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Background: Older patients (≥ 60 years) with acute myeloid leukemia (AML) often have multiple, sequentially acquired, somatic mutations that drive leukemogenesis and are associated with poor outcome. Beat AML is a Leukemia and Lymphoma Society-sponsored, multicenter umbrella study that algorithmically segregates AML patients based upon cytogenetic and dominant molecular abnormalities (variant allele frequencies (VAF) ≥ 0.2) into different cohorts to select for targeted therapies. During the conception of the Beat AML design, a historical dataset was needed to help in the design of the genomic algorithm for patient assignment and serve as the basis for the statistical design of individual genomic treatment substudies for the Beat AML study., Methods: We classified 563 newly diagnosed older AML patients treated with standard intensive chemotherapy on trials conducted by Cancer and Leukemia Group B based on the same genomic algorithm and assessed clinical outcomes., Results: Our classification identified core-binding factor and NPM1-mutated/FLT3-ITD-negative groups as having the best outcomes, with 30-day early death (ED) rates of 0 and 20%, respectively, and median overall survival (OS) of > 1 year and 3-year OS rates of ≥ 20%. All other genomic groups had ED rates of 17-42%, median OS ≤ 1 year and 3-year OS rates of ≤ 15%., Conclusions: By classifying patients through this genomic algorithm, outcomes were poor and not unexpected from a non-algorithmic, non-dominant VAF approach. The exception is 30-day ED rate typically is not available for intensive induction for individual genomic groups and therefore difficult to compare outcomes with targeted therapeutics. This Alliance data supported the use of this algorithm for patient assignment at the initiation of the Beat AML study. This outcome data was also used for statistical design for Beat AML substudies for individual genomic groups to determine goals for improvement from intensive induction and hopefully lead to more rapid approval of new therapies. Trial registration ClinicalTrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00900224 (CALGB 20202), NCT00003190 (CALGB 9720), NCT00085124 (CALGB 10201), NCT00742625 (CALGB 10502), NCT01420926 (CALGB 11002), NCT00039377 (CALGB 10801), and NCT01253070 (CALGB 11001).

The European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML) have become an important tool to assess patients' prognosis and guide treatment. We tested the prognostic impact of the 2017 ELN classification in a large cohort of 863 AML patients aged <60 years similarly treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. Based on multivariable models within each ELN genetic-risk group, we identified additional gene mutations that may refine the 2017 ELN risk classification. BCOR- or SETBP1-mutated favorable-risk patients with non-core-binding factor AML and IDH-mutated adverse-risk patients had intermediate-risk outcomes. Outcomes of NPM1/WT1 co-mutated patients and those of ZRSR2-mutated patients resembled outcome of adverse-risk patients. Moreover, FLT3-ITD high allelic ratio conferred adverse rather than intermediate-risk irrespective of the NPM1 mutation status, and DNMT3A mutations associated with very poor survival. Application of these refinements reclassified 9% of current favorable-risk patients and 53% of current intermediate-risk patients to the adverse-risk group, with similar poor survival as current adverse-risk patients. Furthermore, 4% of current favorable-risk patients and 9% of adverse-risk patients were reclassified to the intermediate-risk group.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

Complex karyotype (CK) with ≥ 3 abnormalities is detected in 10-12% of patients with acute myeloid leukemia (AML) and associated with poor prognosis. The most common unbalanced abnormalities found in CK result in loss of material from the 5q, 7q, and/or 17p chromosome arms. The presence of 5q, 7q, and/or 17p abnormalities denotes typical CK and their absence denotes atypical CK. Since molecular features of CK-AML are not well characterized, we investigated mutational status of 81 leukemia/cancer-associated genes in 160 clinically well-characterized patients. They included 136 patients with ≥ 3 exclusively unbalanced chromosome abnormalities, 96 of whom had a typical CK and 40 atypical CK, and 24 patients with ≥ 1 balanced abnormality in addition to ≥ 2 unbalanced ones. Patients with atypical CK-AML differed from those with typical CK-AML: they carried TP53 mutations less often (P < 0.001) and more often PHF6 (P = 0.008), FLT3-TKD (P = 0.02), MED12 (P = 0.02), and NPM1 (P = 0.02) mutations. They were younger (P = 0.007), had higher WBC (P = 0.001) and percentages of marrow (P < 0.001) and blood (P = 0.006) blasts, higher complete remission rates (P = 0.02), and longer overall survival (P < 0.001), thus indicating that atypical and typical CK-AMLs constitute distinct disease subtypes. We also identified smaller patient subsets within both typical and atypical CK-AML that differed molecularly and clinically.

Targeted mutation assessment of 81 genes in 1021 adults with de novo acute myeloid leukemia (AML) identified recurrent mutations in the neurofibromin 1 (NF1) gene in 52 (5.1%) patients, including 36 (5.2%) younger and 16 (4.8%) older patients, which suggests NF1 belongs to the 20 most frequently mutated genes in adult AML. NF1 mutations were found throughout the gene, and comprised missense, frameshift, and nonsense mutations. One mutation hotspot, at amino acid threonine 676 (Thr676), was found in 27% of AML patients with NF1 mutations. NF1-mutated patients belonged more often to the adverse European LeukemiaNet (ELN) risk category than NF1 wild-type patients. Among patients aged <60 years, the presence of NF1 Thr676 mutations was associated with lower complete remission (CR) rates (P = 0.04) and shorter overall survival (OS; P = 0.01), as was the presence of any NF1 mutation in patients in the adverse ELN risk category (CR, P = 0.05; OS, P < 0.001). CR rates were also lower in NF1-mutated patients aged ≥60 years compared with NF1 wild-type patients (P = 0.001). In summary, our findings provide novel insights into the frequency of NF1 mutations in AML, and are suggestive of an adverse prognostic impact in patients treated with standard chemotherapy.

Thus far, only 5-15% of AML patients aged ≥60 years are cured with chemotherapy. Identification of patients who are less (more) likely to respond to standard chemotherapy might enable early risk stratification toward alternative treatment regimens. We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 older patients with de novo AML. Using variables identified in multivariable models and co-occurring mutations in NPM1-mutated AML, we classified the patients into good-, intermediate-, and poor-risk groups for complete remission (CR) attainment, disease-free (DFS), and overall survival (OS). Whereas 81% of good-risk patients (comprising NPM1-mutated patients harboring mutations in chromatin remodeling, cohesin complex, methylation-related, spliceosome, and/or RAS pathway genes, FLT3-TKD, and/or patients without FLT3-ITD) achieved a CR, only 32% of poor-risk patients (with U2AF1, WT1 mutations and/or complex karyotype) did. Intermediate-risk patients had a 50% CR rate. Similarly, using NPM1 co-mutation patterns and SF1 mutation status, we identified patients with favorable DFS and OS 3-year rates of 46% and 45%, respectively. Patients with adverse genetic features had DFS and OS rates of only 2% and 4%. We show that application of our proposed criteria may refine the 2017 European LeukemiaNet classification for older patients treated with chemotherapy.

Recurrent chromosomal abnormalities and gene mutations detected at the time of diagnosis of acute myeloid leukemia (AML) are associated with particular disease features, treatment response and survival of AML patients, and are used to denote specific disease entities in the World Health Organization classification of myeloid neoplasms and acute leukemia. However, large studies that integrate cytogenetic and comprehensive mutational information are scarce. We created a comprehensive oncoprint of mutations associated with recurrent cytogenetic findings by combining the information on mutational patterns of 80 cancer- and leukemia-associated genes with cytogenetic findings in 1603 adult patients with de novo AML. We show unique differences in the mutational profiles among major cytogenetic subsets, identify novel associations between recurrent cytogenetic abnormalities and both specific gene mutations and gene functional groups, and reveal differences in cytogenetic and mutational features between patients younger than 60 years and those aged 60 years or older. The identified associations between cytogenetic and molecular genetic data may help guide mutation testing in AML, and result in more focused application of targeted therapy in patients with de novo AML.

Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.

Somatic mutation of the DNMT3A gene at the arginine R882 site is common in acute myeloid leukaemia (AML). The prognostic significance of DNMT3A R882 mutation clearance, using traditional diagnostic next generation sequencing (NGS) methods, during complete remission (CR) in AML patients is controversial. We examined the impact of clearing DNMT3A R882 mutations at diagnosis to the detectable threshold of ˂3% during CR on outcome in 56 adult AML patients. Mutational remission, defined as clearance of pre-treatment DNMT3A R882 and all other AML-associated mutations to a variant allele frequency ˂3%, occurred in 14 patients whereas persistent DNMT3A R882 mutations were observed in 42 patients. There were no significant differences in disease-free or overall survival between patients with and without DNMT3A R882 mutation clearance. Patients with persistent DNMT3A R882 who cleared all other AML mutations and did not acquire new mutations (n = 30), trended towards longer disease-free survival (1·6 vs. 0·6 years, P = 0·06) than patients with persistence of DNMT3A R882, in addition to other mutations or acquisition of new AML-associated mutations, such as those in TET2, JAK2, ASXL1 and TP53 (n = 12). These data demonstrate that DNMT3A R882 mutations, as assessed by traditional NGS methods, persist in the majority of AML patients in CR., Competing Interests: of conflicts of interest The authors declare no conflicts of interest., (© 2016 John Wiley & Sons Ltd.)

We investigated whether octogenarian patients with acute myeloid leukemia enrolled onto Cooperative Group clinical trials and treated with intensive induction therapy could be cured, and whether karyotype and selected molecular markers had any prognostic significance in these patients. Among 138 patients with cytogenetic information, normal karyotype was the most common (47.1%) followed by complex karyotype (14.5%) and sole +8 (9.4%). Among these patients, the relapse-free survival rate at 1 year was 37% and 13% at 3 years, and the respective overall survival rates were 24% and 8%. Whereas the 90 patients who survived beyond 30 days had the same relapse-free survival rates, their 1-year and 3-year overall survival rates were 36% and 11%, respectively. Of the 66 patients surviving beyond 30 days who could be classified into European LeukemiaNet genetic groups, those in the intermediate-I group had better overall survival than patients in the adverse group (P=0.01). Among patients with cytogenetically normal acute myeloid leukemia who were tested for the European LeukemiaNet-associated molecular alterations, FLT3-internal tandem duplication and NPM1 mutations, it was found that FLT3-internal tandem duplication (detected in 29% of patients) did not associate with overall survival (P=0.31), whereas NPM1 mutations (30%) were associated with a significantly longer overall survival (P=0.002). We conclude that intensive induction is effective and indicated in selected octogenarians with acute myeloid leukemia, that their overall survival varies among the European LeukemiaNet genetic groups and that NPM1 mutations may be of prognostic significance among octogenarian patients with cytogenetically normal acute myeloid leukemia.

Purpose: To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures., Patients and Methods: Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays., Results: RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation-associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223., Conclusion: RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.

Purpose: To determine the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in primary cytogenetically normal acute myeloid leukemia (CN-AML)., Patients and Methods: Four hundred fifteen previously untreated adults were analyzed for DNMT3A mutations and established prognostic gene mutations and expression markers. Gene- and microRNA-expression profiles were derived using microarrays., Results: Younger (< 60 years; n = 181) and older (≥ 60 years; n = 234) patients had similar frequencies of DNMT3A mutations (35.3% v 33.3%). Missense mutations affecting arginine codon 882 (R882-DNMT3A) were more common (n = 92; 62%) than those affecting other codons (non-R882-DNMT3A). DNMT3A-mutated patients did not differ regarding complete remission rate, but had shorter disease-free survival (DFS; P = .03) and, by trend, overall survival (OS; P = .07) than DNMT3A-wild-type patients. In multivariable analyses, DNMT3A mutations remained associated with shorter DFS (P = .01), but not with shorter OS. When analyzed separately, the two DNMT3A mutation types had different significance by age group. Younger patients with non-R882-DNMT3A mutations had shorter DFS (P = .002) and OS (P = .02), whereas older patients with R882-DNMT3A mutations had shorter DFS (P = .005) and OS (P = .002) after adjustment for other clinical and molecular prognosticators. Gene- and microRNA-expression signatures did not accurately predict DNMT3A mutational status., Conclusion: DNMT3A mutations are frequent in CN-AML, and their clinical significance seems to be age dependent. DNMT3A-R882 mutations are associated with adverse prognosis in older patients, and non-R882-DNMT3A mutations are associated with adverse prognosis in younger patients. Low accuracy of gene- and microRNA-expression signatures in predicting DNMT3A mutation status suggested that the role of these mutations in AML remains to be elucidated.

The associations of mutations in the enhancer of trithorax and polycomb family gene ASXL1 with pretreatment patient characteristics, outcomes, and gene-/microRNA-expression profiles in primary cytogenetically normal acute myeloid leukemia (CN-AML) are unknown. We analyzed 423 adult patients for ASXL1 mutations, other prognostic gene mutations, and gene-/microRNA-expression profiles. ASXL1 mutations were 5 times more common in older (≥ 60 years) patients (16.2%) than those younger than 60 years (3.2%; P < .001). Among older patients, ASXL1 mutations associated with wild-type NPM1 (P < .001), absence of FLT3-internal tandem duplications (P = .002), mutated CEBPA (P = .01), and with inferior complete remission (CR) rate (P = .04), disease-free survival (DFS; P = .03), overall survival (OS; P = .006), and event-free survival (EFS; P = .002). Within the European LeukemiaNet (ELN) genetic categories of older CN-AML, ASXL1 mutations associated with inferior CR rate (P = .02), OS (P < .001), and EFS (P < .001) among ELN Favorable, but not among ELN Intermediate-I patients. Multivariable analyses confirmed associations of ASXL1 mutations with unfavorable CR rate (P = .03), DFS (P < .001), OS (P < .001), and EFS (P < .001) among ELN Favorable patients. We identified an ASXL1 mutation-associated gene-expression signature, but no microRNA-expression signature. This first study of ASXL1 mutations in primary CN-AML demonstrates that ASXL1-mutated older patients, particularly within the ELN Favorable group, have unfavorable outcomes and may be candidates for experimental treatment approaches.