882H +/-or control BM derived cells to FBM resulted in a significant increase in BM mIL-6 secretion when compared to transplants into control, non-FBM mice. mIL-6 was secreted by adipocytes following irradiation regardless of which cells are transplanted. We then transplanted middle-aged DNMT3A-R882H+/- BM derived cells to FBM mice that had been treated intraperitoneally with a neutralizing IL-6 Ab. The administration of neutralizing IL-6 Ab resulted in a significant decrease in engraftment of DNMT3A-R882H+/- BM derived cells, confirming that IL6 contributes to the expansion of the DNMT3a-R882H+/- cells in FBM. In summary, these results demonstrate for the first time that the FBM provides a selective advantage to pre-leukemic cells carrying DNMT3A-R882H. Importantly, we show that IL-6 is a one of the major players in the molecular mechanism that confers the FBM advantage specifically to preL-HSPCs carrying R882H both in vitro and in vivo. Disclosures Mueller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

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Clinical Relevance of Somatic Mutations in Chronic Myelomonocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-121969 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1713-1713 ◽

Cited By ~ 2

Author(s):

Nico Gagelmann ◽

Anita Badbaran ◽

Rashit Bogdanov ◽

Olivier Nibourel ◽

Friedrich Stoelzel ◽

...

Keyword(s):

High Risk ◽

Genetic Risk ◽

Research Funding ◽

Risk Group ◽

Performance Status ◽

High Risk Group ◽

Low Risk ◽

Intermediate Risk ◽

Research Support ◽

Total Cohort

Current risk stratification for newly diagnosed patients with chronic myelomonocytic leukemia (CMML) includes clinical and genetic features accounting for its variable disease course. Allogeneic stem cell transplantation still remains the only curative treatment option, and prognostication of posttransplant outcome may be improved using molecular information. Here, we aim to evaluate the molecular profile and its role on posttransplant outcome in a multicenter CMML cohort. Mutation analysis was performed on DNA from bone marrow mononuclear cells or peripheral granulocytes collected prior to transplant and included previously published CMML-associated genes (i.a. SETBP1, ASXL1, RUNX1, NRAS, KRAS, TET2, CBL, IDH1/2, SF3B1, DNMT3A, EZH2, ZRSR2, U2AF1). Current prognostic models were calculated at time of transplant. Patients with transformation to acute leukemia were excluded. Top predictors of posttransplant outcome were identified using the Random Forest algorithm. Main end points were overall survival (OS) and non-relapse mortality (NRM). The total cohort consisted of 185 patients of whom seven had CMML-0, 100 CMML-1, and 78 CMML-2 at time of transplant. The median follow-up was 74 months and 6-year OS was 37% for the total cohort and differed for CMML-0 (57%), CMML-1 (43%), and CMML-2 (29%). Relapse and NRM were 27% and 44% for the total cohort being 17% and 31% for CMML-0, 23% and 40% for CMML-1, and 34% and 40% for CMML-2. Most frequently mutated genes were: TET2 (55%), ASXL1 (41%), SF3B1 (38%), DNMT3A (27%), ZRSR2 (22%), NRAS (21%), EZH2 (21%), RUNX1 (17%), and SETBP1 (17%). Ninety-two percent of patients showed at least one somatic mutation. More than three mutations were present in 49% of all patients and in 29% of CMML-0, 50% of CMML-1, and 49% of CMML-2 patients. Frequencies according to CMML-specific prognostic scoring system (CPSS) and its molecular refinement (CPSS-mol) were 8% and 6% (low risk), 31% and 18% (intermediate-1 risk), 43% and 40% (intermediate-2 risk), and 18% and 36% (high risk). Transplants were received from matched unrelated (51%), mismatched unrelated (25%), matched related (21%), or mismatched related donors (3%). Conditioning intensity was reduced (49%), myeloablative (43%), or non-myeloablative (8%). Median age of patients was 60 years, 29% were female, 30% had a Karnofsky performance status <90%, and 15% had a comorbidity index >3. In the first step of the OS analysis, the algorithm identified mutations in ASXL1, KRAS, SF3B1, ZRSR2 as high-risk mutations (HRM) predicting worse OS. In addition, the number of the HRMs was associated with worse OS. In the next step, the algorithm automatically stratified this information into three distinct risk groups: the absence of HRMs (reference; low risk), presence of 1-2 HRMs (HR, 1.81; intermediate-risk), and 3-4 HRMs (HR, 2.93; high-risk). Corresponding 6-year OS was 59% for the low-risk, 34% for the intermediate-risk, and 14% for the high-risk group (P<.001; Figure 1A). Furthermore, the absence of HRMs was associated with lower NRM (15%) compared with present HRMs (46%; P=.01). In contrast, the CPSS-mol genetic risk classification including ASXL1, RUNX1, NRAS, and SETBP1 mutations showed no distinct 6-year OS or NRM (P=.15, respectively). Next, we adjusted the impact on OS of the proposed genetic risk for other factors included in the CPSS-mol. Higher genetic risk was independently associated with increased hazard for death (with the low-risk group as reference) showing HRs of 1.70 for the intermediate-risk and 2.83 for the high-risk group (P<.001). This model showed a concordance index of 0.633 versus CPSS-mol (0.597) or the CPSS (0.572) suggesting utility of transplant-specific prognostication. Therefore, we evaluated the multivariable effect on posttransplant outcome including the following independent clinical and molecular predictors: genetic risk, % of peripheral and bone marrow blasts, leukocyte count, and performance status. This model was predictive of OS and NRM (P<.001, respectively), and showed increased prognostic precision for OS, reflected in a concordance index of 0.684. In conclusion, mutations in ASXL1, KRAS, SF3B1, ZRSR2, and the number of these mutations predict OS and NRM in CMML undergoing transplantation. Accounting for these genetic lesions may improve the prognostic precision and patient counseling in the transplant setting. Figure 1 Disclosures Bogdanov: Jazz Pharmaceuticals, MSD.: Other: Travel subsidies. Stoelzel:Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy; Shire: Consultancy, Other: Travel funding. Rautenberg:Jazz Pharmaceuticals: Other: Travel Support; Celgene: Honoraria, Other: Travel Support. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Finke:Riemser: Honoraria, Other: research support, Speakers Bureau; Neovii: Honoraria, Other: research support, Speakers Bureau; Medac: Honoraria, Other: research support, Speakers Bureau. Kobbe:Pfizer: Honoraria, Other: Travel support; Takeda: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support, Research Funding; Jazz: Honoraria, Other: Travel support; Amgen: Honoraria, Other: Travel support, Research Funding; Biotest: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support; Neovii: Honoraria, Other: Travel support; Abbvie: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Medac: Honoraria, Other: Travel support. Platzbecker:Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Robin:Novartis Neovii: Research Funding. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Kroeger:JAZZ: Honoraria; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Sanofi-Aventis: Honoraria; Novartis: Honoraria, Research Funding; Medac: Honoraria; DKMS: Research Funding; Riemser: Research Funding.

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