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Tandem duplications (TDs) of the UBTF gene have been recently described as a recurrent alteration in pediatric acute myeloid leukemia (AML). Here, by screening 1946 newly diagnosed adult AML, we found that UBTF-TDs occur in about 3% of patients aged 18–60 years, in a mutually exclusive pattern with other known AML subtype-defining alterations. The characteristics of 59 adults with UBTF-TD AML included young age (median 37 years), low bone marrow (BM) blast infiltration (median 25%), and high rates of WT1 mutations (61%), FLT3-ITDs (51%) and trisomy 8 (29%). BM morphology frequently demonstrates dysmyelopoiesis albeit modulated by the co-occurrence of FLT3-ITD. UBTF-TD patients have lower complete remission (CR) rates (57% after 1 course and 76% after 2 courses of intensive chemotherapy [ICT]) than UBTF-wild-type patients. In patients enrolled in the ALFA-0702 study (n = 614 patients including 21 with UBTF-TD AML), the 3-year disease-free survival (DFS) and overall survival of UBTF-TD patients were 42.9% (95%CI: 23.4–78.5%) and 57.1% (95%CI: 39.5–82.8%) and did not significantly differ from those of ELN 2022 intermediate/adverse risk patients. Finally, the study of paired diagnosis and relapsed/refractory AML samples suggests that WT1-mutated clones are frequently selected under ICT. This study supports the recognition of UBTF-TD AML as a new AML entity in adults.
Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.
Context. Functional precision medicine is gaining momentum in AML, notably through ex vivo drug sensitivity screening (DSS) of primary patient (pt) cells (Pemovska Cancer Discov 2013, Tyner Nature 2018). The DSS landscape differs across genetic AML subgroups (Tyner Nature 2016), of which NPM1mut is the most frequent (Papaemmanuil NEJM 2016). DSS in AML has mostly been done in standard conditions, with overall viability as unique endpoint. Niche signals, which can be partly mimicked in vitro, convey drug resistance in vivo. Drugs can induce a variety of cell fates in AML. Induction of differentiation rather than killing of blasts, can result in false negative results in global viability assays. Persistence of leukemic stem cells (LSC) represents a major cause of treatment failure. GPR56 is a ubiquitous surface marker enriching for LSCs and stable upon short-term ex vivo culture (Pabst Blood 2016). Objectives. To develop an ex vivo niche-like multiparametric DSS platform for primary AML cells. To validate its clinical relevance in NPM1mut pts treated with conventional DNR-AraC chemotherapy. To discover new sensitizers to DNR-AraC chemotherapy in NPM1mut AML. Results. We designed an MFC panel to count viable blasts and measure their differentiation (CD11b/CD14/CD15) and stemness (GPR56) after exclusion of residual lymphocytes (Figure 1A). We validated GPR56 expression as stemness marker based on increased retention of GPR56+ cells in niche-like coculture combining hypoxia (O2 3%) and MSC compared to standard conditions (p<0.0001, Figure 1B) and limit dilution assays of residual GPR56+ cells at 72h of niche-like culture in 3 NPM1mut AMLs (Figure 1C). Using a limited panel of 14 drugs or combinations at fixed concentrations, our MFC readout after 72h of coculture with MSC+hypoxia revealed the distinct mode of action of different agents or combinations including the differentiation activity of ATO-ATRA, the LSC-sparring cytotoxicity of DNR-AraC and the anti-LSC- activity of VEN (Figure 1D). To further mimic in vivo conditions, we derived a MEMa-based plasma-like medium (PLM) based on targeted metabolomics (Figure 1E) and electro-chemoluminescent cytokine assays of 29 diagnostic AML bone marrow plasma samples compared to conditioned media of primary AML cells cultured in niche-like conditions (MSC, hypoxia). This instructed the design of our custom PLM with dialyzed FBS and defined low-dose (~1 ng/mL range) cytokines (CK) and amino-acid (AA) concentrations. We next investigated the contribution of MSCs, hypoxia, plasma-like AAs and CKs on blasts viability, differentiation, stemness and drug response in 3 NPM1mut AMLs exposed to fixed concentrations of 6 core AML therapies. This analysis uncovered significant interactions between these 4 niche components in dictating blast viability and stemness upon 72h ex vivo culture (Figure 1F) and revealed the distinct contribution of these niche components to drug sensitivity. RNA-seq of primary blasts cultured in niche-like, plasma-like conditions revealed marked enrichment of stemness pathways compared to ex vivo culture in standard conditions. Finally, we explored DNR-AraC (five-point serial dilution) alone or in combination with fixed, clinically relevant concentrations of 24 drugs in 49 primary AML samples (including 34 NPM1mut). Using AUCs of DNR-AraC on lymphocytes as internal control, we first validated our NEXT assay on NPM1 MRD levels in the 34 NPM1mut pts treated frontline with conventional DNR-AraC regimens (Figure 1G). Across all 49 pts, we uncovered 11 different optimal 'third-drugs', stressing the role of our NEXT assay to deploy precision medicine in daily practice. At the population level, we could nominate 3 top combinations, two of which are currently in clinical investigation (Venetoclax and Selinexor). The unpublished sensitizing effect of low dose (0.25µM) Ruxolitinib on DNR-AraC uncovered with our NEXT assay is currently being investigated in PDX models. Conclusion. We designed the NEXT assay, a multiparametric drug screening of AML viability, differentiation and stemness in niche-like culture combining hypoxia, stromal interactions and plasma-like medium. Components of the niche-like culture interact to govern leukemic viability and stemness. Our assay could predict MRD achievement in NPM1mut AML and identifies novel sensitizers to DNR-AraC in these pts. Disclosures Clappier: Amgen: Honoraria, Research Funding. Ades:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; novartis: Research Funding; Celgene/BMS: Research Funding. Itzykson:Amgen: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Stemline: Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Janssen: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS (Celgene): Honoraria; Sanofi: Honoraria; Astellas: Honoraria.
Context . AML patients (pts) with a high number of drivers have a poor prognosis (Papaemmanuil, NEJM 2016). Whether this unfavorable outcome is caused by clonal heterogeneity or by a high mutational load in the dominant clone remains undetermined. So far, the prognostic impact of clonal heterogeneity in AML has only been studied in pts with complex karyotypes, where it worsens prognosis (Bochtler, JCO 2013). We addressed this question in a two-center cohort of AML pts treated with IC. Methods . We retrospectively evaluated AML pts treated with IC from 2 centers with targeted sequencing of a 43-gene myeloid panel (mean depth 1193X). Variant allele frequencies were adjusted for copy number variation to estimate Cancer Cell Fractions (CCFs). Clone size for each mutation was derived from differences in CCF, assuming a linear accumulation of mutations. Relative clone sizes were then used to derive the Shannon Index (SI), a standard metric of genetic diversity (Maley, Nat Rev Cancer 2017, Figure A). Pts with no gene mutation were not evaluable for SI. Pts with a single mutation have a SI of 0, with increasing SI corresponding to greater dispersal of clone sizes, and thus indirectly of CCFs. Results . We included 292 pts (median age 57y, 37% ELN-2017 adverse risk), 269 (92.1%) with ≥ 2 mutations. Median number of drivers, including both mutations and cytogenetic alterations (the latter defined as in [Papaemmanuil, NEJM 2016]) was 4. SI increased with the number of drivers (Figure B, p<10-4). In bivariate analyses adjusted on the number of drivers, SI was higher in adverse risk (p=0.0007) but was not associated with age or type of AML (de novo vs secondary). Median follow-up and EFS were 26.9 and 18.9 months, respectively. In univariate analyses, an increasing number of driver lesions significantly worsened EFS (HR=1.11, p=0.011), while SI as a continuous variable had no impact (HR=0.89, p=0.3). In a multivariate Cox model, the number of drivers (HR=1.74, p=0.002) and SI (HR=0.58, p=0.001), both as continuous variables, had significant but opposite prognostic value independently of age >60y (HR=1.48, p=0.022), WBC > 50x109/L (HR=1.74, p=0.005), and ELN 2017 adverse-risk AML (HR=1.74, p=0.001). There was no significant statistical interaction between number of drivers and SI. OS was also independently poorer in patients with higher number of drivers (HR=1.19, p=0.021) and with lower SI (HR=0.65, p=0.03) in a similar Cox model. Because of the positive correlation between the number of drivers and SI, but inverse prognostic role, we defined pts with high clonal diversity (132 [49.0%] of the 269 pts with ≥2 driver lesions) as those with a SI higher than the median value of SI of all AMLs with the same number of drivers (Figure B). In those 269 pts, median EFS was 11.3 months in pts (n=73) with ≥5 drivers vs 22.2 months in those with <5 drivers (n=196, p=0.04). EFS was significantly shorter in pts with low clonal diversity (median 10.1 vs. 33.4 months in pts with high diversity, p=0.002). Combining these criteria defined 4 groups with significantly different outcome (log-rank p=0.004, Figure C). We then compared our estimation of SI with a 'true' SI accounting for further clonal variegation caused by branching and/or LOH in 29 pts with resolved clonal architecture (Hirsch, Nat Comm 2016). Estimated and 'true' SI were highly correlated (Pearson's correlation 0.73, p<10-5), showing that our estimated SI captures most of the genetic heterogeneity of AML. We finally performed a validation analysis in a publicly available cohort of 1540 AML pts (Papaemmanuil, NEJM 2016). Median OS of pts with low clonal diversity was 20.7 months vs 33.7 months in those with high clonal diversity (p=0.004). In a multivariate Cox model of OS accounting for age >60y, type of AML (de novo vs else) and ELN 2010 risk (FLT3 allele ratio not available for stratification per ELN 2017), the number of drivers (HR=1.17, p<10-5) and SI (HR=0.82, p=0.012), as continuous variables, significantly affected OS in opposite ways. Conclusion . Our results confirm that a higher number of drivers worsens AML prognosis, but also demonstrate that conversely, higher clonal diversity, estimated with Shannon's Index, confers a better outcome. These findings are consistent with a simple model whereby both a higher driver load and a higher expansion of the most mutated clone independently worsen prognosis in AML. Measuring both clonal diversity and dynamics could improve AML risk stratification. Figure Disclosures Thomas: ABBVIE: Honoraria; INCYTE: Honoraria; PFIZER: Honoraria; DAICHI: Honoraria. Ades:Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Fenaux:Aprea: Research Funding; Jazz: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Boissel:NOVARTIS: Consultancy. Dombret:CELGENE: Consultancy, Honoraria; AGIOS: Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding.
Whether the LSC17 gene expression can improve risk stratification in the context of NGS-based risk stratification and measurable residual disease (MRD) in AML patients treated intensively has not been explored. We analyzed LSC17 in 504 adult patients prospectively treated in the ALFA-0702 trial. Multiple (cyto)genetic alterations were associated with changes in LSC17, such as higher LSC17 in patients with RUNX1 or TP53 mutations, and lower scores in those with CEBPA and NPM1 mutations. LSC17-high patients had a lower rate of complete response (CR) or CR with incomplete platelet recovery (CRp) after one induction course in a multivariable analysis (OR=0.41, p=0.0007) accounting for European LeukemiaNet 2022 (ELN22) risk groups, age, and white blood cell (WBC) count. The LSC17-high status was associated with shorter overall survival (OS) (3-year OS: 70.0% versus 52.7% in LSC17-low patients, p<0.0001). In a multivariable analysis considering ELN22, age and WBC count, LSC17-high patients had shorter disease-free survival (DFS) (HR=1.36, p=0.048) compared to LSC17-low patients. In 123 NPM1-mutated patients in CR/CRp with available MRD data, LSC17-high status predicted poorer DFS (HR=2.34, p=0.01) independently of age, WBC count, ELN22 risk, and NPM1-MRD. Combining MRD and LSC17 status identified a subset of 48% of NPM1 patients with LSC17-low status and negative NPM1-MRD with a 3-year OS from CR/CRp of 93.1% compared to 60.7% in those with LSC17-high status and/or positive NPM1-MRD (p=0.0001). Overall, LSC17 assessment refines genetic risk stratification in adult AML patients treated intensively. Combined with MRD, LSC17 identifies a subset of NPM1-mutated AML patients with excellent clinical outcome.
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