Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Prognostic methylation markers for overall survival in cytogenetically normal patients with acute myeloid leukemia treated on SWOG trials.

BACKGROUND: Aberrant DNA methylation is known to occur in patients with acute myeloid leukemia (AML), whereas methylation signatures and prognostic markers have been proposed. The objective of the current study was to evaluate all CpG sites of the genome and identify prognostic methylation markers for overall survival in patients with AML with normal karyotype (AML-NK). METHODS: AML-NK samples from 7 SWOG trials were analyzed using a novel genome-wide approach called "CHARMcox" (comprehensive high-throughput array-based relative methylation analysis combined with the Cox proportional hazards model) controlling for known clinical covariates. CHARMcox was applied to a phase 1 discovery cohort (72 patients) to identify survival-associated methylation regions (SAMRs). Subsequently, using bisulfite pyrosequencing, SAMRs were studied in phase 2 model-building (65 patients) and phase 3 validation (65 patients) cohorts. An independent external cohort from The Cancer Genome Atlas (TCGA) AML study (LAML) was used for further validation (93 patients). RESULTS: Two SAMRs, located at the CpG island shores of leucine zipper tumor suppressor 2 (LZTS2) and nuclear receptor subfamily 6 group a member 1 (NR6A1), respectively, were identified. Multivariable analyses demonstrated that hypomethylation of either LZTS2 or NR6A1 was associated with worse overall survival in the SWOG cohort (P<.001). The prognosis was validated in patients with AML-NK from the TCGA-LAML cohort. Methylation values below the median at both markers predicted worse overall survival (SWOG: hazard ratio, 1.89 [P<.001]; and TCGA-LAML: hazard ratio, 2.08 [P=.006]). The C-statistic was 0.71 for both cohorts, and the impact was independent of the Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) status. CONCLUSIONS: The 2 methylation markers, measurable by clinically applicable assays such as bisulfite pyrosequencing, are promising for risk stratification among patients with AML-NK. Cancer 2017;123:2472-81. © 2017 American Cancer Society.

NUP98/NSD1 and FLT3/ITD coexpression is more prevalent in younger AML patients and leads to induction failure: a COG and SWOG report.

NUP98/NSD1 has recently been reported in association with poor outcome in acute myeloid leukemia (AML). Previous studies also observed a high overlap between NUP98/NSD1 and FLT3/ITD, raising the question as to whether the reported poor outcome is due to NUP98/NSD1 or caused by the co-occurrence of these 2 genetic lesions. We aimed to determine the prognostic significance of NUP98/NSD1 in the context of FLT3/ITD AML. A total of 1421 patients enrolled in 5 consecutive Children's Oncology Group/Children's Cancer Group and SWOG trials were evaluated. NUP98/NSD1 was found in 15% of FLT3/ITD and 7% of cytogenetically normal (CN)-AML. Those with dual FLT3/ITD and NUP98/NSD1 (82% of NUP98/NSD1 patients) had a complete remission rate of 27% vs 69% in FLT3/ITD without NUP98/NSD1 (P < .001). The corresponding 3-year overall survival was 31% vs 48% (P = .011), respectively. In CN-AML, patients with concomitant NUP98/NSD1 and FLT3/ITD had a worse outcome than those harboring NUP98/NSD1 only. In multivariate analysis, the dual NUP98/NSD1 and FLT3/ITD remained an independent predictor of poor outcome, and NUP98/NSD1 without FLT3/ITD lost its prognostic significance. Our study demonstrates that it is the interaction between NUP98/NSD1 and FLT3/ITD that determines the poor outcome of patients with NUP98/NSD1 disease.

Outpatient bendamustine and idarubicin for upfront therapy of elderly acute myeloid leukaemia/myelodysplastic syndrome: a phase I/II study using an innovative statistical design.

Combinations of agents may improve outcomes among elderly acute myeloid leukaemia (AML) and high-risk myelodysplastic syndrome (MDS) patients. We performed an adaptive phase I/II trial for newly-diagnosed AML or high-risk MDS patients aged ≥50 years using a Bayesian approach to determine whether 1 of 3 doses of bendamustine (45, 60, 75 mg/m(2) days 1-3), together with idarubicin (12 mg/m(2) days 1-2), might provide a complete response (CR) rate ≥40% with <30% grade 3-4 non-haematological toxicity. We treated 39 patients (34 AML; five MDS with >10% marrow blasts; median age 73 years). None of the three bendamustine doses in combination with idarubicin met the required CR and toxicity rates; the 75 mg/m(2) dose because of excess toxicity (two of three patients) and the 60 mg/m(2) dose because of low efficacy (CR rate 10/33), although no grade 3-4 non-haematological toxicity was seen at this dose. Median survival was 7·2 months. All patients began treatment as outpatients but hospitalization was required in 90% (35/39). Although we did not find a dose of bendamustine combined with idarubicin that would provide a CR rate of >40% with acceptable toxicity, bendamustine may have activity in AML/MDS patients, suggesting its addition to other regimens may be warranted.

Characteristics and prognostic impact of IDH mutations in AML: a COG, SWOG, and ECOG analysis.

Somatic mutations in isocitrate dehydrogenase (IDH) genes occur frequently in adult acute myeloid leukemia (AML) and less commonly in pediatric AML. The objective of this study was to describe the prevalence, mutational profile, and prognostic significance of IDH mutations in AML across age. Our cohort included 3141 patients aged between <1 month and 88 years treated on Children's Cancer Group/Children's Oncology Group (n = 1872), Southwest Oncology Group (n = 359), Eastern Cooperative Oncology Group (n = 397) trials, and in Beat AML (n = 333) and The Cancer Genome Atlas (n = 180) genomic characterization cohorts. We retrospectively analyzed patients in 4 age groups (age range, n): pediatric (0-17, 1744), adolescent/young adult (18-39, 444), intermediate-age (40-59, 640), older (≥60, 309). IDH mutations (IDHmut) were identified in 9.2% of the total cohort (n = 288; IDH1 [n = 123, 42.7%]; IDH2 [n = 165, 57.3%]) and were strongly correlated with increased age: 3.4% pediatric vs 21% older, P < .001. Outcomes were similar in IDHmut and IDH-wildtype (IDHWT) AML (event-free survival [EFS]: 35.6% vs 40.0%, P = .368; overall survival [OS]: 50.3% vs 55.4%, P = .196). IDH mutations frequently occurred with NPM1 (47.2%), DNMT3A (29.3%), and FLT3-internal tandem duplication (ITD) (22.4%) mutations. Patients with IDHmut AML with NPM1 mutation (IDHmut/NPM1mut) had significantly improved survival compared with the poor outcomes experienced by patients without (IDHmut/NPM1WT) (EFS: 55.1% vs 17.0%, P < .001; OS: 66.5% vs 35.2%, P < .001). DNTM3A or FLT3-ITD mutations in otherwise favorable IDHmut/NPM1mut AML led to inferior outcomes. Age group analysis demonstrated that IDH mutations did not abrogate the favorable prognostic impact of NPM1mut in patients aged <60 years; older patients had poor outcomes regardless of NPM1 status. These trials were registered at www.clinicaltrials.gov as #NCT00070174, #NCT00372593, #NCT01371981, #NCT00049517, and #NCT00085709.

Successful nonmyeloablative allogeneic stem cell transplantation for therapy-related acute myelogenous leukemia in a patient with preexisting visceral fungal infection.

Therapy-related acute myelogenous leukemia (t-AML) is a generally fatal disease with a very poor response to conventional chemotherapy. Allogeneic stem cell transplantation (allo-SCT) has been reported in patients with chemotherapy- responsive t-AML. However its use is limited owing to complications from previous treatments. Nonmyeloablative conditioning provides rapid hematologic engraftment and it is a feasible option for patients who are at increased risk for conventional SCT. There are few data on their use in patients with t-AML. We describe the case of a boy who developed visceral fungal infection with liver abscesses after induction chemotherapy for t-AML. He received allo-SCT with a nonmyeloablative regimen, plus amphotericin B during the transplant procedure. The patient is alive and free of both leukemia and fungal infection 2 years after allo-SCT. Nonmyeloablative allo-SCT may provide durable remission in patients with t-AML, preexisting invasive fungal infections, and a high risk of adverse effects from standard chemotherapy and prolonged cytopenia, without resurgence of the fungal infection.

Prospective trial of mycophenolate mofetil-cyclosporine A prophylaxis for acute GVHD after G-CSF stimulated allogeneic bone marrow transplantation with HLA-identical sibling donors in patients with severe aplastic anemia and hematological malignancies.

The combination of methotrexate and cyclosporine A (MTX-CSA) is the standard regimen for the prevention of graft vs. host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) from HLA-identical siblings. Mycophenolate mofetil and CSA (MMF-CSA) combination has been successfully used for GVHD prophylaxis after non-reduced intensity conditioning (non-RIC) allo-SCT with peripheral blood or non-G-CSF stimulated bone marrow as stem cell source. We report the results of the first prospective trial of the MMF-CSA combination for acute GVHD prophylaxis in 47 patients after non-RIC G-CSF stimulated allo-BMT (G-BMT) from HLA-identical siblings in patients with severe aplastic anemia (SAA) or hematological malignancies. Median age was 28 yr (range, 6-48 yr). Median follow-up was 22 months. The median time to neutrophil and platelets recovery were nine d (range, 8-17) and 16 d (range, 10-28), respectively. Acute GVHD of grade II-IV and chronic GVHD occurred in 51% and 27%, respectively. Overall survival rates at two yr for patients with SAA and hematological malignancies were 87% and 65%, respectively. The event-free survival at two yr for patients with hematological malignancies was 76%. We concluded that MMF-CSA appears equivalent to MTX-CSA for GVHD prophylaxis in patients receiving non-RIC G-BMT from HLA-identical siblings, with a tendency for more rapid neutrophil engraftment.

Decrease in vancomycin-resistant Enterococcus colonization associated with a reduction in carbapenem use as empiric therapy for febrile neutropenia in patients with acute leukemia.

OBJECTIVE: To compare the effects of empiric carbapenems versus cycling cefepime and piperacillin/tazobactam on the rates of vancomycin-resistant Enterococcus (VRE) colonization, bloodstream infections, and outcomes of patients admitted with acute leukemia. DESIGN: Retrospective clinical study with VRE molecular strain typing and gastrointestinal microbiome comparison. SETTING: A regional referral center for acute leukemia. PATIENTS: 342 consecutive patients admitted with newly diagnosed acute leukemia. METHODS: In September 2015, we changed our empiric antibiotic of choice for neutropenic fever from a carbapenem to the cycling regimen. We studied 214 consecutive patients during the carbapenem period and 128 during the cycling period. Surveillance for VRE stool colonization was conducted weekly. Representative stool samples were analyzed for VRE MLST types and changes in the composition and diversity of the fecal microbiota. RESULTS: The change in empiric antibiotics was associated with a significant decrease in VRE colonization (hazard ratio [HR], 0.35; 95% confidence interval [CI], 0.27-0.66), a switch in the dominant VRE MLST types on the unit, and some modifications in the gastrointestinal microbiome. There were no differences in total gram-positive or gram-negative BSIs. During the carbapenem period, we observed higher absolute numbers of Candida spp and fewer ESBL BSIs, but these did not reach statistical significance. Patients during the carbapenem period had longer lengths of stay and durations of severe neutropenia and 10% higher hospital cost. CONCLUSIONS: Carbapenem-sparing empiric antibiotic regimens may have advantages related to VRE ecology, gastrointestinal dysbiosis, duration of neutropenia, cost and length of stay.

Transcriptome Profiling of Pediatric Core Binding Factor AML.

The t(8;21) and Inv(16) translocations disrupt the normal function of core binding factors alpha (CBFA) and beta (CBFB), respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML) patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq) to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21) (N = 17), Inv(16) (N = 14), and normal karyotype (NK, N = 33) were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21) and 474 genes in Inv(16) were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10(-30)) are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs) differentially expressed across subtypes, with 337 t(8;21)-specific and 407 Inv(16)-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5 x 10(-51) and p = 1.8 x 10(-54) for the two subsets). In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six patients. Clustering of differentially expressed genes indicated that the homeobox (HOX) gene family, including two transcription factors (MEIS1 and NKX2-3) were down-regulated in CBF compared to NK samples. This finding supports existing data that the dysregulation of HOX genes play a central role in biology CBF-AML hematopoiesis. These data provide comprehensive transcriptome profiling of CBF-AML and delineate genes and pathways that are differentially expressed, providing insights into the shared biology as well as differences in the two CBF subsets.

Prognostic significance of NPM1 mutations in the absence of FLT3-internal tandem duplication in older patients with acute myeloid leukemia: a SWOG and UK National Cancer Research Institute/Medical Research Council report.

PURPOSE: Younger patients with acute myeloid leukemia (AML) harboring NPM1 mutations without FLT3-internal tandem duplications (ITDs; NPM1-positive/FLT3-ITD-negative genotype) are classified as better risk; however, it remains uncertain whether this favorable classification can be applied to older patients with AML with this genotype. Therefore, we examined the impact of age on the prognostic significance of NPM1-positive/FLT3-ITD-negative status in older patients with AML. PATIENTS AND METHODS: Patients with AML age ≥ 55 years treated with intensive chemotherapy as part of Southwest Oncology Group (SWOG) and UK National Cancer Research Institute/Medical Research Council (NCRI/MRC) trials were evaluated. A comprehensive analysis first examined 156 patients treated in SWOG trials. Validation analyses then examined 1,258 patients treated in MRC/NCRI trials. Univariable and multivariable analyses were used to determine the impact of age on the prognostic significance of NPM1 mutations, FLT3-ITDs, and the NPM1-positive/FLT3-ITD-negative genotype. RESULTS: Patients with AML age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype treated in SWOG trials had a significantly improved 2-year overall survival (OS) as compared with those without this genotype (70% v 32%; P < .001). Moreover, patients age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype had a significantly improved 2-year OS as compared with those age > 65 years with this genotype (70% v 27%; P < .001); any potential survival benefit of this genotype in patients age > 65 years was marginal (27% v 16%; P = .33). In multivariable analysis, NPM1-positive/FLT3-ITD-negative genotype remained independently associated with an improved OS in patients age 55 to 65 years (P = .002) but not in those age > 65 years (P = .82). These results were confirmed in validation analyses examining the NCRI/MRC patients. CONCLUSION: NPM1-positive/FLT3-ITD-negative genotype remains a relatively favorable prognostic factor for patients with AML age 55 to 65 years but not in those age > 65 years.

Comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation.

PURPOSE: Age has long been used as a major factor for assessing suitability for allogeneic hematopoietic cell transplantation (HCT). The HCT-comorbidity index (HCT-CI) was developed as a measure of health status to predict mortality risk after HCT. Whether age, comorbidities, or both should guide decision making for HCT is unknown. PATIENTS AND METHODS: Data from 3,033 consecutive recipients of HLA-matched grafts from five institutions contributed to this analysis. Patients were randomly divided into a training set to develop weights for age intervals and a validation set to assess the performance of prognostic models. RESULTS: In the training set, patients age 20 to 39 years, 40 to 49 years, 50 to 59 years, and ≥ 60 years had hazard ratios for nonrelapse mortality (NRM) of 1.21 (P = .29), 1.48 (P = .04), 1.75 (P = .004), and 1.84 (P = .005), respectively, compared with those age younger than 20 years. Consequently, age ≥ 40 years was assigned a weight of 1 to be added to the HCT-CI to constitute a composite comorbidity/age index. In the validation set, the composite comorbidity/age score had statistically significantly higher c-statistic estimates for prediction of NRM (0.664 v 0.556; P < .001) and survival (0.682 v 0.560; P < .001) compared with age, respectively. Patients with comorbidity/age scores of 0 to 2 had comparable mortality risks regardless of conditioning regimens. Patients with scores of 3 to 4 and ≥ 5 had statistically significant higher mortality risks after high-dose versus nonmyeloablative regimens. CONCLUSION: Age is a poor prognostic factor. The proposed composite measure allows integration of both comorbidities and age into clinical decision making and comparative-effectiveness research of HCT.

The role of quizartinib in the treatment of acute myeloid leukemia.

INTRODUCTION: Approximately one-third of the patients with acute myeloid leukemia (AML) harbor internal tandem duplication (ITD) in the gene encoding FMS-like tyrosine kinase 3 (FLT3-ITD), which is associated with poor prognosis. Over the course of the last decade, several FLT3 inhibitors have been developed. Nevertheless, the pharmacokinetic limitations of some of these compounds as well as their potency have limited their therapeutic efficacy. Quizartinib (AC220) is a second-generation FLT3 inhibitor that has shown promising activity in AML in Phase II clinical trials. AREAS COVERED: The pharmacokinetic, mechanism of action and resistance as well as clinical studies of quizartinib in AML are reported here in detail. EXPERT OPINION: Quizartinib is potent and selective FLT3 tyrosine kinase inhibitor with significant activity in both FLT3-mutant and wild-type AML. The quality and duration of achievable response thus far seen with this agent is suboptimal. Quizartinib in combination with chemotherapy might result in improved outcome and results of these trials are eagerly awaited. In addition, quizartinib in combination with other agents tackling the bone marrow microenvironment and FLT3 cooperative pathways may enhance response to quizartinib.

The prognostic significance of IRF8 transcripts in adult patients with acute myeloid leukemia.

Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis, such that disruption of IRF8 activity promotes leukemogenesis. We and others have identified aberrant expression of IRF8 transcripts, including novel splice variants, in acute myeloid leukemia (AML), but studies have not investigated the prognostic significance of these transcripts. Therefore, we developed and optimized quantitative expression assays for both, the wild type, or the reference sequence (WT-IRF8) and novel splice variants (SV-IRF8). These assays were used to quantify IRF8 transcript levels in 194 adult patients with AML, and multivariate analyses investigated the prognostic significance of these expression levels. After adjusting for known prognostic factors, expression levels of WT- or SV-IRF8 transcripts were not significantly associated with complete responses or overall survival. However, increased expression of WT-IRF8 was associated with decreased relapse-free survival (RFS) in both univariate (P = 0.010) and multivariate (P = 0.019) analyses. Similarly, increased expression of SV-IRF8 was associated with a decreased RFS (univariate, P = 0.026 and multivariate, P = 0.021). These studies show for the first time that WT-IRF8 and SV-IRF8 are independent adverse prognostic factors for patients with AML. Additional studies are planned to examine the prognostic significance of IRF8 transcripts in other populations of AML patients.

Normal hematopoietic function and multiple bone marrow clonal abnormalities in a patient with acute myeloid leukemia after two mismatched stem-cell transplants with graft failure and autologous reconstitution.

A 13-year-old male with primary refractory acute myelogenous leukemia (AML-M0), underwent two mismatched stem cell transplantations (SCT) and experienced graft failure after both procedures. Of interest, his peripheral blood cell counts are normal 7 years after his first SCT, his bone marrow is morphologically normal, however, cytogenetic analysis reveals multiple recurring cytogenetic abnormalities. This is the third case of chromosomal instability with morphological normal marrow and peripheral blood to be reported, these rare cases suggest that hematopoietic stem cells must have compensating mechanisms that allow normal function despite extensive chromosomal damage, supporting the notion that normal marrow function is possible even with extensive chromosomal damage.