William D. Shannon

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.

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

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.

Effect of a nursing-implemented sedation protocol on the duration of mechanical ventilation.

OBJECTIVE To compare a practice of protocol-directed sedation during mechanical ventilation implemented by nurses with traditional non-protocol-directed sedation administration. DESIGN Randomized, controlled clinical trial. SETTING Medical intensive care unit (19 beds) in an urban teaching hospital. PATIENTS Patients requiring mechanical ventilation (n = 321). INTERVENTIONS Patients were randomly assigned to receive either protocol-directed sedation (n = 162) or non-protocol-directed sedation (n = 159). MEASUREMENTS AND MAIN RESULTS The median duration of mechanical ventilation was 55.9 hrs (95% confidence interval, 41.0-90.0 hrs) for patients managed with protocol-directed sedation and 117.0 hrs (95% confidence interval, 96.0-155.6 hrs) for patients receiving non-protocol-directed sedation. Kaplan-Meier analysis demonstrated that patients in the protocol-directed sedation group had statistically shorter durations of mechanical ventilation than patients in the non-protocol-directed sedation group (chi-square = 7.00, p = .008, log rank test; chi-square = 8.54, p = .004, Wilcoxons test; chi-square = 9.18, p = .003, -2 log test). Lengths of stay in the intensive care unit (5.7+/-5.9 days vs. 7.5+/-6.5 days; p = .013) and hospital (14.0+/-17.3 days vs. 19.9+/-24.2 days; p < .001) were also significantly shorter among patients in the protocol-directed sedation group. Among the 132 patients (41.1%) receiving continuous intravenous sedation, those in the protocol-directed sedation group (n = 66) had a significantly shorter duration of continuous intravenous sedation than those in the non-protocol-directed sedation group (n = 66) (3.5+/-4.0 days vs. 5.6+/-6.4 days; p = .003). Patients in the protocol-directed sedation group also had a significantly lower tracheostomy rate compared with patients in the non-protocol-directed sedation group (10 of 162 patients [6.2%] vs. 21 of 159 patients [13.2%], p = .038). CONCLUSIONS The use of protocol-directed sedation can reduce the duration of mechanical ventilation, the intensive care unit and hospital lengths of stay, and the need for tracheostomy among critically ill patients with acute respiratory failure.

A randomized, controlled trial of protocol-directed versus physician-directed weaning from mechanical ventilation.

OBJECTIVE To compare a practice of protocol-directed weaning from mechanical ventilation implemented by nurses and respiratory therapists with traditional physician-directed weaning. DESIGN Randomized, controlled trial. SETTING Medical and surgical intensive care units in two university-affiliated teaching hospitals. PATIENTS Patients requiring mechanical ventilation (n = 357). INTERVENTIONS Patients were randomly assigned to receive either protocol-directed (n = 179) or physician-directed (n = 178) weaning from mechanical ventilation. MEASUREMENTS AND MAIN RESULTS The primary outcome measure was the duration of mechanical ventilation from tracheal intubation until discontinuation of mechanical ventilation. Other outcome measures included need for reintubation, length of hospital stay, hospital mortality rate, and hospital costs. The median duration of mechanical ventilation was 35 hrs for the protocol-directed group (first quartile 15 hrs; third quartile 114 hrs) compared with 44 hrs for the physician-directed group (first quartile 21 hrs; third quartile 209 hrs). Kaplan-Meier analysis demonstrated that patients randomized to protocol-directed weaning had significantly shorter durations of mechanical ventilation compared with patients randomized to physician-directed weaning (chi 2 = 3.62, p = .057, log-rank test; chi 2 = 5.12, p = .024, Wilcoxon test). Cox proportional-hazards regression analysis, adjusting for other covariates, showed that the rate of successful weaning was significantly greater for patients receiving protocol-directed weaning compared with patients receiving physician-directed weaning (risk ratio 1.31; 95% confidence interval 1.15 to 1.50; p = .039). The hospital mortality rates for the two treatment groups were similar (protocol-directed 22.3% vs. physician-directed 23.6%; p = .779). Hospital cost savings for patients in the protocol-directed group were

Clinical predictors and outcomes for patients requiring tracheostomy in the intensive care unit

42,960 compared with hospital costs for patients in the physician-directed group. CONCLUSION Protocol-guided weaning of mechanical ventilation, as performed by nurses and respiratory therapists, is safe and led to extubation more rapidly than physician-directed weaning.

Effect of zoledronic acid on disseminated tumour cells in women with locally advanced breast cancer: an open label, randomised, phase 2 trial

OBJECTIVE To identify clinical predictors for tracheostomy among patients requiring mechanical ventilation in the intensive care unit (ICU) setting and to describe the outcomes of patients receiving a tracheostomy. DESIGN Prospective cohort study. SETTING Intensive care units of Barnes-Jewish Hospital, an urban teaching hospital. PATIENTS 521 patients requiring mechanical ventilation in an ICU for >12 hours. INTERVENTIONS Prospective patient surveillance and data collection. MEASUREMENTS AND MAIN RESULTS The main variables studied were hospital mortality, duration of mechanical ventilation, length of stay in the ICU and the hospital, and acquired organ-system derangements. Fifty-one (9.8%) patients received a tracheostomy. The hospital mortality of patients with a tracheostomy was statistically less than the hospital mortality of patients not receiving a tracheostomy (13.7% vs. 26.4%; p = .048), despite having a similar severity of illness at the time of admission to the ICU (Acute Physiology and Chronic Health Evaluation [APACHE] II scores, 19.2 +/- 6.1 vs. 17.8 +/- 7.2; p = .173). Patients receiving a tracheostomy had significantly longer durations of mechanical ventilation (19.5 +/- 15.7 days vs. 4.1 +/- 5.3 days; p < .001) and hospitalization (30.9 +/- 18.1 days vs. 12.8 +/- 10.1 days; p < .001) compared with patients not receiving a tracheostomy. Similarly, the average duration of intensive care was significantly longer among the hospital nonsurvivors receiving a tracheostomy (n = 7) compared with the hospital nonsurvivors without a tracheostomy (n = 124; 30.9 +/- 16.3 days vs. 7.9 +/- 7.3 days; p < .001). Multiple logistic regression analysis demonstrated that the development of nosocomial pneumonia (adjusted odds ratio [AOR], 4.72; 95% confidence interval [CI], 3.24-6.87; p < .001), the administration of aerosol treatments (AOR, 3.00; 95% CI, 2.184.13; p < .001), having a witnessed aspiration event (AOR, 3.79; 95% CI, 2.30-6.24; p = .008), and requiring reintubation (AOR, 2.21; 95% CI, 1.54-3.18; p = .028) were variables independently associated with patients undergoing tracheostomy and receiving prolonged ventilatory support. Among the 44 survivors receiving a tracheostomy in the ICU, 38 (86.4%) were alive 30 days after hospital discharge and 31 (70.5%) were living at home. CONCLUSIONS Despite having longer lengths of stay in the ICU and hospital, patients with respiratory failure who received a tracheostomy had favorable outcomes compared with patients who did not receive a tracheostomy. These data suggest that physicians are capable of selecting critically ill patients who most likely will benefit from placement of a tracheostomy. Additionally, specific clinical variables were identified as risk factors for prolonged ventilatory assistance and the need for tracheostomy.

A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome

BACKGROUND Treatment with bisphosphonates decreases bone loss and can increase disease-free survival in patients with breast cancer. The aim of our study was to assess the effect of zoledronic acid on clearance of disseminated tumour cells (DTCs) from the bone marrow in women undergoing neoadjuvant chemotherapy for breast cancer. METHODS Patients were recruited for this open-label, phase 2 randomised trial between March 17, 2003, and May 19, 2006, at a single centre. Eligible patients had clinical stage II-III (> or = T2 and/or > or = N1) newly diagnosed breast cancer, Eastern Cooperative Oncology Group performance status of 0 or 1, and normal cardiac, renal, and liver function. 120 women were randomly assigned, using allocation concealment, to receive 4 mg zoledronic acid intravenously every 3 weeks (n=60), or no zoledronic acid (n=60), for 1 year concomitant with four cycles of neoadjuvant epirubicin (75 mg/m(2)) plus docetaxel (75 mg/m(2)) and two cycles of adjuvant epirubicin plus docetaxel. The primary endpoint was the number of patients with detectable DTCs at 3 months. Final analysis was done 1 year after the last patient was enrolled. Analyses were done for all patients with available data at 3 months. This study is registered with ClinicalTrials.gov, number NCT00242203. FINDINGS Of the 120 patients initially enrolled, one withdrew after signing consent and one patients baseline bone marrow was not available. Both of these patients were in the control group. At 3 months, 109 bone-marrow samples were available for analysis. In the zoledronic acid group, bone marrow was not collected from one patient because of disease progression, one patient was taken off study because of severe diarrhoea, and two patients had not consented at the time of surgery. In the control group, bone marrow was not collected from two patients because of disease progression, one patient withdrew consent, and three patients were not consented at the time of surgery. At baseline, DTCs were detected in 26 of 60 patients in the zoledronic acid group and 28 of 58 patients in the control group. At 3 months, 17 of 56 patients receiving zoledronic acid versus 25 of 53 patients who did not receive zoledronic acid had detectable DTCs (p=0.054). The most common grade 3-4 toxicities were infection (five of 60 patients in the zoledronic acid group and six of 59 in the control group) and thrombosis (five of 60 in the zoledronic acid and two of 59 in the control group). There was one documented case of osteonecrosis in the zoledronic acid group. INTERPRETATION Zoledronic acid administered with chemotherapy resulted in a decreased proportion of patients with DTCs detected in the bone marrow at the time of surgery. Our study supports the hypothesis that the antimetastatic effects of zoledronic acid may be through effects on DTCs. FUNDING Novartis Pharmaceuticals and Pfizer Inc.

Acquired copy number alterations in adult acute myeloid leukemia genomes

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia

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.