Supplementary Table 2. Characteristics of the AML patients according to FLT3 and NPM1 gene mutational status.

Supplementary Figure 3. MPFC and molecular analysis of UPN 241 bone marrow samples at diagnosis, during follow-up and at relapse.

Supplementary Figure 2. MPFC detection limit.

Supplementary Table 5. Longitudinal analysis of FLT3-ITDs using patient-specific RQPCR in four AML patients.

Supplementary Figure 1. ROC curve analysis using CD34CD123CD25+ve immunophenotype.

Supplementary Table 1. anti-human antibodies used.

Abstract Purpose: We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in acute myeloid leukemia (AML). Experimental Design: Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Results: Within the CD34+ cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics showed that, within the CD34+ cell fraction a percentage of CD123/CD99/CD25+ cells ≥11.7% predicted FLT3–ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25+ clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1+ AML who relapsed with FLT3-ITD/NPM1+ AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. Conclusions: Our results suggest that the CD34/CD25/CD123/CD99+ LAIP is strictly associated with FLT3-ITD–positive cells. Clin Cancer Res; 21(17); 3977–85. ©2015 AACR.

Abstract Background The ZBTB16‐RARA fusion gene, resulting from the reciprocal translocation between ZBTB16 on chromosome 11 and RARA genes on chromosome 17 [t(11;17)(q23;q21)], is rarely observed in acute myeloid leukemia (AML), and accounts for about 1% of retinoic acid receptor‐α (RARA) rearrangements. AML with this rare translocation shows unusual bone marrow (BM) morphology, with intermediate aspects between acute promyelocytic leukemia (APL) and AML with maturation. Patients may have a high incidence of disseminated intravascular coagulation at diagnosis, are poorly responsive to all‐trans retinoic acid (ATRA) and arsenic tryoxyde, and are reported to have an overall poor prognosis. Aims The mutational profile of ZBTB16‐RARA rearranged AML has not been described so far. Materials and methods We performed targeted next‐generation sequencing of 24 myeloid genes in BM diagnostic samples from seven ZBTB16‐RARA+AML, 103 non‐RARA rearranged AML, and 46 APL. The seven ZBTB16‐RARA‐positive patients were then screened for additional mutations using whole exome sequencing (n = 3) or an extended cancer panel including 409 genes (n = 4). Results ZBTB16‐RARA+AML showed an intermediate number of mutations per patient and involvement of different genes, as compared to APL and other AMLs. In particular, we found a high incidence of ARID1A mutations in ZBTB16‐RARA+AML (five of seven cases, 71%). Mutations in ARID2 and SMARCA4, other tumor suppressor genes also belonging to SWI/SNF chromatin remodeling complexes, were also identified in one case (14%). Discussion and conclusion Our data suggest the association of mutations of the ARID1A gene and of the other members of the SWI/SNF chromatin remodeling complexes with ZBTB16‐RARA+AMLs, where they may support the peculiar disease phenotype.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10 −8 ; KMT5B ). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10 −10 ; HLA ). Our results inform on AML etiology and identify putative functional genes operating in histone methylation ( KMT5B ) and immune function ( HLA ).

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17–11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

NRF2 (NF-E2 p45-related factor 2) orchestrates cellular adaptive responses to stress. Its quantity and subcellular location is controlled through a complex network and its activity increases during redox perturbation, inflammation, growth factor stimulation, and energy fluxes. Even before all-trans retinoic acid (ATRA) treatment era it was a common experience that acute promyelocytic leukemia (APL) cells are highly sensitive to first line chemotherapy. Since we demonstrated how high doses of ascorbate (ASC) preferentially kill leukemic blast cells from APL patients, we aimed to define the underlying mechanism and found that promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NRF2 function, impedes its transfer to the nucleus and enhances its degradation in the cytoplasm. Such loss of NRF2 function alters cell metabolism, demarcating APL tissue from both normal promyelocytes and other acute myeloide leukemia (AML) blast cells. Resistance to ATRA/arsenic trioxide (ATO) treatment is rare but grave and the metabolically-oriented treatment with high doses of ASC, which is highly effective on APL cells and harmless on normal hematopoietic stem cells (HSCs), could be of use in preventing clonal evolution and in rescuing APL-resistant patients.

Abstract Introduction: Internal tandem duplication of the FLT3 gene (FLT3-ITD), resulting in duplication of 3 to more than hundreds of nucleotides, are present in approximately 25% of adults with newly diagnosed AML. Several studies have shown that ITD mutations are associated with poor prognosis due to a high relapse rate, in particular in case of a high mutant to wild-type allele ratio and/or insertion site in the beta1-sheet of the tyrosine kinase domain-1 (beta1-sheet). Aims: To investigate the relationship between ITD insertion site and patient outcome, Roche 454 next generation sequencing (NGS) was performed in 452/555 (81.4%) FLT3-ITD positive patients (pts) enrolled into the RATIFY trial (NCT00651261). Results: NGS identified 908 ITDs with up to 9 ITDs per case (1 ITD: n=210, 46.5%; 2 ITDs: n=131, 29.0%; 3 ITDs: n=58, 12.8%; 4 ITDs: n=24, 5.3%; 5 ITDs: n=18, 4.0%; 6 ITDs: n=3, 0.7%; 7 ITDs: n=7, 1.5%; 9 ITDs: n=1, 0.2%). Median ITD-size was 45 nucleotides (range, 6-246); all ITDs were in-frame with direct head-to-tail orientation. According to the 4 functional groups, 488 ITDs (53.7%) were located within the juxtamembrane domain (JMD), 155 ITDs (17.1%) within the hinge region, 211 ITDs (23.2%) within the beta1-sheet, and 54 ITDs (5.9%) 3´of beta1-sheet. ITD size strongly correlated with insertion site, in that the more C-terminal the insertion site, the longer the size of the inserted fragment (P NPM1 mutations (NPM1mut) were present in 203/358 pts (56.7%). Correlation of ITD insertion site with NPM1mut revealed a significantly lower incidence of NPM1mut in pts with insertion located within the hinge region (50/106, 47.2% vs 153/252, 60.7%; P=.02) and 3´of beta1-sheet (14/41, 34.1% vs 189/317, 59.6%; P=.002), whereas NPM1mut were significantly more frequent in pts with insertions affecting JMD (143/235, 60.9% vs 60/123, 48.8%; P=.03). Clinical characteristics differing among the 4 functional ITD groups were gender and WBC. Pts with insertions 3´of beta1-sheet were predominantly male (28/46, 60.9% vs 178/406, 43.8%; P=.03); pts with JMD insertions exhibited lower WBC (median 36.5 vs 52.7 x109/L; P=.03). Complete remission (CR) was achieved within 60 days in 248/452 pts (54.9%). To evaluate the impact of ITD insertion site on response to induction, a logistic regression model was used. ITD insertion sites were categorized in (i) only in beta1-sheet, (ii) in beta1-sheet and other sites, and (iii) outside the beta1-sheet. Other variables were ITD mutant to wild-type allelic ratio (fragment analysis, cutoff at 0.5), number of ITDs per patient, log2 of WBC counts, age, NPM1mut, and midostaurin treatment. In this model, only number of ITDs predicted lower CR rate (OR, 0.72; 95% CI, 0.57-0.90), while NPM1mut was a favorable marker for CR (OR, 2.69; 95% CI, 1.70-4.28). Median follow-up for survival was 60.6 months (mo); median event free survival (EFS) and overall survival (OS) were 3.9 mo and 24.4 mo, respectively. The 4-year EFS and OS rates were 21.0% (95% CI, 17.2%-24.8%) and 42.6% (95% CI, 37.9%-47.4%), respectively. Survival analysis according to categorized insertion site groups showed that pts exhibiting insertion exclusively in the beta1-sheet had significantly inferior OS (P=.014) compared to the other two groups. Multivariate models for OS and EFS including hematopoietic stem-cell transplantation (HSCT) as a time-dependent covariate revealed WBC counts as unfavorable and NPM1mut as favorable for both endpoints; further unfavorable factors were older age and exclusive insertion in the beta1-sheet (HR, 1.49; 95% CI, 1.01-2.20) for OS and number of ITDs (HR, 1.15; 95% CI, 1.04-1.28) for EFS; HSCT was a favorable factor only for EFS (HR, 0.66; 95% CI, 0.44-0.99). Midostaurin treatment was associated with in trend improved EFS (HR, 0.81; 95% CI, 0.63-1.03) and OS (HR, 0.77; 95% CI, 0.57-1.02). Conclusions: In this large cohort of 452 FLT3-ITD mutated AML treated within the RATIFY trial the negative prognostic impact of beta1-sheet insertion site of FLT3-ITD could be confirmed. Further analyses to investigate potential predictive effects of midostaurin treatment are ongoing. Support: U10CA180821, U10CA180882, U24CA196171, (CCSRI) #704970. Disclosures Du: Novartis: Employment. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Tallman:Orsenix: Other: Advisory board; Cellerant: Research Funding; AROG: Research Funding; AbbVie: Research Funding; BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. de Witte:Amgen: Consultancy, Research Funding; Novartis: Research Funding; Celgene: Honoraria, Research Funding. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau. Döhner:Pfizer: Research Funding; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership.

Relapses are generally thought to derive from the persistence of a few leukemic cells at the end of the therapeutic treatment. In fact, it is generally assumed that if even a few leukemic cells are able to survive, they subsequently may grow out and appear again above the detection level. This phenomenon is generally referred to as "minimal residual disease" (MRD). A preliminary retrospective multicentric study conducted in Italy analyzed the presence of MRD at variable intervals in patients treated with different modalities, including all-trans-retinoic acid, chemotherapy and/or bone marrow transplantation. This study and successive reports indicated that PCR positivity detected after consolidation strongly predicts clinical relapse, while patients whose test is repeatedly negative are likely to remain disease free. The efforts aimed at developing more sensitive and specific methods to detect MRD in leukemia will certainly further increase in the next future.

The results from the RATIFY trial (ClinicalTrials.gov: NCT00651261; CALGB 10603) showed that midostaurin combined with standard chemotherapy significantly improved outcomes in patients with FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML), compared with placebo. In this post hoc subgroup analysis from the trial, we evaluated the impact of midostaurin in 163 patients with FLT3-tyrosine kinase domain (TKD) mutations. At a median follow-up of 60.7 months (95% CI, 55.0-70.8), the 5-year event-free survival (EFS) rate was significantly higher in patients treated with midostaurin than in those treated with placebo (45.2% vs 30.1%; P = .044). A trend toward improved disease-free survival was also observed with midostaurin (67.3% vs 53.4%; P = .089), whereas overall survival (OS) was similar in the 2 groups. Patients with AML and NPM1mut/FLT3-TKDmut or core binding factor (CBF)-rearranged/FLT3-TKDmut genotypes had significantly prolonged OS with or without censoring at hematopoietic cell transplantation (HCT), compared with NPM1WT/CBF-negative AMLs. The multivariable model for OS and EFS adjusted for allogeneic HCT in first complete remission as a time-dependent covariable, revealed NPM1 mutations and CBF rearrangements as significant favorable factors. These data show that NPM1 mutations or CBF rearrangements identify favorable prognostic groups in patients with FLT3-TKD AMLs, independent of other factors, also in the context of midostaurin treatment.

Acute myeloid leukemia (AML) with FLT3 -ITD mutations ( FLT3 -ITD mut ) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3 -ITD-positivity. The aim of this study was to characterize the distribution of FLT3 -ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3 -ITD mut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3 -ITD mut AML ( n = 12). A higher FLT3 -ITD mut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) ( p = 0.0005) and mononuclear cells (MNCs) ( p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3 -ITD mut burden was also observed in LPCs of AML patients with a small FLT3 -ITD mut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3- ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3 -ITD mut AML.

Background: Patients (pts) with AML harboring FLT3 internal tandem duplications (ITD) have poor outcomes, in particular pts with a high ITD mutant to wild-type (wt) allelic ratio (AR; ≥0.5). The 2017 update of the European LeukemiaNet (ELN) recommendations addressed the importance of the ITD AR in the genetic risk stratification and defined distinct NPM1/FLT3-ITD genotypes. Aims: In this exploratory post-hoc analysis from randomized pts treated within the RATIFY trial evaluating the addition of midostaurin to standard chemotherapy, we investigated the prognostic impact of the NPM1/FLT3-ITD genotypes according to the 2017 ELN risk categories. Methods: In total, 319 of 717 pts with FLT3-ITD positive AML were included who gave informed consent for biomarker analyses and who could be categorized according to 2017 ELN genetic risk stratification. Median age was 47.9 years (range, 18–60); median follow-up time was 4.8 years. Results: Pts were categorized as follows: (i) favorable-risk (n = 85), NPM1mut/FLT3-ITDlow, irrespective of secondary chromosome abnormalities and concurrent gene mutations; (ii) intermediate-risk (n = 111), NPM1mut/FLT3-ITDhigh and NPM1wt/FLT3-ITDlow, both subgroups without adverse cytogenetics and without concurrent RUNX1, ASXL1, TP53 mutations; (iii) adverse-risk (n = 123), NPM1wt/FLT3-ITDhigh; as well as NPM1mut/FLT3-ITDhigh and NPM1wt/FLT3-ITDlow with adverse-risk cytogenetic and/or molecular markers; frequencies of RUNX1, ASXL1, and TP53 mutations in adverse-risk AML were 29.2%, 19.8%, and 1.9%, respectively. Rates of allogeneic transplantation (alloSCT) in first complete remission (CR) were not different among the groups, with 29.4%, 35.1%, and 35.0% in pts. with favorable-, intermediate-, and adverse-risk AML, respectively. Rates of CR (including responses after induction 1 and 2) were negatively associated with adverse-risk genetics (51.2% vs 69.4% and 64.0% in favorable- and intermediate-risk, respectively; p = 0.02); multivariable logistic regression analysis revealed adverse-risk as significantly different compared to favorable-risk (odds ratio [OR] 0.534, 95% confidence interval [CI] 0.287–0.979; p = 0.044), whereas no difference was seen between the two other risk groups; treatment with midostaurin had no impact (p = 0.32). Overall survival (OS) differed among the 3 ELN-risk groups; 5-year survival rates were 63%, 43%, 33% for favorable, intermediate- and adverse-risk groups, respectively. The OS curves did not show differential effects of midostaurin among the 3 risk groups (Figure 1), with a benefical effect seen for midostaurin vs placebo in all 3 groups with hazard ratios (HR) estimated as HR = 0.52, HR = 0.51, and HR = 0.55 for favorable-, intermediate-, and adverse-risk groups, respectively. A multivariable Cox model adjusted for age, WBC, sex, and bone marrow blasts was used to test the effects of treatment and risk classification. The resulting model revealed an increasing HR with increasing risk category (p < 0.001; intermediate vs. favorable, HR = 1.70, 95% CI 1.08–2.68; adverse vs. favorable risk, HR = 2.48, 95% CI 1.59–3.86), while treatment with midostaurin significantly reduced risk compared to placebo (p < 0.001; HR = 0.53, 95% CI 0.38–0.72).Summary/Conclusion: Data from this post-hoc exploratory analysis confirm the prognostic value of the 2017 ELN risk categories also among AML pts with the distinct NPM1/FLT3-ITD genotypes. A Cox model revealed a significant effect for midostaurin compared to placebo independent of the ELN risk groups. Analysis with regard to potential confounding effects of allogenic transplantation are ongoing.

Despite the high probability of cure of patients with acute promyelocytic leukemia (APL), mechanisms of relapse are still largely unclear. Mutational profiling at diagnosis and/or relapse may help to identify APL patients needing frequent molecular monitoring and early treatment intervention. Using an NGS approach including a 31 myeloid gene-panel, we tested BM samples of 44 APLs at the time of diagnosis, and of 31 at relapse. Mutations in PML and RARA genes were studied using a customized-NGS-RNA panel. Patients relapsing after ATRA-chemotherapy rarely had additional mutations (P = .009). In patients relapsing after ATRA/ATO, the PML gene was a preferential mutation target. We then evaluated the predictive value of mutations at APL diagnosis. A median of two mutations was detectable in 9/11 patients who later relapsed, vs one mutation in 21/33 patients who remained in CCR (P = .0032). This corresponded to a significantly lower risk of relapse in patients with one or less mutations (HR 0.046; 95% CI 0.011-0.197; P < .0001). NGS-analysis at the time of APL diagnosis may inform treatment decisions, including alternative treatments for cases with an unfavorable mutation profile.

Arsenic trioxide (ATO) and all-trans retinoic acid have become the front-line treatments for patients with acute promyelocytic leukemia (APL). Despite the long wait for an oral arsenic drug, a commercially available agent, "Realgar-Indigo naturalis formula (RIF)", was not launched in China until 2009. Since then, over 5000 patients with APL have been treated with oral RIF in China. Oral arsenic not only shows a clinical efficacy comparable to that of IV formulations but also displays a better safety profile, improved quality of life and lower medical costs for patients. The promising results promote incorporating an outpatient postremission therapy model into clinical practice for both low-risk and high-risk APL patients in China. In this review, we discuss the evolution of oral arsenic RIF in the treatment of APL, with a special focus on how to address the related complications during induction therapy.