Publications by authors named "Catrina Fronick"

57 Publications

A domestic cat whole exome sequencing resource for trait discovery.

Sci Rep 2021 Mar 30;11(1):7159. Epub 2021 Mar 30.

Department of Animal Sciences, College of Agriculture, Department of Surgery, School of Medicine, Institute for Data Science and Informatics, University of Missouri, Columbia, MO, 65211, USA.

Over 94 million domestic cats are susceptible to cancers and other common and rare diseases. Whole exome sequencing (WES) is a proven strategy to study these disease-causing variants. Presented is a 35.7 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. Whole exome sequencing was conducted on 41 cats with known and unknown genetic diseases and traits, of which ten cats had matching whole genome sequence (WGS) data available, used to validate WES performance. At 80 × mean exome depth of coverage, 96.4% of on-target base coverage had a sequencing depth > 20-fold, while over 98% of single nucleotide variants (SNVs) identified by WGS were also identified by WES. Platform-specific SNVs were restricted to sex chromosomes and a small number of olfactory receptor genes. Within the 41 cats, we identified 31 previously known causal variants and discovered new gene candidate variants, including novel missense variance for polycystic kidney disease and atrichia in the Peterbald cat. These results show the utility of WES to identify novel gene candidate alleles for diseases and traits for the first time in a feline model.
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http://dx.doi.org/10.1038/s41598-021-86200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009874PMC
March 2021

Draft Genome Sequences of Two Polycyclic Tetramate Macrolactam Producers, sp. Strains JV180 and SP18CM02.

Microbiol Resour Announc 2020 Dec 10;9(50). Epub 2020 Dec 10.

Department of Biology, Washington University in St. Louis, St. Louis, Missouri, USA

Here, we report the draft genome sequences of two related sp. strains, JV180 and SP18CM02. Despite their isolation from soils in Connecticut and Missouri (USA), respectively, they are strikingly similar in gene content. Both belong to the clade and harbor several secondary metabolite biosynthetic gene clusters.
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http://dx.doi.org/10.1128/MRA.01066-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729405PMC
December 2020

Sequence analysis in reveals pervasiveness of X-Y arms races in mammalian lineages.

Genome Res 2020 12 18;30(12):1716-1726. Epub 2020 Nov 18.

Whitehead Institute, Cambridge, Massachusetts 02142, USA.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.
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http://dx.doi.org/10.1101/gr.269902.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706723PMC
December 2020

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Clin Chem 2021 01;67(2):415-424

Department of Genetics, Washington University School of Medicine, St. Louis, MO.

Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.

Methods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples.

Results: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%.

Conclusions: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.
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http://dx.doi.org/10.1093/clinchem/hvaa267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665435PMC
January 2021

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Cell 2020 08 11;182(4):886-900.e17. Epub 2020 Aug 11.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2 mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1 and CXCR1 macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.
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http://dx.doi.org/10.1016/j.cell.2020.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485282PMC
August 2020

Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing To Define Immune Cell Chimerism in the Rejecting Kidney Transplant.

J Am Soc Nephrol 2020 09 15;31(9):1977-1986. Epub 2020 Jul 15.

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

Background: In solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells.

Methods: Whole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples.

Results: Analysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation.

Conclusions: Analysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.
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http://dx.doi.org/10.1681/ASN.2020030326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461682PMC
September 2020

The clonal evolution of metastatic colorectal cancer.

Sci Adv 2020 Jun 10;6(24):eaay9691. Epub 2020 Jun 10.

The Alvin J. Siteman Comprehensive Cancer Center, Washington University in St. Louis, St. Louis, MO, USA.

Tumor heterogeneity and evolution drive treatment resistance in metastatic colorectal cancer (mCRC). Patient-derived xenografts (PDXs) can model mCRC biology; however, their ability to accurately mimic human tumor heterogeneity is unclear. Current genomic studies in mCRC have limited scope and lack matched PDXs. Therefore, the landscape of tumor heterogeneity and its impact on the evolution of metastasis and PDXs remain undefined. We performed whole-genome, deep exome, and targeted validation sequencing of multiple primary regions, matched distant metastases, and PDXs from 11 patients with mCRC. We observed intricate clonal heterogeneity and evolution affecting metastasis dissemination and PDX clonal selection. Metastasis formation followed both monoclonal and polyclonal seeding models. In four cases, metastasis-seeding clones were not identified in any primary region, consistent with a metastasis-seeding-metastasis model. PDXs underrepresented the subclonal heterogeneity of parental tumors. These suggest that single sample tumor sequencing and current PDX models may be insufficient to guide precision medicine.
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http://dx.doi.org/10.1126/sciadv.aay9691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286679PMC
June 2020

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

medRxiv 2020 May 11. Epub 2020 May 11.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 10^2 viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples are needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.
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http://dx.doi.org/10.1101/2020.05.07.20093542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273276PMC
May 2020

Remethylation of hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing.

Proc Natl Acad Sci U S A 2020 02 29;117(6):3123-3134. Epub 2020 Jan 29.

Division of Oncology, Section of Stem Cell Biology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110;

Mutations in the DNA methyltransferase 3A () gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine bone marrow cells. To determine whether the hypomethylation phenotype of hematopoietic cells is reversible, we developed an inducible transgene to restore expression of in transplanted bone marrow cells from mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function mutations.
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http://dx.doi.org/10.1073/pnas.1918611117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022185PMC
February 2020

A general approach for detecting expressed mutations in AML cells using single cell RNA-sequencing.

Nat Commun 2019 08 14;10(1):3660. Epub 2019 Aug 14.

Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA.

Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5' Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous.
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http://dx.doi.org/10.1038/s41467-019-11591-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694122PMC
August 2019

Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

Nat Immunol 2019 08 17;20(8):980-991. Epub 2019 Jun 17.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.
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http://dx.doi.org/10.1038/s41590-019-0425-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685551PMC
August 2019

Author Correction: The prognostic effects of somatic mutations in ER-positive breast cancer.

Nat Commun 2018 11 14;9(1):4850. Epub 2018 Nov 14.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis 63108, MO, USA.

The original version of this Article contained errors in the depiction of confidence intervals in the NF1 BCSS data illustrated in Figure 3b. These have now been corrected in both the PDF and HTML versions of the Article. The incorrect version of Figure 3b is presented in the associated Author Correction.
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http://dx.doi.org/10.1038/s41467-018-07407-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235964PMC
November 2018

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

High-Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune-Checkpoint Cancer Therapy.

Cell 2018 11 18;175(4):1014-1030.e19. Epub 2018 Oct 18.

Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Electronic address:

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.
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http://dx.doi.org/10.1016/j.cell.2018.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501221PMC
November 2018

Mutation Clearance after Transplantation for Myelodysplastic Syndrome.

N Engl J Med 2018 Sep;379(11):1028-1041

From the Department of Pathology and Immunology (E.J.D.), the Department of Medicine, Division of Oncology (M.A.J., C.A.M., N.E., J.S., K.E., J.R., S.E.H., K.B., L.D.W., M.J.C., I.P., J.S.W., G.L.U., D.C.L., J.F.D., P.W., T.J.L., M.J.W.), the McDonnell Genome Institute (G.S.C., C.A.M., H.A., R.S.F., C.C.F., M.O.), the Department of Medicine, Division of Hospital Medicine (R.S.), and Siteman Biostatistics Shared Resource, Siteman Cancer Center (K.T.), Washington University School of Medicine in St. Louis, St. Louis; and Massachusetts General Hospital Cancer Center, Boston (T.A.G.).

Background: Allogeneic hematopoietic stem-cell transplantation is the only curative treatment for patients with myelodysplastic syndrome (MDS). The molecular predictors of disease progression after transplantation are unclear.

Methods: We sequenced bone marrow and skin samples from 90 adults with MDS who underwent allogeneic hematopoietic stem-cell transplantation after a myeloablative or reduced-intensity conditioning regimen. We detected mutations before transplantation using enhanced exome sequencing, and we evaluated mutation clearance by using error-corrected sequencing to genotype mutations in bone marrow samples obtained 30 days after transplantation. In this exploratory study, we evaluated the association of a mutation detected after transplantation with disease progression and survival.

Results: Sequencing identified at least one validated somatic mutation before transplantation in 86 of 90 patients (96%); 32 of these patients (37%) had at least one mutation with a maximum variant allele frequency of at least 0.5% (equivalent to 1 heterozygous mutant cell in 100 cells) 30 days after transplantation. Patients with disease progression had mutations with a higher maximum variant allele frequency at 30 days than those who did not (median maximum variant allele frequency, 0.9% vs. 0%; P<0.001). The presence of at least one mutation with a variant allele frequency of at least 0.5% at day 30 was associated with a higher risk of progression (53.1% vs. 13.0%; conditioning regimen-adjusted hazard ratio, 3.86; 95% confidence interval [CI], 1.96 to 7.62; P<0.001) and a lower 1-year rate of progression-free survival than the absence of such a mutation (31.3% vs. 59.3%; conditioning regimen-adjusted hazard ratio for progression or death, 2.22; 95% CI, 1.32 to 3.73; P=0.005). The rate of progression-free survival was lower among patients who had received a reduced-intensity conditioning regimen and had at least one persistent mutation with a variant allele frequency of at least 0.5% at day 30 than among patients with other combinations of conditioning regimen and mutation status (P≤0.001). Multivariate analysis confirmed that patients who had a mutation with a variant allele frequency of at least 0.5% detected at day 30 had a higher risk of progression (hazard ratio, 4.48; 95% CI, 2.21 to 9.08; P<0.001) and a lower 1-year rate of progression-free survival than those who did not (hazard ratio for progression or death, 2.39; 95% CI, 1.40 to 4.09; P=0.002).

Conclusions: The risk of disease progression was higher among patients with MDS in whom persistent disease-associated mutations were detected in the bone marrow 30 days after transplantation than among those in whom these mutations were not detected. (Funded by the Leukemia and Lymphoma Society and others.).
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http://dx.doi.org/10.1056/NEJMoa1804714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309244PMC
September 2018

The prognostic effects of somatic mutations in ER-positive breast cancer.

Nat Commun 2018 09 4;9(1):3476. Epub 2018 Sep 4.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, 63108, MO, USA.

Here we report targeted sequencing of 83 genes using DNA from primary breast cancer samples from 625 postmenopausal (UBC-TAM series) and 328 premenopausal (MA12 trial) hormone receptor-positive (HR+) patients to determine interactions between somatic mutation and prognosis. Independent validation of prognostic interactions was achieved using data from the METABRIC study. Previously established associations between MAP3K1 and PIK3CA mutations with luminal A status/favorable prognosis and TP53 mutations with Luminal B/non-luminal tumors/poor prognosis were observed, validating the methodological approach. In UBC-TAM, NF1 frame-shift nonsense (FS/NS) mutations were also a poor outcome driver that was validated in METABRIC. For MA12, poor outcome associated with PIK3R1 mutation was also reproducible. DDR1 mutations were strongly associated with poor prognosis in UBC-TAM despite stringent false discovery correction (q = 0.0003). In conclusion, uncommon recurrent somatic mutations should be further explored to create a more complete explanation of the highly variable outcomes that typifies ER+ breast cancer.
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http://dx.doi.org/10.1038/s41467-018-05914-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123466PMC
September 2018

A multiple myeloma-specific capture sequencing platform discovers novel translocations and frequent, risk-associated point mutations in IGLL5.

Blood Cancer J 2018 03 21;8(3):35. Epub 2018 Mar 21.

Department of Medicine, Washington University School of Medicine, St. Louis, 63110, MO, USA.

Multiple myeloma (MM) is a disease of copy number variants (CNVs), chromosomal translocations, and single-nucleotide variants (SNVs). To enable integrative studies across these diverse mutation types, we developed a capture-based sequencing platform to detect their occurrence in 465 genes altered in MM and used it to sequence 95 primary tumor-normal pairs to a mean depth of 104×. We detected cases of hyperdiploidy (23%), deletions of 1p (8%), 6q (21%), 8p (17%), 14q (16%), 16q (22%), and 17p (4%), and amplification of 1q (19%). We also detected IGH and MYC translocations near expected frequencies and non-silent SNVs in NRAS (24%), KRAS (21%), FAM46C (17%), TP53 (9%), DIS3 (9%), and BRAF (3%). We discovered frequent mutations in IGLL5 (18%) that were mutually exclusive of RAS mutations and associated with increased risk of disease progression (p = 0.03), suggesting that IGLL5 may be a stratifying biomarker. We identified novel IGLL5/IGH translocations in two samples. We subjected 15 of the pairs to ultra-deep sequencing (1259×) and found that although depth correlated with number of mutations detected (p = 0.001), depth past ~300× added little. The platform provides cost-effective genomic analysis for research and may be useful in individualizing treatment decisions in clinical settings.
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http://dx.doi.org/10.1038/s41408-018-0062-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862875PMC
March 2018

Subclones dominate at MDS progression following allogeneic hematopoietic cell transplant.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Department of Medicine, Division of Oncology.

Allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative treatment for myelodysplastic syndromes (MDS), but patients who relapse after transplant have poor outcomes. In order to understand the contribution of tumor clonal evolution to disease progression,we applied exome and error-corrected targeted sequencing coupled with copy number analysis to comprehensively define changes in the clonal architecture of MDS in response to therapy using 51 serially acquired tumor samples from 9 patients who progressed after an alloHCT. We show that small subclones before alloHCT can drive progression after alloHCT. Notably, at least one subclone expanded or emerged at progression in all patients. Newly acquired structural variants (SVs) were present in an emergent/expanding subclone in 8 of 9 patients at progression, implicating the acquisition of SVs as important late subclonal progression events. In addition, pretransplant therapy with azacitidine likely influenced the mutation spectrum and evolution of emergent subclones after alloHCT. Although subclone evolution is common, founding clone mutations are always present at progression and could be detected in the bone marrow as early as 30 and/or 100 days after alloHCT in 6 of 8 (75%) patients, often prior to clinical progression. In conclusion, MDS progression after alloHCT is characterized by subclonal expansion and evolution, which can be influenced by pretransplant therapy.
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http://dx.doi.org/10.1172/jci.insight.98962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922277PMC
March 2018

An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies.

Leukemia 2018 09 20;32(9):1970-1983. Epub 2018 Feb 20.

Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA.

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.
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http://dx.doi.org/10.1038/s41375-018-0065-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102094PMC
September 2018

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

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Nat Commun 2017 05 16;8:15451. Epub 2017 May 16.

The McDonnell Genome Institute, Washington University, Saint Louis, Missouri 63108 USA.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.
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http://dx.doi.org/10.1038/ncomms15451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440852PMC
May 2017

CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression.

Cell 2017 02 16;168(5):801-816.e13. Epub 2017 Feb 16.

Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; The McDonnell Genome Institute, Washington University, St. Louis, MO 63110, USA. Electronic address:

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3A, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3A mutations. Non-leukemic hematopoietic cells with DNMT3A displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3A mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3A, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.
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http://dx.doi.org/10.1016/j.cell.2017.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328582PMC
February 2017

Mutant U2AF1-expressing cells are sensitive to pharmacological modulation of the spliceosome.

Nat Commun 2017 01 9;8:14060. Epub 2017 Jan 9.

Division of Oncology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations-drug and mutation-compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.
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http://dx.doi.org/10.1038/ncomms14060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5227701PMC
January 2017

Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma.

Blood 2017 01 14;129(4):473-483. Epub 2016 Nov 14.

McDonnell Genome Institute, Department of Medicine.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment.
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http://dx.doi.org/10.1182/blood-2016-07-729954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270390PMC
January 2017

TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes.

N Engl J Med 2016 11;375(21):2023-2036

the Department of Internal Medicine, Division of Oncology (J.S.W., L.D.W., G.L.U., A.G., M.H.T., I.P., R.R., T.A.F., K.E.S.-G., R.V., S.T.O., C.N.A., A.F.C., M.A.S., M.A.J., S.E.H., K.L., M.R.J., M.J.W., P.W., D.C.L., J.F.D., T.J.L.), and the Division of Biostatistics (J.D.B.), and the Department of Pathology and Immunology (E.J.D., B.T., Y.-S.L.), Washington University School of Medicine, and McDonnell Genome Institute, Washington University in St. Louis (A.A.P., C.A.M., C.C.F., M.O., R.S.F., R.K.W., L.D.W., T.J.L.) - both in St. Louis; the Departments of Internal Medicine (A.H., N.K., M.J.S., W.S.) and Pharmacy (R.W.K.), University of Chicago, Chicago; and the Department of Internal Medicine (T.A.G.), Massachusetts General Hospital, Boston.

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 .).
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http://dx.doi.org/10.1056/NEJMoa1605949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217532PMC
November 2016