Charles Gawad

All Publications

• Defining heritability, plasticity, and transition dynamics of cellular phenotypes in somatic evolution. Nature genetics Schiffman, J. S., D'Avino, A. R., Prieto, T., Pang, Y., Fan, Y., Rajagopalan, S., Potenski, C., Hara, T., Suvà, M. L., Gawad, C., Landau, D. A. 2024

  Abstract

  Single-cell sequencing has characterized cell state heterogeneity across diverse healthy and malignant tissues. However, the plasticity or heritability of these cell states remains largely unknown. To address this, we introduce PATH (phylogenetic analysis of trait heritability), a framework to quantify cell state heritability versus plasticity and infer cell state transition and proliferation dynamics from single-cell lineage tracing data. Applying PATH to a mouse model of pancreatic cancer, we observed heritability at the ends of the epithelial-to-mesenchymal transition spectrum, with higher plasticity at more intermediate states. In primary glioblastoma, we identified bidirectional transitions between stem- and mesenchymal-like cells, which use the astrocyte-like state as an intermediary. Finally, we reconstructed a phylogeny from single-cell whole-genome sequencing in B cell acute lymphoblastic leukemia and delineated the heritability of B cell differentiation states linked with genetic drivers. Altogether, PATH replaces qualitative conceptions of plasticity with quantitative measures, offering a framework to study somatic evolution.

  View details for DOI 10.1038/s41588-024-01920-6

  View details for PubMedID 39317739

  View details for PubMedCentralID 8100173

• scMicrobe PTA: near complete genomes from single bacterial cells. ISME communications Bowers, R. M., Gonzalez-Pena, V., Wardhani, K., Goudeau, D., Blow, M. J., Udwary, D., Klein, D., Vill, A. C., Brito, I. L., Woyke, T., Malmstrom, R. R., Gawad, C. 2024; 4 (1): ycae085

  Abstract

  Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

  View details for DOI 10.1093/ismeco/ycae085

  View details for PubMedID 39021442

• Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia. Genome medicine Mumme, H. L., Raikar, S. S., Bhasin, S. S., Thomas, B. E., Lawrence, T., Weinzierl, E. P., Pang, Y., DeRyckere, D., Gawad, C., Wechsler, D. S., Porter, C. C., Castellino, S. M., Graham, D. K., Bhasin, M. 2023; 15 (1): 83

  Abstract

  Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape.We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes.B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples.We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

  View details for DOI 10.1186/s13073-023-01241-z

  View details for PubMedID 37845689

  View details for PubMedCentralID PMC10577904

• Single-cell Retrieval from Clinical Cytology Slides Under Morphologic Guidance Facilitates Future Comprehensive Genomic Profiling from Paucicellular Samples Zhu, Y., Aragon, A., Wang, A., Gonzalez-pena, V., Gawad, C., Lowe, A. ELSEVIER SCIENCE INC. 2023: S388

  View details for Web of Science ID 000990969800408

• Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia. HemaSphere Pilheden, M., Ahlgren, L., Hyrenius-Wittsten, A., Gonzalez-Pena, V., Sturesson, H., Hansen Marquart, H. V., Lausen, B., Castor, A., Pronk, C. J., Barbany, G., Pokrovskaja Tamm, K., Fogelstrand, L., Lohi, O., Noren-Nystrom, U., Asklin, J., Chen, Y., Song, G., Walsh, M., Ma, J., Zhang, J., Saal, L. H., Gawad, C., Hagstrom-Andersson, A. K. 2022; 6 (10): e785

  Abstract

  Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3 D835H or NRAS G13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

  View details for DOI 10.1097/HS9.0000000000000785

  View details for PubMedID 36204688

• Single-cell genome sequencing of human neurons identifies somatic point mutation and indel enrichment in regulatory elements. Nature genetics Luquette, L. J., Miller, M. B., Zhou, Z., Bohrson, C. L., Zhao, Y., Jin, H., Gulhan, D., Ganz, J., Bizzotto, S., Kirkham, S., Hochepied, T., Libert, C., Galor, A., Kim, J., Lodato, M. A., Garaycoechea, J. I., Gawad, C., West, J., Walsh, C. A., Park, P. J. 2022

  Abstract

  Accurate somatic mutation detection from single-cell DNA sequencing is challenging due to amplification-related artifacts. To reduce this artifact burden, an improved amplification technique, primary template-directed amplification (PTA), was recently introduced. We analyzed whole-genome sequencing data from 52 PTA-amplified single neurons using SCAN2, a new genotyper we developed to leverage mutation signatures and allele balance in identifying somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) in PTA data. Our analysis confirms an increase in nonclonal somatic mutation in single neurons with age, but revises the estimated rate of this accumulation to 16 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels increase by at least three per year per neuron and are enriched in functional regions of the genome such as enhancers and promoters. Our data suggest that indels in gene-regulatory elements have a considerable effect on genome integrity in human neurons.

  View details for DOI 10.1038/s41588-022-01180-2

  View details for PubMedID 36163278

• Simultaneous monitoring of disease and microbe dynamics through plasma DNA sequencing in pediatric patients with acute lymphoblastic leukemia. Science advances Barsan, V., Xia, Y., Klein, D., Gonzalez-Pena, V., Youssef, S., Inaba, Y., Mahmud, O., Natarajan, S., Agarwal, V., Pang, Y., Autry, R., Pui, C. H., Inaba, H., Evans, W., Gawad, C. 2022; 8 (16): eabj1360

  Abstract

  Treatment of acute lymphoblastic leukemia (ALL) necessitates continuous risk assessment of leukemic disease burden and infections that arise in the setting of immunosuppression. This study was performed to assess the feasibility of a hybrid capture next-generation sequencing panel to longitudinally measure molecular leukemic disease clearance and microbial species abundance in 20 pediatric patients with ALL throughout induction chemotherapy. This proof of concept helps establish a technical and conceptual framework that we anticipate will be expanded and applied to additional patients with leukemia, as well as extended to additional cancer types. Molecular monitoring can help accelerate the attainment of insights into the temporal biology of host-microbe-leukemia interactions, including how those changes correlate with and alter anticancer therapy efficacy. We also anticipate that fewer invasive bone marrow examinations will be required, as these methods improve with standardization and are validated for clinical use.

  View details for DOI 10.1126/sciadv.abj1360

  View details for PubMedID 35442732

• Bringing precision oncology to cellular resolution with single-cell genomics. Clinical & experimental metastasis Xia, Y., Gawad, C. 2021

  Abstract

  Single-cell sequencing technologies have undergone rapid development and adoption by the scientific community in the past 5years, fueling discoveries about the etiology, pathogenesis, and treatment responsiveness of individual tumor cells within cancer ecosystems. Most of the advancements in our understanding of cancer with these new technologies have focused on basic tumor biology. However, the knowledge produced by these and other studies are beginning to provide biomarkers and drug targets for clinically-relevant subpopulations within a tumor, creating opportunities for the development of biologically-informed, clone-specific combination treatment strategies. Here we provide an overview of the development of the field of single-cell cancer sequencing and provide a roadmap for shepherding these technologies from research tools to diagnostic instruments that provide high-resolution, treatment-directing details of tumors to clinical oncologists.

  View details for DOI 10.1007/s10585-021-10129-4

  View details for PubMedID 34807338

• Accurate genomic variant detection in single cells with primary template-directed amplification. Proceedings of the National Academy of Sciences of the United States of America Gonzalez-Pena, V., Natarajan, S., Xia, Y., Klein, D., Carter, R., Pang, Y., Shaner, B., Annu, K., Putnam, D., Chen, W., Connelly, J., Pruett-Miller, S., Chen, X., Easton, J., Gawad, C. 2021; 118 (24)

  Abstract

  Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

  View details for DOI 10.1073/pnas.2024176118

  View details for PubMedID 34099548

• Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia. Nature cancer Autry, R. J., Paugh, S. W., Carter, R. n., Shi, L. n., Liu, J. n., Ferguson, D. C., Lau, C. E., Bonten, E. J., Yang, W. n., McCorkle, J. R., Beard, J. A., Panetta, J. C., Diedrich, J. D., Crews, K. R., Pei, D. n., Coke, C. J., Natarajan, S. n., Khatamian, A. n., Karol, S. E., Lopez-Lopez, E. n., Diouf, B. n., Smith, C. n., Gocho, Y. n., Hagiwara, K. n., Roberts, K. G., Pounds, S. n., Kornblau, S. M., Stock, W. n., Paietta, E. M., Litzow, M. R., Inaba, H. n., Mullighan, C. G., Jeha, S. n., Pui, C. H., Cheng, C. n., Savic, D. n., Yu, J. n., Gawad, C. n., Relling, M. V., Yang, J. J., Evans, W. E. 2020; 1 (3): 329–44

  Abstract

  Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.

  View details for DOI 10.1038/s43018-020-0037-3

  View details for PubMedID 32885175

  View details for PubMedCentralID PMC7467080

• Evaluation of Plasma Microbial Cell-Free DNA Sequencing to Predict Bloodstream Infection in Pediatric Patients With Relapsed or Refractory Cancer. JAMA oncology Goggin, K. P., Gonzalez-Pena, V. n., Inaba, Y. n., Allison, K. J., Hong, D. K., Ahmed, A. A., Hollemon, D. n., Natarajan, S. n., Mahmud, O. n., Kuenzinger, W. n., Youssef, S. n., Brenner, A. n., Maron, G. n., Choi, J. n., Rubnitz, J. E., Sun, Y. n., Tang, L. n., Wolf, J. n., Gawad, C. n. 2019

  Abstract

  Bloodstream infection (BSI) is a common, life-threatening complication of treatment for cancer. Predicting BSI before onset of clinical symptoms would enable preemptive therapy, but there is no reliable screening test.To estimate sensitivity and specificity of plasma microbial cell-free DNA sequencing (mcfDNA-seq) for predicting BSI in patients at high risk of life-threatening infection.A prospective pilot cohort study of mcfDNA-seq for predicting BSI in pediatric patients (<25 years of age) with relapsed or refractory cancers at St Jude Children's Research Hospital, a specialist quaternary pediatric hematology-oncology referral center. Remnant clinical blood samples were collected during chemotherapy and hematopoietic cell transplantation. Samples collected during the 7 days before and at onset of BSI episodes, along with negative control samples from study participants, underwent blinded testing using a mcfDNA-seq test in a Clinical Laboratory Improvement Amendments/College of American Pathologists-approved laboratory.The primary outcomes were sensitivity of mcfDNA-seq for detecting a BSI pathogen during the 3 days before BSI onset and specificity of mcfDNA-seq in the absence of fever or infection in the preceding or subsequent 7 days.Between August 9, 2017, and June 4, 2018, 47 participants (27 [57%] male; median age [IQR], 10 [5-14] years) were enrolled; 19 BSI episodes occurred in 12 participants, and predictive samples were available for 16 episodes, including 15 bacterial BSI episodes. In the 3 days before the onset of infection, predictive sensitivity of mcfDNA-seq was 75% for all BSIs (12 of 16; 95% CI, 51%-90%) and 80% (12 of 15; 95% CI, 55%-93%) for bacterial BSIs. The specificity of mcfDNA-seq, evaluated on 33 negative control samples from enrolled participants, was 82% (27 of 33; 95% CI, 66%-91%) for any bacterial or fungal organism and 91% (30 of 33; 95% CI, 76%-97%) for any common BSI pathogen, and the concentration of pathogen DNA was lower in control than predictive samples.A clinically relevant pathogen can be identified by mcfDNA-seq days before the onset of BSI in a majority of episodes, potentially enabling preemptive treatment. Clinical application appears feasible pending further study.ClinicalTrials.gov identifier: NCT03226158.

  View details for DOI 10.1001/jamaoncol.2019.4120

  View details for PubMedID 31855231

• Sequencing the Genomes of Single Cells. Methods in molecular biology (Clifton, N.J.) Gonzalez-Pena, V. n., Gawad, C. n. 2019; 1979: 227–34

  Abstract

  Single-cell genome sequencing can detect low-frequency genetic alterations present in complex tissues. However, the experimental procedures are technically challenging. This includes dissociation of the tissue, isolation of single cells, whole-genome amplification, sequencing library preparation, and an optional target enrichment. Here we describe how to perform each of these processes to obtain high-quality single-cell genome sequencing data.

  View details for DOI 10.1007/978-1-4939-9240-9_14

  View details for PubMedID 31028641

• Single-cell RNA sequencing reveals the impact of chromosomal instability on glioblastoma cancer stem cells. BMC medical genomics Zhao, Y. n., Carter, R. n., Natarajan, S. n., Varn, F. S., Compton, D. A., Gawad, C. n., Cheng, C. n., Godek, K. M. 2019; 12 (1): 79

  Abstract

  Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. A major source of genetic heterogeneity comes from DNA sequence differences and/or whole chromosome and focal copy number variations (CNVs). Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Accordingly, CIN causes constantly changing karyotypes that result in extensive cell-to-cell genetic heterogeneity. How the genetic heterogeneity caused by CIN influences gene expression in individual cells remains unknown.We performed single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line to investigate the impact of CNV due to CIN on gene expression. From the gene expression data, we computationally inferred large-scale CNVs in single cells. Also, we performed copy number adjusted differential gene expression analysis between NSCs and glioblastoma CSCs to identify copy number dependent and independent differentially expressed genes.Here, we demonstrate that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we show that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival.These results demonstrate that CIN is directly responsible for gene expression changes and contributes to both genetic and transcriptional heterogeneity among glioblastoma CSCs. These results also demonstrate that the expression of some genes is buffered against changes in copy number, thus preserving some consistency in gene expression levels from cell-to-cell despite the continuous change in karyotype driven by CIN. Importantly, a gene signature derived from the subset of genes whose expression is buffered against copy number alterations correlates with tumor grade and is prognostic for patient survival that could facilitate patient diagnosis and treatment.

  View details for DOI 10.1186/s12920-019-0532-5

  View details for PubMedID 31151460

  View details for PubMedCentralID PMC6545015

• Resolving medulloblastoma cellular architecture by single-cell genomics. Nature Hovestadt, V. n., Smith, K. S., Bihannic, L. n., Filbin, M. G., Shaw, M. L., Baumgartner, A. n., DeWitt, J. C., Groves, A. n., Mayr, L. n., Weisman, H. R., Richman, A. R., Shore, M. E., Goumnerova, L. n., Rosencrance, C. n., Carter, R. A., Phoenix, T. N., Hadley, J. L., Tong, Y. n., Houston, J. n., Ashmun, R. A., DeCuypere, M. n., Sharma, T. n., Flasch, D. n., Silkov, A. n., Ligon, K. L., Pomeroy, S. L., Rivera, M. N., Rozenblatt-Rosen, O. n., Rusert, J. M., Wechsler-Reya, R. J., Li, X. N., Peyrl, A. n., Gojo, J. n., Kirchhofer, D. n., Lötsch, D. n., Czech, T. n., Dorfer, C. n., Haberler, C. n., Geyeregger, R. n., Halfmann, A. n., Gawad, C. n., Easton, J. n., Pfister, S. M., Regev, A. n., Gajjar, A. n., Orr, B. A., Slavc, I. n., Robinson, G. W., Bernstein, B. E., Suvà, M. L., Northcott, P. A. 2019; 572 (7767): 74–79

  Abstract

  Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

  View details for DOI 10.1038/s41586-019-1434-6

  View details for PubMedID 31341285

• Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs. Nature communications Ganuza, M. n., Chabot, A. n., Tang, X. n., Bi, W. n., Natarajan, S. n., Carter, R. n., Gawad, C. n., Kang, G. n., Cheng, Y. n., McKinney-Freeman, S. n. 2018; 9 (1): 5405

  Abstract

  The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.

  View details for DOI 10.1038/s41467-018-07769-8

  View details for PubMedID 30573729

  View details for PubMedCentralID PMC6302089

• Measurable residual disease detection by high-throughput sequencing improves risk stratification for pediatric B-ALL. Blood Wood, B. n., Wu, D. n., Crossley, B. n., Dai, Y. n., Williamson, D. n., Gawad, C. n., Borowitz, M. J., Devidas, M. n., Maloney, K. W., Larsen, E. n., Winick, N. n., Raetz, E. n., Carroll, W. L., Hunger, S. P., Loh, M. L., Robins, H. n., Kirsch, I. n. 2018; 131 (12): 1350–59

  Abstract

  Early response to induction chemotherapy is an important prognostic factor in B-lymphoblastic leukemia (B-ALL). Here, we compare high-throughput sequencing (HTS) of IGH and TRG genes vs flow cytometry (FC) for measurable residual disease (MRD) detection at the end of induction chemotherapy in pediatric patients with newly diagnosed B-ALL. Six hundred nineteen paired pretreatment and end-of-induction bone marrow samples from Children's Oncology Group studies AALL0331 (clinicaltrials.gov #NCT00103285) (standard risk [SR]; with MRD by FC at any level) and AALL0232 (clinicaltrials.gov #NCT00075725) (high risk; with day 29 MRD <0.1% by FC) were evaluated by HTS and FC for event-free (EFS) and overall survival (OS). HTS and FC showed similar 5-year EFS and OS for MRD-positive and -negative patients using an MRD threshold of 0.01%. However, there was a high discordant rate with HTS identifying 55 (38.7%) more patients MRD positive at this threshold. These discrepant patients have worse outcomes than FC MRD-negative patients. In addition, the increased analytic sensitivity of HTS permitted identification of 19.9% of SR patients without MRD at any detectable level who had excellent 5-year EFS (98.1%) and OS (100%). The higher analytic sensitivity and lower false-negative rate of HTS improves upon FC for MRD detection in pediatric B-ALL by identifying a novel subset of patients at end of induction who are essentially cured using current chemotherapy and identifying MRD at 0.01% in up to one-third of patients who are missed at the same threshold by FC.

  View details for DOI 10.1182/blood-2017-09-806521

  View details for PubMedID 29284596

  View details for PubMedCentralID PMC5865233

• Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours. Nature Ma, X. n., Liu, Y. n., Liu, Y. n., Alexandrov, L. B., Edmonson, M. N., Gawad, C. n., Zhou, X. n., Li, Y. n., Rusch, M. C., Easton, J. n., Huether, R. n., Gonzalez-Pena, V. n., Wilkinson, M. R., Hermida, L. C., Davis, S. n., Sioson, E. n., Pounds, S. n., Cao, X. n., Ries, R. E., Wang, Z. n., Chen, X. n., Dong, L. n., Diskin, S. J., Smith, M. A., Guidry Auvil, J. M., Meltzer, P. S., Lau, C. C., Perlman, E. J., Maris, J. M., Meshinchi, S. n., Hunger, S. P., Gerhard, D. S., Zhang, J. n. 2018; 555 (7696): 371–76

  Abstract

  Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.

  View details for DOI 10.1038/nature25795

  View details for PubMedID 29489755

  View details for PubMedCentralID PMC5854542

• High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor. PLoS genetics Yamashita, T. n., Zheng, F. n., Finkelstein, D. n., Kellard, Z. n., Carter, R. n., Rosencrance, C. D., Sugino, K. n., Easton, J. n., Gawad, C. n., Zuo, J. n. 2018; 14 (7): e1007552

  Abstract

  In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.

  View details for DOI 10.1371/journal.pgen.1007552

  View details for PubMedID 30063705

  View details for PubMedCentralID PMC6086484

• A Single-Cell Transcriptional Atlas of the Developing Murine Cerebellum. Current biology : CB Carter, R. A., Bihannic, L. n., Rosencrance, C. n., Hadley, J. L., Tong, Y. n., Phoenix, T. N., Natarajan, S. n., Easton, J. n., Northcott, P. A., Gawad, C. n. 2018; 28 (18): 2910–20.e2

  Abstract

  The cerebellum develops from a restricted number of cell types that precisely organize to form the circuitry that controls sensory-motor coordination and some higher-order cognitive processes. To acquire an enhanced understanding of the molecular processes that mediate cerebellar development, we performed single-cell RNA-sequencing of 39,245 murine cerebellar cells at twelve critical developmental time points. Using recognized lineage markers, we confirmed that the single-cell data accurately recapitulate cerebellar development. We then followed distinct populations from emergence through migration and differentiation, and determined the associated transcriptional cascades. After identifying key lineage commitment decisions, focused analyses uncovered waves of transcription factor expression at those branching points. Finally, we created Cell Seek, a flexible online interface that facilitates exploration of the dataset. Our study provides a transcriptional summarization of cerebellar development at single-cell resolution that will serve as a valuable resource for future investigations of cerebellar development, neurobiology, and disease.

  View details for DOI 10.1016/j.cub.2018.07.062

  View details for PubMedID 30220501

• LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance. Cell Cunha, L. D., Yang, M. n., Carter, R. n., Guy, C. n., Harris, L. n., Crawford, J. C., Quarato, G. n., Boada-Romero, E. n., Kalkavan, H. n., Johnson, M. D., Natarajan, S. n., Turnis, M. E., Finkelstein, D. n., Opferman, J. T., Gawad, C. n., Green, D. R. 2018; 175 (2): 429–41.e16

  Abstract

  Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.

  View details for DOI 10.1016/j.cell.2018.08.061

  View details for PubMedID 30245008

  View details for PubMedCentralID PMC6201245

• Genome-wide segregation of single nucleotide and structural variants into single cancer cells. BMC genomics Easton, J. n., Gonzalez-Pena, V. n., Yergeau, D. n., Ma, X. n., Gawad, C. n. 2017; 18 (1): 906

  Abstract

  Single-cell genome sequencing provides high-resolution details of the clonal genomic modifications that occur during cancer initiation, progression, and ongoing evolution as patients undergo treatment. One limitation of current single-cell sequencing strategies is a suboptimal capacity to detect all classes of single-nucleotide and structural variants in the same cells.Here we present a new approach for determining comprehensive variant profiles of single cells using a microfluidic amplicon-based strategy to detect structural variant breakpoint sequences instead of using relative read depth to infer copy number changes. This method can reconstruct the clonal architecture and mutational history of a malignancy using all classes and sizes of somatic variants, providing more complete details of the temporal changes in mutational classes and processes that led to the development of a malignant neoplasm. Using this approach, we interrogated cells from a patient with leukemia, determining that processes producing structural variation preceded single nucleotide changes in the development of that malignancy.All classes and sizes of genomic variants can be efficiently detected in single cancer cells using our new method, enabling the ordering of distinct classes of mutations during tumor evolution.

  View details for DOI 10.1186/s12864-017-4286-1

  View details for PubMedID 29178827

  View details for PubMedCentralID PMC5702214

• Early somatic mosaicism is a rare cause of long-QT syndrome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Priest, J. R., Gawad, C., Kahlig, K. M., Yu, J. K., O'Hara, T., Boyle, P. M., Rajamani, S., Clark, M. J., Garcia, S. T., Ceresnak, S., Harris, J., Boyle, S., Dewey, F. E., Malloy-Walton, L., Dunn, K., Grove, M., Perez, M. V., Neff, N. F., Chen, R., Maeda, K., Dubin, A., Belardinelli, L., West, J., Antolik, C., Macaya, D., Quertermous, T., Trayanova, N. A., Quake, S. R., Ashley, E. A. 2016; 113 (41): 11555-11560

  Abstract

  Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

  View details for DOI 10.1073/pnas.1607187113

  View details for PubMedID 27681629

• Single-cell genome sequencing: current state of the science NATURE REVIEWS GENETICS Gawad, C., Koh, W., Quake, S. R. 2016; 17 (3): 175-188

  Abstract

  The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years.

  View details for DOI 10.1038/nrg.2015.16

  View details for Web of Science ID 000371739200012

  View details for PubMedID 26806412

• Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells. PloS one Koh, W., Gonzalez, V., Natarajan, S., Carter, R., Brown, P. O., Gawad, C. 2016; 11 (10)

  Abstract

  Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.

  View details for DOI 10.1371/journal.pone.0164085

  View details for PubMedID 27736885

  View details for PubMedCentralID PMC5063410

• Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proceedings of the National Academy of Sciences of the United States of America Gawad, C., Koh, W., Quake, S. R. 2014; 111 (50): 17947-17952

  Abstract

  Many cancers have substantial genomic heterogeneity within a given tumor, and to fully understand that diversity requires the ability to perform single cell analysis. We performed targeted sequencing of a panel of single nucleotide variants (SNVs), deletions, and IgH sequences in 1,479 single tumor cells from six acute lymphoblastic leukemia (ALL) patients. By accurately segregating groups of cooccurring mutations into distinct clonal populations, we identified codominant clones in the majority of patients. Evaluation of intraclonal mutation patterns identified clone-specific punctuated cytosine mutagenesis events, showed that most structural variants are acquired before SNVs, determined that KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and identified clones within the same patient that are arrested at varied stages in B-cell development. Taken together, these data order the sequence of genetic events that underlie childhood ALL and provide a framework for understanding the development of the disease at single-cell resolution.

  View details for DOI 10.1073/pnas.1420822111

  View details for PubMedID 25425670

• Noninvasive in vivo monitoring of tissue-specific global gene expression in humans. Proceedings of the National Academy of Sciences of the United States of America Koh, W., Pan, W., Gawad, C., Fan, H. C., Kerchner, G. A., Wyss-Coray, T., Blumenfeld, Y. J., El-Sayed, Y. Y., Quake, S. R. 2014; 111 (20): 7361-7366

  Abstract

  Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer's disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

  View details for DOI 10.1073/pnas.1405528111

  View details for PubMedID 24799715

• A quantitative comparison of single-cell whole genome amplification methods. PloS one de Bourcy, C. F., De Vlaminck, I., Kanbar, J. N., Wang, J., Gawad, C., Quake, S. R. 2014; 9 (8)

  Abstract

  Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

  View details for DOI 10.1371/journal.pone.0105585

  View details for PubMedID 25136831

  View details for PubMedCentralID PMC4138190

• Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia BLOOD Gawad, C., Pepin, F., Carlton, V. E., Klinger, M., Logan, A. C., Miklos, D. B., Faham, M., Dahl, G., Lacayo, N. 2012; 120 (22): 4407-4417

  Abstract

  The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

  View details for DOI 10.1182/blood-2012-05-429811

  View details for Web of Science ID 000313111300023

  View details for PubMedID 22932801

  View details for PubMedCentralID PMC3507147

• Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLOS ONE Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. 2012; 7 (2)

  Abstract

  Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells.

  View details for DOI 10.1371/journal.pone.0030733

  View details for Web of Science ID 000301977500016

  View details for PubMedID 22319583

  View details for PubMedCentralID PMC3270023

• Gene Expression Arrays in Pancreatic Cancer Drug Discovery Research DRUG DISCOVERY IN PANCREATIC CANCER: MODELS AND TECHNIQUES Gawad, C., Han, H., Grippo, P. 2010: 113-134

  View details for DOI 10.1007/978-1-4419-1160-5_6

  View details for Web of Science ID 000276882000006

• Towards molecular medicine - A case for a biological periodic table AMERICAN JOURNAL OF PHARMACOGENOMICS Gawad, C. 2005; 5 (4): 207-211

  Abstract

  The recently amplified pace of development in the technologies to study both normal and aberrant cellular physiology has allowed for a transition from the traditional reductionist approaches to global interrogations of human biology. This transformation has created the anticipation that we will soon more effectively treat or contain most types of diseases through a 'systems-based' approach to understanding and correcting the underlying etiology of these processes. However, to accomplish these goals, we must first have a more comprehensive understanding of all the elements involved in human cellular physiology, as well as why and how they interact. With the vast number of biological components that have and are being discovered, creating methods with modern computational techniques to better organize biological elements is the next requisite step in this process. This article aims to articulate the importance of the organization of chemical elements into a periodic table had on the conversion of chemistry into a quantitative, translatable science, as well as how we can apply the lessons learned in that transition to the current transformation taking place in biology.

  View details for Web of Science ID 000231842000001

  View details for PubMedID 16078857