Publications by authors named "Jeffery M Klco"

57 Publications

Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators.

Blood Cancer Discov 2021 Nov 9;2(6):586-599. Epub 2021 Sep 9.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by -ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell-like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification.

Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. .
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http://dx.doi.org/10.1158/2643-3230.BCD-21-0049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8580615PMC
November 2021

Serial assessment of measurable residual disease in medulloblastoma liquid biopsies.

Cancer Cell 2021 Nov 21;39(11):1519-1530.e4. Epub 2021 Oct 21.

Division of Neuro-Oncology, Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.
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http://dx.doi.org/10.1016/j.ccell.2021.09.012DOI Listing
November 2021

Rational biomarker development for the early and minimally invasive monitoring of AML.

Blood Adv 2021 11;5(21):4515-4520

Children's Hospital of Philadelphia, Comprehensive Bone Marrow Failure Center, Philadelphia, PA.

Recurrent disease remains the principal cause for treatment failure in acute myeloid leukemia (AML) across age groups. Reliable biomarkers of AML relapse risk and disease burden have been problematic, as symptoms appear late and current monitoring relies on invasive and cost-ineffective serial bone marrow (BM) surveillance. In this report, we discover a set of unique microRNA (miRNA) that circulates in AML-derived vesicles in the peripheral blood ahead of the general dissemination of leukemic blasts and symptomatic BM failure. Next-generation sequencing of extracellular vesicle-contained small RNA in 12 AML patients and 12 controls allowed us to identify a panel of differentially incorporated miRNA. Proof-of-concept studies using a murine model and patient-derived xenografts demonstrate the feasibility of developing miR-1246, as a potential minimally invasive AML biomarker.
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http://dx.doi.org/10.1182/bloodadvances.2021004621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8579272PMC
November 2021

Genomes for Kids: The Scope of Pathogenic Mutations in Pediatric Cancer Revealed by Comprehensive DNA and RNA Sequencing.

Cancer Discov 2021 Jul 23. Epub 2021 Jul 23.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor-normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. SIGNIFICANCE: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1631DOI Listing
July 2021

Enhancer Hijacking Drives Oncogenic Expression in Lineage-Ambiguous Stem Cell Leukemia.

Cancer Discov 2021 Nov 8;11(11):2846-2867. Epub 2021 Jun 8.

Department of Data Science, Dana-Farber Cancer Institute, Boston, Massachusetts.

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of , a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to . Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed allele and association of with activated hematopoietic progenitor cell -regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. SIGNIFICANCE: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of driven by diverse structural alterations, including superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries..
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http://dx.doi.org/10.1158/2159-8290.CD-21-0145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8563395PMC
November 2021

Activity of venetoclax against relapsed acute undifferentiated leukemia.

Cancer 2021 Aug 6;127(15):2608-2611. Epub 2021 Apr 6.

Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee.

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http://dx.doi.org/10.1002/cncr.33531DOI Listing
August 2021

Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells.

Leukemia 2021 Nov 17;35(11):3232-3244. Epub 2021 Mar 17.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.
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http://dx.doi.org/10.1038/s41375-021-01212-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446103PMC
November 2021

The acquisition of molecular drivers in pediatric therapy-related myeloid neoplasms.

Nat Commun 2021 02 12;12(1):985. Epub 2021 Feb 12.

St. Jude Children's Research Hospital, Department of Pathology, Memphis, TN, US.

Pediatric therapy-related myeloid neoplasms (tMN) occur in children after exposure to cytotoxic therapy and have a dismal prognosis. The somatic and germline genomic alterations that drive these myeloid neoplasms in children and how they arise have yet to be comprehensively described. We use whole exome, whole genome, and/or RNA sequencing to characterize the genomic profile of 84 pediatric tMN cases (tMDS: n = 28, tAML: n = 56). Our data show that Ras/MAPK pathway mutations, alterations in RUNX1 or TP53, and KMT2A rearrangements are frequent somatic drivers, and we identify cases with aberrant MECOM expression secondary to enhancer hijacking. Unlike adults with tMN, we find no evidence of pre-existing minor tMN clones (including those with TP53 mutations), but rather the majority of cases are unrelated clones arising as a consequence of cytotoxic therapy. These studies also uncover rare cases of lineage switch disease rather than true secondary neoplasms.
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http://dx.doi.org/10.1038/s41467-021-21255-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880998PMC
February 2021

SequencErr: measuring and suppressing sequencer errors in next-generation sequencing data.

Genome Biol 2021 01 25;22(1):37. Epub 2021 Jan 25.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Background: There is currently no method to precisely measure the errors that occur in the sequencing instrument/sequencer, which is critical for next-generation sequencing applications aimed at discovering the genetic makeup of heterogeneous cellular populations.

Results: We propose a novel computational method, SequencErr, to address this challenge by measuring the base correspondence between overlapping regions in forward and reverse reads. An analysis of 3777 public datasets from 75 research institutions in 18 countries revealed the sequencer error rate to be ~ 10 per million (pm) and 1.4% of sequencers and 2.7% of flow cells have error rates > 100 pm. At the flow cell level, error rates are elevated in the bottom surfaces and > 90% of HiSeq and NovaSeq flow cells have at least one outlier error-prone tile. By sequencing a common DNA library on different sequencers, we demonstrate that sequencers with high error rates have reduced overall sequencing accuracy, and removal of outlier error-prone tiles improves sequencing accuracy. We demonstrate that SequencErr can reveal novel insights relative to the popular quality control method FastQC and achieve a 10-fold lower error rate than popular error correction methods including Lighter and Musket.

Conclusions: Our study reveals novel insights into the nature of DNA sequencing errors incurred on DNA sequencers. Our method can be used to assess, calibrate, and monitor sequencer accuracy, and to computationally suppress sequencer errors in existing datasets.
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http://dx.doi.org/10.1186/s13059-020-02254-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829059PMC
January 2021

Advances in germline predisposition to acute leukaemias and myeloid neoplasms.

Nat Rev Cancer 2021 02 16;21(2):122-137. Epub 2020 Dec 16.

Department of Pathology and the Hematological Malignancies Program, St. Jude Children's Research Hospital, Memphis, TN, USA.

Although much work has focused on the elucidation of somatic alterations that drive the development of acute leukaemias and other haematopoietic diseases, it has become increasingly recognized that germline mutations are common in many of these neoplasms. In this Review, we highlight the different genetic pathways impacted by germline mutations that can ultimately lead to the development of familial and sporadic haematological malignancies, including acute lymphoblastic leukaemia, acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Many of the genes disrupted by somatic mutations in these diseases (for example, TP53, RUNX1, IKZF1 and ETV6) are the same as those that harbour germline mutations in children and adolescents who develop these malignancies. Moreover, the presumption that familial leukaemias only present in childhood is no longer true, in large part due to the numerous studies demonstrating germline DDX41 mutations in adults with MDS and AML. Lastly, we highlight how different cooperating events can influence the ultimate phenotype in these different familial leukaemia syndromes.
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http://dx.doi.org/10.1038/s41568-020-00315-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8404376PMC
February 2021

Safety, pharmacokinetics, and pharmacodynamics of panobinostat in children, adolescents, and young adults with relapsed acute myeloid leukemia.

Cancer 2020 11 18;126(21):4800-4805. Epub 2020 Aug 18.

St. Jude Children's Research Hospital, Memphis, Tennessee.

Background: Novel therapies are urgently needed for pediatric patients with relapsed acute myeloid leukemia (AML).

Methods: To determine whether the histone deacetylase inhibitor panobinostat could be safely given in combination with intensive chemotherapy, a phase 1 trial was performed in which 17 pediatric patients with relapsed or refractory AML received panobinostat (10, 15, or 20 mg/m ) before and in combination with fludarabine and cytarabine.

Results: All dose levels were tolerated, with no dose-limiting toxicities observed at any dose level. Pharmacokinetic studies demonstrated that exposure to panobinostat was proportional to the dose given, with no associations between pharmacokinetic parameters and age, weight, or body surface area. Among the 9 patients who had sufficient (>2%) circulating blasts on which histone acetylation studies could be performed, 7 demonstrated at least 1.5-fold increases in acetylation. Although no patients had a decrease in circulating blasts after single-agent panobinostat, 8 of the 17 patients (47%), including 5 of the 6 patients treated at dose level 3, achieved complete remission. Among the 8 complete responders, 6 (75%) attained negative minimal residual disease status.

Conclusions: Panobinostat can be safely administered with chemotherapy and results in increased blast histone acetylation. This suggests that it should be further studied in AML.
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http://dx.doi.org/10.1002/cncr.33156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722063PMC
November 2020

Mechanistic insights and potential therapeutic approaches for NUP98-rearranged hematologic malignancies.

Blood 2020 11;136(20):2275-2289

Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN.

Nucleoporin 98 (NUP98) fusion oncoproteins are observed in a spectrum of hematologic malignancies, particularly pediatric leukemias with poor patient outcomes. Although wild-type full-length NUP98 is a member of the nuclear pore complex, the chromosomal translocations leading to NUP98 gene fusions involve the intrinsically disordered and N-terminal region of NUP98 with over 30 partner genes. Fusion partners include several genes bearing homeodomains or having known roles in transcriptional or epigenetic regulation. Based on data in both experimental models and patient samples, NUP98 fusion oncoprotein-driven leukemogenesis is mediated by changes in chromatin structure and gene expression. Multiple cofactors associate with NUP98 fusion oncoproteins to mediate transcriptional changes possibly via phase separation, in a manner likely dependent on the fusion partner. NUP98 gene fusions co-occur with a set of additional mutations, including FLT3-internal tandem duplication and other events contributing to increased proliferation. To improve the currently dire outcomes for patients with NUP98-rearranged malignancies, therapeutic strategies have been considered that target transcriptional and epigenetic machinery, cooperating alterations, and signaling or cell-cycle pathways. With the development of more faithful experimental systems and continued study, we anticipate great strides in our understanding of the molecular mechanisms and therapeutic vulnerabilities at play in NUP98-rearranged models. Taken together, these studies should lead to improved clinical outcomes for NUP98-rearranged leukemia.
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http://dx.doi.org/10.1182/blood.2020007093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702474PMC
November 2020

Molecular basis of ETV6-mediated predisposition to childhood acute lymphoblastic leukemia.

Blood 2021 01;137(3):364-373

Department of Pharmaceutical Sciences and.

There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.
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http://dx.doi.org/10.1182/blood.2020006164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819760PMC
January 2021

Venetoclax in combination with cytarabine with or without idarubicin in children with relapsed or refractory acute myeloid leukaemia: a phase 1, dose-escalation study.

Lancet Oncol 2020 04 11;21(4):551-560. Epub 2020 Mar 11.

Department of Oncology, St Jude Children's Research Hospital, Memphis, TN, USA. Electronic address:

Background: Outcomes for children with relapsed or refractory acute myeloid leukaemia remain poor. The BCL-2 inhibitor, venetoclax, has shown promising activity in combination with hypomethylating agents and low-dose cytarabine in older adults for whom chemotherapy is not suitable with newly diagnosed acute myeloid leukaemia. We aimed to determine the safety and explore the activity of venetoclax in combination with standard and high-dose chemotherapy in paediatric patients with relapsed or refractory acute myeloid leukaemia.

Methods: We did a phase 1, dose-escalation study at three research hospitals in the USA. Eligible patients were aged 2-22 years with relapsed or refractory acute myeloid leukaemia or acute leukaemia of ambiguous lineage with adequate organ function and performance status. During dose escalation, participants received venetoclax orally once per day in continuous 28-day cycles at either 240 mg/m or 360 mg/m, in combination with cytarabine received intravenously every 12 h at either 100 mg/m for 20 doses or 1000 mg/m for eight doses, with or without intravenous idarubicin (12 mg/m) as a single dose, using a rolling-6 accrual strategy. The primary endpoint was the recommended phase 2 dose of venetoclax plus chemotherapy and the secondary endpoint was the proportion of patients treated at the recommended phase 2 dose who achieved complete remission or complete remission with incomplete haematological recovery. Analyses were done on patients who received combination therapy. The study is registered with ClinicalTrials.gov (NCT03194932) and is now enrolling to address secondary and exploratory objectives.

Findings: Between July 1, 2017, and July 2, 2019, 38 patients were enrolled (aged 3-22 years; median 10 [IQR 7-13]), 36 of whom received combination therapy with dose escalation, with a median follow-up of 7·1 months (IQR 5·1-11·2). The recommended phase 2 dose of venetoclax was found to be 360 mg/m (maximum 600 mg) combined with cytarabine (1000 mg/m per dose for eight doses), with or without idarubicin (12 mg/m as a single dose). Overall responses were observed in 24 (69%) of the 35 patients who were evaluable after cycle 1. Among the 20 patients treated at the recommended phase 2 dose, 14 (70%, 95% CI 46-88) showed complete response with or without complete haematological recovery, and two (10%) showed partial response. The most common grade 3-4 adverse events were febrile neutropenia (22 [66%]), bloodstream infections (six [16%]), and invasive fungal infections (six [16%]). Treatment-related death occurred in one patient due to colitis and sepsis.

Interpretation: The safety and activity of venetoclax plus chemotherapy in paediatric patients with heavily relapsed and refractory acute myeloid leukaemia suggests that this combination should be tested in newly diagnosed paediatric patients with high-risk acute myeloid leukaemia.

Funding: US National Institutes of Health, American Lebanese Syrian Associated Charities, AbbVie, and Gateway for Cancer Research.
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http://dx.doi.org/10.1016/S1470-2045(20)30060-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153631PMC
April 2020

Immune Escape of Relapsed AML Cells after Allogeneic Transplantation.

N Engl J Med 2018 12 31;379(24):2330-2341. Epub 2018 Oct 31.

From the Division of Oncology, Department of Internal Medicine (M.J.C., A.A.P., M.P.R., C.A.M., E.C., N.M.H., L.D.W., J.S.W., S.E.H., D.C.L., M.J.W., P.W., T.J.L., J.F.D.), the McDonnell Genome Institute (A.A.P., C.A.M., M.O., C.C.F., R.S.F., L.D.W., T.J.L.), the Department of Pathology and Immunology (E.J.D., J.E.P.), and the Division of Biostatistics (J.D.B.), Washington University in St. Louis, St. Louis; the Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN (J.M.K.); the Institute for Genomic Medicine, Nationwide Children's Hospital, Columbus, OH (R.K.W.); and the Center for Cancer Research, Massachusetts General Hospital, Boston (T.A.G.).

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.).
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http://dx.doi.org/10.1056/NEJMoa1808777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322675PMC
December 2018

Clinical cancer genomic profiling by three-platform sequencing of whole genome, whole exome and transcriptome.

Nat Commun 2018 09 27;9(1):3962. Epub 2018 Sep 27.

Pediatric Cancer Genome Project, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97-99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing.
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http://dx.doi.org/10.1038/s41467-018-06485-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160438PMC
September 2018

Donor-derived MDS/AML in families with germline mutation.

Blood 2018 11 19;132(18):1994-1998. Epub 2018 Sep 19.

Hematology Section, Department of Laboratory Medicine, Clinical Center, National Institutes of Health (NIH), Bethesda, MD.

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http://dx.doi.org/10.1182/blood-2018-07-861070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213320PMC
November 2018

Germline SAMD9 and SAMD9L mutations are associated with extensive genetic evolution and diverse hematologic outcomes.

JCI Insight 2018 07 26;3(14). Epub 2018 Jul 26.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Germline SAMD9 and SAMD9L mutations cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with somatic monosomy 7. Here, we describe 16 siblings, the majority of which were phenotypically normal, from 5 families diagnosed with myelodysplasia and leukemia syndrome with monosomy 7 (MLSM7; OMIM 252270) who primarily had onset of hematologic abnormalities during the first decade of life. Molecular analyses uncovered germline SAMD9L (n = 4) or SAMD9 (n = 1) mutations in these families. Affected individuals had a highly variable clinical course that ranged from mild and transient dyspoietic changes in the bone marrow to a rapid progression of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with monosomy 7. Expression of these gain-of-function SAMD9 and SAMD9L mutations reduces cell cycle progression, and deep sequencing demonstrated selective pressure favoring the outgrowth of clones that have either lost the mutant allele or acquired revertant mutations. The myeloid malignancies of affected siblings acquired cooperating mutations in genes that are also altered in sporadic cases of AML characterized by monosomy 7. These data have implications for understanding how SAMD9 and SAMD9L mutations contribute to myeloid transformation and for recognizing, counseling, and treating affected families.
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http://dx.doi.org/10.1172/jci.insight.121086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124395PMC
July 2018

Clonal dynamics of donor-derived myelodysplastic syndrome after unrelated hematopoietic cell transplantation for high-risk pediatric B-lymphoblastic leukemia.

Cold Spring Harb Mol Case Stud 2018 10 1;4(5). Epub 2018 Oct 1.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Donor-derived hematologic malignancies are rare complications of hematopoietic cell transplantation (HCT). Although these are commonly either a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), in general, they are a heterogeneous group of diseases, and a unified mechanism for their development has remained elusive. Here we report next-generation sequencing, including whole-exome sequencing (WES), whole-genome sequencing (WGS), and targeted sequencing, of a case of donor-derived MDS (dMDS) following HCT for high-risk B-lymphoblastic leukemia (B-ALL) in an adolescent. Through interrogation of single-nucleotide polymorphisms (SNPs) in the WGS data, we unequivocally prove that the MDS is donor-derived. Additionally, we sequenced 15 samples from 12 time points, including the initial B-ALL diagnostic sample through several post-HCT remission samples, the dMDS, and representative germline samples from both patient and donor, to show that the MDS-related pathologic mutations, including a canonical (p.Y700*) mutation, were detectable nearly 3 yr prior to the morphological detection of MDS. Furthermore, these MDS mutations were not detectable immediately following, and for >1 yr post-, HCT. These data support the clinical utility of comprehensive sequencing following HCT to detect donor-derived malignancies, while providing insights into the clonal progression of dMDS over a 4-yr period.
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http://dx.doi.org/10.1101/mcs.a002980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169831PMC
October 2018

TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation.

J Exp Med 2018 04 2;215(4):1023-1034. Epub 2018 Mar 2.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN

The NOD-like receptor (NLR)-P3 inflammasome is a global sensor of infection and stress. Elevated NLRP3 activation levels are associated with human diseases, but the mechanisms controlling NLRP3 inflammasome activation are largely unknown. Here, we show that TGF-β activated kinase-1 (TAK1) is a central regulator of NLRP3 inflammasome activation and spontaneous cell death. Absence of TAK1 in macrophages induced spontaneous activation of the NLRP3 inflammasome without requiring toll-like receptor (TLR) priming and subsequent activating signals, suggesting a distinctive role for TAK1 in maintaining NLRP3 inflammasome homeostasis. Autocrine tumor necrosis factor (TNF) signaling in the absence of TAK1 induced spontaneous RIPK1-dependent NLRP3 inflammasome activation and cell death. We further showed that TAK1 suppressed homeostatic NF-κB and extracellular signal-related kinase (ERK) activation to limit spontaneous TNF production. Moreover, the spontaneous inflammation resulting from TAK1-deficient macrophages drives myeloid proliferation in mice, and was rescued by RIPK1 deficiency. Overall, these studies identify a critical role for TAK1 in maintaining NLRP3 inflammasome quiescence and preserving cellular homeostasis and survival.
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http://dx.doi.org/10.1084/jem.20171922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881469PMC
April 2018

The genomic landscape of pediatric myelodysplastic syndromes.

Nat Commun 2017 11 16;8(1):1557. Epub 2017 Nov 16.

Department of Pathology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 342, Memphis, TN, 38105, USA.

Myelodysplastic syndromes (MDS) are uncommon in children and have a poor prognosis. In contrast to adult MDS, little is known about the genomic landscape of pediatric MDS. Here, we describe the somatic and germline changes of pediatric MDS using whole exome sequencing, targeted amplicon sequencing, and/or RNA-sequencing of 46 pediatric primary MDS patients. Our data show that, in contrast to adult MDS, Ras/MAPK pathway mutations are common in pediatric MDS (45% of primary cohort), while mutations in RNA splicing genes are rare (2% of primary cohort). Surprisingly, germline variants in SAMD9 or SAMD9L were present in 17% of primary MDS patients, and these variants were routinely lost in the tumor cells by chromosomal deletions (e.g., monosomy 7) or copy number neutral loss of heterozygosity (CN-LOH). Our data confirm that adult and pediatric MDS are separate diseases with disparate mechanisms, and that SAMD9/SAMD9L mutations represent a new class of MDS predisposition.
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http://dx.doi.org/10.1038/s41467-017-01590-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691144PMC
November 2017

Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies.

J Clin Invest 2017 Oct 5;127(10):3657-3674. Epub 2017 Sep 5.

Department of Medicine, Division of Oncology, Section of Stem Cell Biology, Washington University School of Medicine, St. Louis, Missouri, USA.

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.
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http://dx.doi.org/10.1172/JCI93041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617681PMC
October 2017

Comprehensive discovery of noncoding RNAs in acute myeloid leukemia cell transcriptomes.

Exp Hematol 2017 11 28;55:19-33. Epub 2017 Jul 28.

The McDonnell Genome Institute, Washington University, St. Louis, MO; Department of Medicine, Washington University, St. Louis, MO; Siteman Cancer Center, Washington University, St. Louis, MO; Department of Biomedical Engineering, Washington University, St. Louis, MO. Electronic address:

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses.
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http://dx.doi.org/10.1016/j.exphem.2017.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772960PMC
November 2017

The genomic landscape of core-binding factor acute myeloid leukemias.

Nat Genet 2016 12 31;48(12):1551-1556. Epub 2016 Oct 31.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.
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http://dx.doi.org/10.1038/ng.3709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508996PMC
December 2016

Comprehensive genomic analysis reveals FLT3 activation and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic leukemia.

Exp Hematol 2016 Jul 13;44(7):603-13. Epub 2016 May 13.

Siteman Cancer Center, Washington University, St. Louis, MO, USA; Department of Medicine, Washington University, St. Louis, MO, USA.

The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome.
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http://dx.doi.org/10.1016/j.exphem.2016.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914477PMC
July 2016

Optimizing cancer genome sequencing and analysis.

Cell Syst 2015 Sep;1(3):210-223

The McDonnell Genome Institute, Washington University, St. Louis, MO, USA, 63108 ; Department of Genetics, Washington University, St. Louis, MO, USA, 63108 ; Siteman Cancer Center, Washington University, St. Louis, MO, USA, 63108 ; Department of Medicine, Washington University, St. Louis, MO, USA, 63108.

Tumors are typically sequenced to depths of 75-100× (exome) or 30-50× (whole genome). We demonstrate that current sequencing paradigms are inadequate for tumors that are impure, aneuploid or clonally heterogeneous. To reassess optimal sequencing strategies, we performed ultra-deep (up to ~312×) whole genome sequencing (WGS) and exome capture (up to ~433×) of a primary acute myeloid leukemia, its subsequent relapse, and a matched normal skin sample. We tested multiple alignment and variant calling algorithms and validated ~200,000 putative SNVs by sequencing them to depths of ~1,000×. Additional targeted sequencing provided over 10,000× coverage and ddPCR assays provided up to ~250,000× sampling of selected sites. We evaluated the effects of different library generation approaches, depth of sequencing, and analysis strategies on the ability to effectively characterize a complex tumor. This dataset, representing the most comprehensively sequenced tumor described to date, will serve as an invaluable community resource (dbGaP accession id phs000159).
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http://dx.doi.org/10.1016/j.cels.2015.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669575PMC
September 2015

Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML.

Blood 2016 Feb 2;127(7):893-7. Epub 2015 Dec 2.

Division of Oncology, Siteman Cancer Center, Washington University, St. Louis, MO; and.

There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy.
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http://dx.doi.org/10.1182/blood-2015-10-677021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760092PMC
February 2016

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

JAMA 2015 Aug;314(8):811-22

Division of Oncology, Department of Medicine, Washington University School of Medicine, St Louis, Missouri.

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.
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http://dx.doi.org/10.1001/jama.2015.9643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621257PMC
August 2015
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