Publications by authors named "Robert S Fulton"

117 Publications

Co-evolution of tumor and immune cells during progression of multiple myeloma.

Nat Commun 2021 05 7;12(1):2559. Epub 2021 May 7.

Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA.

Multiple myeloma (MM) is characterized by the uncontrolled proliferation of plasma cells. Despite recent treatment advances, it is still incurable as disease progression is not fully understood. To investigate MM and its immune environment, we apply single cell RNA and linked-read whole genome sequencing to profile 29 longitudinal samples at different disease stages from 14 patients. Here, we collect 17,267 plasma cells and 57,719 immune cells, discovering patient-specific plasma cell profiles and immune cell expression changes. Patients with the same genetic alterations tend to have both plasma cells and immune cells clustered together. By integrating bulk genomics and single cell mapping, we track plasma cell subpopulations across disease stages and find three patterns: stability (from precancer to diagnosis), and gain or loss (from diagnosis to relapse). In multiple patients, we detect "B cell-featured" plasma cell subpopulations that cluster closely with B cells, implicating their cell of origin. We validate AP-1 complex differential expression (JUN and FOS) in plasma cell subpopulations using CyTOF-based protein assays, and integrated analysis of single-cell RNA and CyTOF data reveals AP-1 downstream targets (IL6 and IL1B) potentially leading to inflammation regulation. Our work represents a longitudinal investigation for tumor and microenvironment during MM progression and paves the way for expanding treatment options.
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http://dx.doi.org/10.1038/s41467-021-22804-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105337PMC
May 2021

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

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

N Engl J Med 2021 03;384(10):924-935

From the Department of Pathology and Immunology (E.J.D., M.C.S., A.E.O.H., J.N., J.E.P., D.H.S.), McDonnell Genome Institute (M.O., R.W., S.M., A.B., S.K., J.G., F.D., R.S.F., D.H.S.), and the Divisions of Oncology (J.R., E.H., S.E.H., M.J.C., M.A.J., G.L.U., C.A.M., D.C.L., M.J.W., P.W., J.F.D., T.J.L., D.H.S.) and Biostatistics (J.D.B.), Department of Medicine, Washington University School of Medicine, St. Louis.

Background: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated.

Methods: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing.

Results: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different.

Conclusions: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.).
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http://dx.doi.org/10.1056/NEJMoa2024534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130455PMC
March 2021

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

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

Exome sequencing of Finnish isolates enhances rare-variant association power.

Nature 2019 08 31;572(7769):323-328. Epub 2019 Jul 31.

Center for Life Course Health Research, Faculty of Medicine, University of Oulu, Oulu, Finland.

Exome-sequencing studies have generally been underpowered to identify deleterious alleles with a large effect on complex traits as such alleles are mostly rare. Because the population of northern and eastern Finland has expanded considerably and in isolation following a series of bottlenecks, individuals of these populations have numerous deleterious alleles at a relatively high frequency. Here, using exome sequencing of nearly 20,000 individuals from these regions, we investigate the role of rare coding variants in clinically relevant quantitative cardiometabolic traits. Exome-wide association studies for 64 quantitative traits identified 26 newly associated deleterious alleles. Of these 26 alleles, 19 are either unique to or more than 20 times more frequent in Finnish individuals than in other Europeans and show geographical clustering comparable to Mendelian disease mutations that are characteristic of the Finnish population. We estimate that sequencing studies of populations without this unique history would require hundreds of thousands to millions of participants to achieve comparable association power.
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http://dx.doi.org/10.1038/s41586-019-1457-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697530PMC
August 2019

No evidence that G6PD deficiency affects the efficacy or safety of daunorubicin in acute lymphoblastic leukemia induction therapy.

Pediatr Blood Cancer 2019 06 7;66(6):e27681. Epub 2019 Mar 7.

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

Background/objectives: Anthracyclines are used in induction therapy of pediatric acute lymphoblastic leukemia (ALL) and are known to generate oxidative stress; whether this translates into enhanced antileukemic activity or hemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency is unknown.

Design/methods: Among 726 pediatric patients with newly diagnosed ALL treated at St. Jude Children's Research Hospital, 22 had deficient G6PD activity. We compared the prevalence of positive minimal residual disease (MRD) ≥1% at Day 15/Day 19 of induction or ≥0.01% at Day 42/Day 46 (end of induction) and the number of red blood cell (RBC) transfusions after daunorubicin in induction between patients with or without G6PD deficiency, adjusting for ALL risk group, treatment protocol, age, and gender.

Results: There was no difference in Day 15/19 (P = 1) or end of induction MRD (P = 0.76) nor in the number of RBC transfusions (P = 0.73); the lack of association with MRD was confirmed in a dataset of 1192 newly diagnosed male patients enrolled in a Children's Oncology Group trial (P = 0.78).

Conclusion: We found no evidence that G6PD deficiency affects daunorubicin activity during induction treatment for ALL.
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http://dx.doi.org/10.1002/pbc.27681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518412PMC
June 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

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer.

Nat Commun 2018 09 17;9(1):3787. Epub 2018 Sep 17.

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

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.
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http://dx.doi.org/10.1038/s41467-018-06162-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141466PMC
September 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

High-resolution comparative analysis of great ape genomes.

Science 2018 06;360(6393)

Bionano Genomics, San Diego, CA 92121, USA.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.
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http://dx.doi.org/10.1126/science.aar6343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178954PMC
June 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

Resistance-promoting effects of ependymoma treatment revealed through genomic analysis of multiple recurrences in a single patient.

Cold Spring Harb Mol Case Stud 2018 04 2;4(2). Epub 2018 Apr 2.

Institute for Genomic Medicine, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio 43205, USA.

As in other brain tumors, multiple recurrences after complete resection and irradiation of supratentorial ependymoma are common and frequently result in patient death. This standard-of-care treatment was established in the pregenomic era without the ability to evaluate the effect that mutagenic therapies may exert on tumor evolution and in promoting resistance, recurrence, and death. We seized a rare opportunity to characterize treatment effects and the evolution of a single patient's ependymoma across four recurrences after different therapies. A combination of high-depth whole-genome and exome-based DNA sequencing of germline and tumor specimens, RNA sequencing of tumor specimens, and advanced computational analyses were used. Treatment with radiation and chemotherapies resulted in a substantial increase in mutational burden and diversification of the tumor subclonal architecture without eradication of the founding clone. Notable somatic alterations included a driver, several epigenetic modifiers, and therapy-induced mutations that impacted multiple other cancer-relevant pathways and altered the neoantigen landscape. These genomic data provided new mechanistic insights into the genesis of ependymoma and pathways of resistance. They also revealed that radiation and chemotherapy were significant forces in shaping the increased subclonal complexity of each tumor recurrence while also failing to eradicate the founding clone. This raises the question of whether standard-of-care treatments have similar consequences in other patients with ependymoma and other types of brain tumors. If so, the perspective obtained by real-time genomic characterization of a tumor may be essential for making effective patient-specific and adaptive clinical decisions.
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http://dx.doi.org/10.1101/mcs.a002444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880262PMC
April 2018

Clinical outcomes and differential effects of PI3K pathway mutation in obese versus non-obese patients with cervical cancer.

Oncotarget 2018 Jan 23;9(3):4061-4073. Epub 2017 Dec 23.

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

The purpose of this study was to evaluate the effect of obesity and obesity-associated factors on the outcomes of patients with cervical cancer. Outcomes were evaluated in 591 patients with FIGO Ib to IV cervical cancer treated uniformly with definitive radiation. Patients were stratified into 3 groups based upon pretreatment Body Mass Index (BMI): A ≤ 18.5; B 18.6 - 34.9; and C ≥ 35. The 5-year freedom from failure rates were 58, 59, and 73% for BMI groups A, B, and C (p = 0.01). Overall survival rates were 50, 59, and 68%, respectively (p = 0.02). High expression of phosphorylated AKT (pAKT) was associated with poor outcomes only in non-obese patients. Obese patients with PI3K pathway mutant tumors had a trend toward favorable outcomes, while a similar effect was not observed in non-obese patients. Compared to similar tumors from non-obese hosts, and mutant tumors from obese patients failed to express high levels of phosphorylated AKT and its downstream targets. These results show that patients with obesity at the time of diagnosis of cervical cancer exhibit improved outcomes after radiation. PI3K/AKT pathway mutations are common in obese patients, but are not associated with activation of AKT signaling.
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http://dx.doi.org/10.18632/oncotarget.23664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790521PMC
January 2018

Cellular stressors contribute to the expansion of hematopoietic clones of varying leukemic potential.

Nat Commun 2018 01 31;9(1):455. Epub 2018 Jan 31.

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

Hematopoietic clones harboring specific mutations may expand over time. However, it remains unclear how different cellular stressors influence this expansion. Here we characterize clonal hematopoiesis after two different cellular stressors: cytotoxic therapy and hematopoietic transplantation. Cytotoxic therapy results in the expansion of clones carrying mutations in DNA damage response genes, including TP53 and PPM1D. Analyses of sorted populations show that these clones are typically multilineage and myeloid-biased. Following autologous transplantation, most clones persist with stable chimerism. However, DNMT3A mutant clones often expand, while PPM1D mutant clones often decrease in size. To assess the leukemic potential of these expanded clones, we genotyped 134 t-AML/t-MDS samples. Mutations in non-TP53 DNA damage response genes are infrequent in t-AML/t-MDS despite several being commonly identified after cytotoxic therapy. These data suggest that different hematopoietic stressors promote the expansion of distinct long-lived clones, carrying specific mutations, whose leukemic potential depends partially on the mutations they harbor.
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http://dx.doi.org/10.1038/s41467-018-02858-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792556PMC
January 2018

Somatic mutations and clonal hematopoiesis in congenital neutropenia.

Blood 2018 01 1;131(4):408-416. Epub 2017 Nov 1.

Division of Oncology, Department of Internal Medicine.

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls ( = NS). Clonal hematopoiesis due to mutations in was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, < .001) or patients with SCN (0/40, < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple mutations. Conversely, clonal hematopoiesis due to mutations of was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand -mutated HSPCs, suggesting that acquisition of mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.
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http://dx.doi.org/10.1182/blood-2017-08-801985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790127PMC
January 2018

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

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

Genome Res 2017 05 10;27(5):849-864. Epub 2017 Apr 10.

Pacific Biosciences, Menlo Park, California 94025, USA.

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.
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http://dx.doi.org/10.1101/gr.213611.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411779PMC
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