Peter Westervelt

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.

Clonal Architecture of Secondary Acute Myeloid Leukemia

BACKGROUND The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood. METHODS We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations. RESULTS Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene. CONCLUSIONS Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.).

Peripheral-Blood Stem Cells versus Bone Marrow from Unrelated Donors

BACKGROUND Randomized trials have shown that the transplantation of filgrastim-mobilized peripheral-blood stem cells from HLA-identical siblings accelerates engraftment but increases the risks of acute and chronic graft-versus-host disease (GVHD), as compared with the transplantation of bone marrow. Some studies have also shown that peripheral-blood stem cells are associated with a decreased rate of relapse and improved survival among recipients with high-risk leukemia. METHODS We conducted a phase 3, multicenter, randomized trial of transplantation of peripheral-blood stem cells versus bone marrow from unrelated donors to compare 2-year survival probabilities with the use of an intention-to-treat analysis. Between March 2004 and September 2009, we enrolled 551 patients at 48 centers. Patients were randomly assigned in a 1:1 ratio to peripheral-blood stem-cell or bone marrow transplantation, stratified according to transplantation center and disease risk. The median follow-up of surviving patients was 36 months (interquartile range, 30 to 37). RESULTS The overall survival rate at 2 years in the peripheral-blood group was 51% (95% confidence interval [CI], 45 to 57), as compared with 46% (95% CI, 40 to 52) in the bone marrow group (P=0.29), with an absolute difference of 5 percentage points (95% CI, -3 to 14). The overall incidence of graft failure in the peripheral-blood group was 3% (95% CI, 1 to 5), versus 9% (95% CI, 6 to 13) in the bone marrow group (P=0.002). The incidence of chronic GVHD at 2 years in the peripheral-blood group was 53% (95% CI, 45 to 61), as compared with 41% (95% CI, 34 to 48) in the bone marrow group (P=0.01). There were no significant between-group differences in the incidence of acute GVHD or relapse. CONCLUSIONS We did not detect significant survival differences between peripheral-blood stem-cell and bone marrow transplantation from unrelated donors. Exploratory analyses of secondary end points indicated that peripheral-blood stem cells may reduce the risk of graft failure, whereas bone marrow may reduce the risk of chronic GVHD. (Funded by the National Heart, Lung, and Blood Institute-National Cancer Institute and others; ClinicalTrials.gov number, NCT00075816.).

Recurrent DNMT3A mutations in patients with myelodysplastic syndromes

Alterations in DNA methylation have been implicated in the pathogenesis of myelodysplastic syndromes (MDS), although the underlying mechanism remains largely unknown. Methylation of CpG dinucleotides is mediated by DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. DNMT3A mutations have recently been reported in patients with de novo acute myeloid leukemia (AML), providing a rationale for examining the status of DNMT3A in MDS samples. In this study, we report the frequency of DNMT3A mutations in patients with de novo MDS, and their association with secondary AML. We sequenced all coding exons of DNMT3A using DNA from bone marrow and paired normal cells from 150 patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12/150 patients (8.0%). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/13 mutations. DNMT3A mutations were expressed in the majority of cells in all tested mutant samples regardless of myeloblast counts, suggesting that DNMT3A mutations occur early in the course of MDS. Patients with DNMT3A mutations had worse overall survival compared with patients without DNMT3A mutations (P=0.005) and more rapid progression to AML (P=0.007), suggesting that DNMT3A mutation status may have prognostic value in de novo MDS.

RECURRENT MUTATIONS IN THE U2AF1 SPLICING FACTOR IN MYELODYSPLASTIC SYNDROMES

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3′ end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

Impact of Mobilization and Remobilization Strategies on Achieving Sufficient Stem Cell Yields for Autologous Transplantation

The purpose of this article was to examine historic institutional autologous stem cell mobilization practices and evaluate factors influencing mobilization failure and kinetics. In this retrospective study we analyzed clinical records of 1834 patients who underwent stem cell mobilization for autologous transplantation from November 1995 to October 2006 at the Washington University in St. Louis. Successful mobilization was defined as collection of > or =2 x 10(6) CD34(+) cells/kg. From 1834 consecutive patients, 1040 met our inclusion criteria (502 non-Hodgkins lymphoma [NHL], 137 Hodgkins lymphoma, and 401 multiple myeloma [MM]). A total of 976 patients received granulocyte colony-stimulating factor (G-CSF) and 64 received G-CSF plus chemotherapy (G/C) for the initial mobilization. Although the median CD34(+) cell yield was higher in G/C group than in G-CSF alone group, the failure rates were similar: 18.8% and 18.6%, respectively. Overall, 53% of patients collected > or =2 x 10(6) CD34(+) cells/kg during the first apheresis with either mobilization regimen. Regardless of mobilization regimen used, MM patients had the highest total CD34(+) cell yield and required less aphereses to collect > or =2 x 10(6) CD34(+) cells/kg. Mobilized, preapheresis, peripheral blood CD34(+) count correlated with first day apheresis yield (r = .877, P < .001) and 20 cells/microL was the minimum threshold needed for a successful day 1 collection. For the remobilization analysis we included patients from the whole database. A total of 269 of 1834 patients underwent remobilization using G/C, G-CSF, and/or GM-CSF, and G-CSF plus plerixafor. Only 23% of remobilized patients achieved > or =2 x 10(6) CD34(+) cells/kg and 29.7% failed to pool sufficient number of stem cells from both collections. Patients receiving G-CSF plus plerixafor had lowest failure rates, P = .03. NHL patients remobilized with G-CSF who waited > or =25 days before remobilization had lower CD34(+) cell yield than those who waited < or =16 days, P = .023. Current mobilization regimens are associated with a substantial failure rate irrespective of underlying disease. Patients who fail initial mobilization are more likely to fail remobilization. These findings suggest that there is a need for more effective first-line mobilization agents.

Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction

Allografts from HLA-matched sibling donors were mobilized and collected without granulocyte colony-stimulating factor (G-CSF) using AMD3100, a direct antagonist of CXCR4/stromal-derived factor 1 (SDF-1/CXCL12). Donors (N = 25) were treated with AMD3100 at a dose of 240 mug/kg by subcutaneous injection, and leukapheresis was then initiated just 4 hours later. Two-thirds of the donors collected an allograft with a CD34(+) cell dose sufficient for transplantation after just one dose of AMD3100. No donor experienced more than grade 1 toxicity. After a myeloablative regimen, 20 patients with hematologic malignancies received allografts collected after AMD3100 alone. All patients engrafted neutrophils (median day 10) and platelets (median day 12) promptly. Acute graft-versus-host disease (GVHD) grades 2 through 4 occurred in 35% of patients. One patient died due to complications related to acute GVHD. No unexpected adverse events were observed in any of the recipients. All 14 patients surviving in remission have robust trilineage hematopoiesis and are transfusion-free with a median follow-up of 277 days (range, 139-964 days). Direct antagonism of CXCR4 by AMD3100 may provide a more rapid and possibly less toxic and cumbersome alternative to traditional G-CSF-based mobilization in normal donors. This trial was registered as no. NCT00241358 at www.ClinicalTrials.gov.

A phase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor in relapsed or refractory acute myeloid leukemia

The interaction of acute myeloid leukemia (AML) blasts with the leukemic microenvironment is postulated to be an important mediator of resistance to chemotherapy and disease relapse. We hypothesized that inhibition of the CXCR4/CXCL12 axis by the small molecule inhibitor, plerixafor, would disrupt the interaction of leukemic blasts with the environment and increase the sensitivity of AML blasts to chemotherapy. In this phase 1/2 study, 52 patients with relapsed or refractory AML were treated with plerixafor in combination with mitoxantrone, etoposide, and cytarabine. In phase 1, plerixafor was escalated to a maximum of 0.24 mg/kg/d without any dose-limiting toxicities. In phase 2, 46 patients were treated with plerixafor 0.24 mg/kg/d in combination with chemotherapy with an overall complete remission and complete remission with incomplete blood count recovery rate (CR + CRi) of 46%. Correlative studies demonstrated a 2-fold mobilization in leukemic blasts into the peripheral circulation. No evidence of symptomatic hyperleukocytosis or delayed count recovery was observed with the addition of plerixafor. We conclude that the addition of plerixafor to cytotoxic chemotherapy is feasible in AML, and results in encouraging rates of remission with correlative studies demonstrating in vivo evidence of disruption of the CXCR4/CXCL12 axis.