The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers.

BACKGROUND: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated. METHODS: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing. RESULTS: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. CONCLUSIONS: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.).

Immunosuppression and outcomes in adult patients with de novo acute myeloid leukemia with normal karyotypes.

Acute myeloid leukemia (AML) patients rarely have long first remissions (LFRs; >5 y) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, standard remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing, and functional immunologic studies, we characterized 28 normal karyotype (NK)-AML patients with >5 y first remissions after chemotherapy (LFRs) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 y (standard first remissions [SFRs]). Our combined analyses indicated that genetic-risk profiling at presentation (as defined by European LeukemiaNet [ELN] 2017 criteria) was not sufficient to explain the outcomes of many SFR cases. Single-cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells or blocking the inhibitory major histocompatibility complex class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

Immune Escape of Relapsed AML Cells after Allogeneic Transplantation.

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes ( HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.).

Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia.

Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.

Somatic mutations and clonal hematopoiesis in congenital neutropenia.

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls (P = NS). Clonal hematopoiesis due to mutations in TP53 was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, P < .001) or patients with SCN (0/40, P < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple TP53 mutations. Conversely, clonal hematopoiesis due to mutations of CSF3R was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand TP53-mutated HSPCs, suggesting that acquisition of TP53 mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.

TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes.

BACKGROUND: The molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear. METHODS: We enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols. RESULTS: Of the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile (29 of 43 patients [67%] vs. 24 of 71 patients [34%], P<0.001) and among patients with TP53 mutations than among patients with wild-type TP53 (21 of 21 [100%] vs. 32 of 78 [41%], P<0.001). Previous studies have consistently shown that patients with an unfavorable-risk cytogenetic profile and TP53 mutations who receive conventional chemotherapy have poor outcomes. However, in this study of 10-day courses of decitabine, neither of these risk factors was associated with a lower rate of overall survival than the rate of survival among study patients with intermediate-risk cytogenetic profiles. CONCLUSIONS: Patients with AML and MDS who had cytogenetic abnormalities associated with unfavorable risk, TP53 mutations, or both had favorable clinical responses and robust (but incomplete) mutation clearance after receiving serial 10-day courses of decitabine. Although these responses were not durable, they resulted in rates of overall survival that were similar to those among patients with AML who had an intermediate-risk cytogenetic profile and who also received serial 10-day courses of decitabine. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT01687400 .).

A Pilot Study Evaluating the Effect of Cooler Dialysate Temperature on Hemodynamic Stability During Prolonged Intermittent Renal Replacement Therapy in Acute Kidney Injury.

OBJECTIVES: Acute kidney injury requiring renal replacement therapy is associated with high morbidity and mortality. Complications of renal replacement therapy include hemodynamic instability with ensuing shortened treatments, inadequate ultrafiltration, and delay in renal recovery. Studies have shown that lowering dialysate temperature in patients with end-stage renal disease is associated with a decrease in the frequency of intradialytic hypotension. However, data regarding mitigation of hypotension by lowering dialysate temperature in patients with acute kidney injury are scarce. We conducted a prospective, randomized, cross-over pilot study to evaluate the effect of lower dialysate temperature on hemodynamic status of critically ill patients with acute kidney injury during prolonged intermittent renal replacement therapy. DESIGN: Single-center prospective, randomized, cross-over study. SETTING: ICUs and a step down unit in a tertiary referral center. PATIENTS: Acute kidney injury patients undergoing prolonged intermittent renal replacement therapy. INTERVENTIONS: Participants were randomized to start prolonged intermittent renal replacement therapy with dialysate temperature of 35°C or dialysate temperature of 37°C. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the number of hypotensive events, as defined by any of the following: decrease in systolic blood pressure greater than or equal to 20 mm Hg, decrease in mean arterial pressure greater than or equal to 10 mm Hg, decrease in ultrafiltration, or increase in vasopressor requirements. The number of events was analyzed by Poisson regression and other outcomes with repeated-measures analysis of variance. Twenty-one patients underwent a total of 78 prolonged intermittent renal replacement therapy sessions, 39 in each arm. The number of hypotensive events was twice as high during treatments with dialysate temperature of 37°C, compared with treatments with the cooler dialysate (1.49 ± 1.12 vs 0.72 ± 0.69; incidence rate ratio, 2.06; p ≤ 0.0001). Treatment sessions with cooler dialysate were more likely to reach prescribed ultrafiltration targets. CONCLUSIONS: Patients with acute kidney injury undergoing prolonged intermittent renal replacement therapy with cooler dialysate experienced significantly less hypotension during treatment. Prevention of hemodynamic instability during renal replacement therapy helped to achieve ultrafiltration goals and may help to prevent volume overload in critically ill patients.

Effect of sleep on overnight cerebrospinal fluid amyloid ß kinetics.

Sleep disturbances are associated with future risk of Alzheimer disease. Disrupted sleep increases soluble amyloid ß, suggesting a mechanism for sleep disturbances to increase Alzheimer disease risk. We tested this response in humans using indwelling lumbar catheters to serially sample cerebrospinal fluid while participants were sleep-deprived, treated with sodium oxybate, or allowed to sleep normally. All participants were infused with 13 C6 -leucine to measure amyloid ß kinetics. We found that sleep deprivation increased overnight amyloid ß38, amyloid ß40, and amyloid ß42 levels by 25 to 30% via increased overnight amyloid ß production relative to sleeping controls. These findings suggest that disrupted sleep increases Alzheimer disease risk via increased amyloid ß production. Ann Neurol 2018;83:197-204.

Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.

BACKGROUND: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

A Population-Based Analysis of Time to Surgery and Travel Distances for Brachial Plexus Surgery.

PURPOSE: Despite the importance of timely evaluation for patients with brachial plexus injuries (BPIs), in clinical practice we have noted delays in referral. Because the published BPI experience is largely from individual centers, we used a population-based approach to evaluate the delivery of care for patients with BPI. METHODS: We used statewide administrative databases from Florida (2007-2013), New York (2008-2012), and North Carolina (2009-2010) to create a cohort of patients who underwent surgery for BPI (exploration, repair, neurolysis, grafting, or nerve transfer). Emergency department and inpatient records were used to determine the time interval between the injury and surgical treatment. Distances between treating hospitals and between the patient's home ZIP code and the surgical hospital were recorded. A multivariable logistic regression model was used to determine predictors for time from injury to surgery exceeding 365 days. RESULTS: Within the 222 patients in our cohort, median time from injury to surgery was 7.6 months and exceeded 365 days in 29% (64 of 222 patients) of cases. Treatment at a smaller hospital for the initial injury was significantly associated with surgery beyond 365 days after injury. Patient insurance type, travel distance for surgery, distance between the 2 treating hospitals, and changing hospitals between injury and surgery did not significantly influence time to surgery. CONCLUSIONS: Nearly one third of patients in Florida, New York, and North Carolina underwent BPI surgery more than 1 year after the injury. Patients initially treated at smaller hospitals are at risk for undergoing delayed BPI surgery. CLINICAL RELEVANCE: These findings can inform administrative and policy efforts to expedite timely referral of patients with BPI to experienced centers.

Randomized trial to evaluate azithromycin's effects on serum and upper airway IL-8 levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Respiratory syncytial virus (RSV) bronchiolitis in infancy is a major risk factor for recurrent wheezing and asthma. Because azithromycin attenuated neutrophilic airway inflammation in a murine viral bronchiolitis model, demonstration of similar effects in human subjects might provide a strategy for the prevention of postbronchiolitis recurrent wheezing. OBJECTIVES: We sought to investigate whether azithromycin treatment during RSV bronchiolitis reduces serum and nasal lavage IL-8 levels and the occurrence of postbronchiolitis recurrent wheezing. METHOD: We performed a randomized, double-masked, placebo-controlled proof-of-concept trial in 40 otherwise healthy infants hospitalized with RSV bronchiolitis who were treated with azithromycin or placebo for 14 days. IL-8 levels were measured in nasal lavage fluid and serum on randomization, day 8, and day 15 (nasal lavage only). The occurrence of wheezing episodes was assessed monthly over the ensuing 50 weeks. RESULTS: Compared with placebo, azithromycin treatment did not reduce serum IL-8 levels at day 8 (P = .6) but resulted in a greater decrease in nasal lavage fluid IL-8 levels by day 15 (P = .03). Twenty-two percent of azithromycin-treated participants experienced at least 3 wheezing episodes compared with 50% of participants in the placebo group (P = .07). Azithromycin treatment resulted in prolonged time to the third wheezing episode (P = .048) and in fewer days with respiratory symptoms over the subsequent year in comparison with placebo (36.7 vs 70.1 days, P = .01). CONCLUSION: In this proof-of-concept study azithromycin treatment during RSV bronchiolitis reduced upper airway IL-8 levels, prolonged the time to the third wheezing episode, and reduced overall respiratory morbidity over the subsequent year.

Remodeling Characteristics and Collagen Distributions of Biologic Scaffold Materials Biopsied From Postmastectomy Breast Reconstruction Sites.

OBJECTIVE: The study purpose was to evaluate the associations between patient characteristics and the histologic remodeling scores of acellular dermal matrices (ADMs) biopsied from breast reconstruction sites in the first attempt to generate a multivariable risk prediction model of nonconstructive remodeling. It was hypothesized that host characteristics and surgical site assessments predict the degree of graft remodeling for ADMs used during breast reconstruction. METHODS: The ADMs were biopsied from the breast reconstruction sites of n = 62 patients during a subsequent breast procedure, stained with hematoxylin-eosin, and evaluated according to a semi-quantitative scoring system for remodeling characteristics (cell types, cell infiltration, extracellular matrix deposition, scaffold degradation, fibrous encapsulation, and neovascularization) and a mean composite score. Biopsies were stained with Sirius Red and Fast Green, and analyzed to determine the collagen I:III ratio. Based on univariate analyses between subject clinical characteristics and the histologic remodeling scores, cohort variables were selected for multivariable regression models using a P value of 0.20 or less. RESULTS: The composite score model yielded 3 variables: pack-year history, corticosteroid use, and radiation timing (r pseudo = 0.81). The model for collagen I yielded 2 variables: corticosteroid use and reason for reoperation (r pseudo = 0.78). The model for collagen III yielded 1 variable: reason for reoperation (r pseudo = 0.35). CONCLUSIONS: These preliminary results constitute the first steps in generating a risk prediction model that predicts the patients and clinical circumstances most likely to experience nonconstructive remodeling of biologic grafts used to reconstruct the breast.

Association Between Mutation Clearance After Induction Therapy and Outcomes in Acute Myeloid Leukemia.

IMPORTANCE: Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. OBJECTIVES: To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. DESIGN, SETTING, AND PARTICIPANTS: Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. EXPOSURES: Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. MAIN OUTCOMES AND MEASURES: Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. RESULTS: Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free and overall survival vs the 26 who cleared all mutations. Patients with intermediate cytogenetic risk profiles had similar findings. [table: see text]. CONCLUSIONS AND RELEVANCE: The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome.

Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the past two decades, because most of the genetic events that initiate the disease remain undiscovered. Whole-genome sequencing is now possible at a reasonable cost and timeframe to use this approach for the unbiased discovery of tumour-specific somatic mutations that alter the protein-coding genes. Here we present the results obtained from sequencing a typical acute myeloid leukaemia genome, and its matched normal counterpart obtained from the same patient's skin. We discovered ten genes with acquired mutations; two were previously described mutations that are thought to contribute to tumour progression, and eight were new mutations present in virtually all tumour cells at presentation and relapse, the function of which is not yet known. Our study establishes whole-genome sequencing as an unbiased method for discovering cancer-initiating mutations in previously unidentified genes that may respond to targeted therapies.

DNMT3A mutations in acute myeloid leukemia.

BACKGROUND: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS: Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS: A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS: DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

Recurring mutations found by sequencing an acute myeloid leukemia genome.

BACKGROUND: The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known. METHODS: We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome. RESULTS: We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis. CONCLUSIONS: By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Acquired copy number alterations in adult acute myeloid leukemia genomes.

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10-29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions <5 megabases in size. A total of 8 uniparental disomy (UPD) segments were identified in the 86 genomes; 6 of 8 UPD calls occurred in samples with a normal karyotype. Collectively, 34 of 86 AML genomes (40%) contained alterations not found with cytogenetics, and 98% of these regions contained genes. Of 86 genomes, 43 (50%) had no CNA or UPD at this level of resolution. In this study of 86 adult AML genomes, the use of an unbiased high-resolution genomic screen identified many genes not previously implicated in AML that may be relevant for pathogenesis, along with many known oncogenes and tumor suppressor genes.

The brain recovery core: building a system of organized stroke rehabilitation and outcomes assessment across the continuum of care.

This Special Interest article describes a multidisciplinary, interinstitutional effort to build an organized system of stroke rehabilitation and outcomes measurement across the continuum of care. This system is focused on a cohort of patients who are admitted with the diagnosis of stroke to our acute facility, are discharged to inpatient and/or outpatient rehabilitation at our free-standing facility, and are then discharged to the community. This article first briefly explains the justification, goals, and purpose of the Brain Recovery Core system. The next sections describe its development and implementation, with details on the aspects related to physical therapy. The article concludes with an assessment of how the Brain Recovery Core system has changed and improved delivery of rehabilitation services. It is hoped that the contents of this article will be useful in initiating discussions and potentially facilitating similar efforts among other centers.