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Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules (‘single duplexes’) to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10−8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. Significance: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.

Supplementary Table from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Supplementary Figure from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.Significance:Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.See related commentary by Beytagh and Weiss, p. 2730.See related article by Mo et al., p. 2906.This article is highlighted in the In This Issue feature, p. 2711

NXT2 expression is associated with NXT1 dependency

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Supplementary Figure S1. Genome-scale CRISPR-Cas9 screening identifies a strong correlation between copy number and sensitivity to CRISPR-Cas9 genome editing. Supplementary Figure S2. Global summary of the relationship of CRISPR-Cas9 guide scores to genomic copy number for all 33 cell lines screened. Supplementary Figure S3. Amplified genes represent the strongest perceived dependencies in pooled CRISPR-Cas9 screening data. Figure S4. Evaluation of the influence of copy number on CRISPR-Cas9 dependency scores in published data from Hart et al. (25). Figure S5. Representative examples of structural variations leading to copy number amplification underlying the gene-independent anti-proliferative response to CRISPRCas9 targeting of loci within these regions.

This file contains supplementary methods, supplementary references, supplementary figures S1-S6, supplementary tables S1, S3-S6, S8-S15, and S17, and legends for all supplementary figures and tables. Supplementary Figure S1. Summary of Ewing sarcoma tumor and cell line cohorts. Supplementary Figure S2. RNASeq validation of mutations identified by WES and WGS. Supplementary Figure S3. Power calculations for the detection of significantly mutated genes. Supplementary Figure S4. Copy number variants in Ewing sarcoma tumors. Supplementary Figure S5. ETS1 deletions associated with EWS/FLI rearrangements. Supplementary Figure S6. Copy number variants in diagnostic vs. treated Ewing sarcoma tumors. Supplementary Figure S7. SCNAs in paired Ewing sarcoma tumor samples. Supplementary Table S1. Tissue samples. Supplementary Table S3. Multiply mutated genes. Supplementary Table S4. Rearrangements and fusions detected by WGS and RNASeq. Supplementary Table S5. Mutational significance for tumor/normal pairs. Supplementary Table S6. Rate of coding mutations in Ewing sarcoma tumors. Supplementary Table S8. Mutational significance for all tumors. Supplementary Table S9. Gene set enrichment analyses of mutated genes. Supplementary Table S10. Significance of arm-level SCNAs. Supplementary Table S11. Multiply mutated genes of interest in all sample cohorts. Supplementary Table S12. Immunohistochemical staining of STAG2 in Ewing sarcoma tumors. Supplementary Table S13. Gene set enrichment analyses of STAG2 expressing vs. STAG2 loss. Supplementary Table S14. Gene and gene-fragment RPKM Values for EWS and ETS genes. Supplementary Table S15. ETS1 copy-number changes. Supplementary Table S17. Gene set enrichment analyses of tumors vs. cell lines.

The efficacy of radiotherapy in many tumor types is limited by normal tissue toxicity and by intrinsic or acquired radioresistance. Therefore, it is essential to understand the molecular network responsible for regulating radiosensitivity/resistance. Here, an unbiased functional screen identified four microRNAs (miR1, miR125a, miR150, and miR425) that induce radioresistance. Considering the clinical importance of radiotherapy for patients with glioblastoma, the impact of these miRNAs on glioblastoma radioresistance was investigated. Overexpression of miR1, miR125a, miR150, and/or miR425 in glioblastoma promotes radioresistance through upregulation of the cell-cycle checkpoint response. Conversely, antagonizing with antagomiRs sensitizes glioblastoma cells to irradiation, suggesting their potential as targets for inhibiting therapeutic resistance. Analysis of glioblastoma datasets from The Cancer Genome Atlas (TCGA) revealed that these miRNAs are expressed in glioblastoma patient specimens and correlate with TGFβ signaling. Finally, it is demonstrated that expression of miR1 and miR125a can be induced by TGFβ and antagonized by a TGFβ receptor inhibitor. Together, these results identify and characterize a new role for miR425, miR1, miR125, and miR150 in promoting radioresistance in glioblastomas and provide insight into the therapeutic application of TGFβ inhibitors in radiotherapy.Implications: Systematic identification of miRs that cause radioresistance in gliomas is important for uncovering predictive markers for radiotherapy or targets for overcoming radioresistance. Mol Cancer Res; 12(12); 1767–78. ©2014 AACR.

Pool composition of in vivo screen.

CRISPR guide sequences

Variants identified from WES of 92 tumors after filtering.

Sample Information: Table with cell line identities and relevant information for this manuscript.

Supplementary Figure S1. Distribution of barcode read representation in pre-expansion and pre-injection samples. Supplementary Figure S2. Tumor composition of in vivo pooled screen, excluding the pools shown in Figure 2. Supplementary Figure S3. Gene expression differentiates functional alleles. Supplementary Figure S4. Validation of rare oncogenic alleles, excluding the ones shown in Figure 4. Supplementary Figure S5. Gene expression signatures of NFE2L2 wild type and gain-offunction mutants are correlated. Supplementary Figure S6. Comparison to in silico methods.

S1A. List of miRNAs enriched independent of IR. S1B. Clonogenic survival assay was performed with 2 Gy IR in 12 different GBM lines. Bar graphs were generated with Mean {plus minus} S.D. of 3 independent experiments. S2A. IR sensitivity of LN229 with individual stable expression of miR-1, miR-125a, miR-150 and miR-425 was determined by clonogenic assay. Curves were generated with Mean {plus minus} S.D. of 3 independent experiments. For statistical analysis the areas under the curves were calculated by approximating them to trapezoids. Comparison was done with one-way ANOVA (p < 0.05) and Tukey's post hoc (p < 0.05) test. S2B. Expression of fused miR-1, miR-125a, miR-150 and miR-425 from pcDNA3.1 vector. S3. A) Illustration shows the construct of GFP-fused miRNA sponges. GFP is fused at the 3' end of 4 identical repeats of miRNA sponges in tandem. The sequence of each miRNA sponge is shown below. (B) Inhibition of GFP expression fused to miRNA sponges was tested by transfecting corresponding miRNA mimics. Comparison was done with one-way ANOVA (p < 0.05) and Tukey's post hoc (p < 0.05) test. S4A. Impact of individual miR-scr, miR-1, miR-125a, miR-150 and miR-425 and combined miR-4x' on apoptosis in LN229 cells. Cells were exposed to 10 Gy γ-IR and the activation of Caspase 3 and 7 was determined with Caspase-Glo 3/7 Assay System. Comparison was done with one-way ANOVA (p < 0.05) and Tukey's post hoc (p < 0.05) test. S4B. Phosphorylation of Chk1 S345 in LN229 cells transfected with indicated miRNA upon exposure to 10 Gy IR. The mean {plus minus} S.D. of 3 independent experiments is demonstrated. Comparison was done with one-way ANOVA (p < 0.05) and Tukey's post hoc (p

Pediatric Ewing sarcoma is characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, representing a paradigm for studying cancers driven by transcription factor rearrangements. In this study, we describe the somatic landscape of pediatric Ewing sarcoma. These tumors are among the most genetically normal cancers characterized to date, with only EWS–ETS rearrangements identified in the majority of tumors. STAG2 loss, however, is present in more than 15% of Ewing sarcoma tumors; occurs by point mutation, rearrangement, and likely nongenetic mechanisms; and is associated with disease dissemination. Perhaps the most striking finding is the paucity of mutations in immediately targetable signal transduction pathways, highlighting the need for new therapeutic approaches to target EWS–ETS fusions in this disease.Significance: We performed next-generation sequencing of Ewing sarcoma, a pediatric cancer involving bone, characterized by expression of EWS–ETS fusions. We found remarkably few mutations. However, we discovered that loss of STAG2 expression occurs in 15% of tumors and is associated with metastatic disease, suggesting a potential genetic vulnerability in Ewing sarcoma. Cancer Discov; 4(11); 1326–41. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1243

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity.Significance:We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1–NXT2 paralog relationship.See related commentary by Wang and Abdel-Wahab, p. 2129.This article is highlighted in the In This Issue feature, p. 2113

Supplementary Methods

Verification of detection power calculation. Comparison between the predicted detection power of 18 replicates and the actual fraction of those replicates that were called MRD+. 18 independent samples from a 1:100k dilution were probed with a 488 SNV panel (see methods). Replicates had their duplex consensus reads downsampled in increments of 10% and detection power was recalculated. Reported are the median and range of predicted detection powers for all replicates at each downsampling.

Early stage breast cancer cohort. (A) Overview of subtype distribution of cohort as well as the timing of blood draws relative to surgery and treatment. (B) REMARK diagram of the early stage breast cancer cohort. (C) Distribution of the number of mutations tracked for each patient.

Purpose:Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing.Experimental Design:We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2− metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples.Results:Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2–346). Clinical sensitivity was 81% (n = 13/16) in newly diagnosed MBC, 23% (n = 7/30) at postoperative and 19% (n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3–58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4–39.2 months).Conclusions:Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.

Overview schematic of MRD assay. MRD assay workflow from characterizing a primary tumor to determining if a blood draw contains evidence of residual disease.

Purpose: More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate.Experimental Design: We present the two largest whole-genome expression studies from FFPE tissues to date, comprising 1,003 colorectal cancer (CRC) and 168 breast cancer samples, combined with a meta-analysis of 14 new and published FFPE microarray datasets. We develop and validate quality control (QC) methods through technical replication, independent samples, comparison to results from fresh-frozen tissue, and recovery of expected associations between gene expression and protein abundance.Results: Archival tissues from large, multicenter studies showed a much wider range of transcriptional data quality relative to smaller or frozen tissue studies and required stringent QC for subsequent analysis. We developed novel methods for such QC of archival tissue expression profiles based on sample dynamic range and per-study median profile. This enabled validated identification of gene signatures of microsatellite instability and additional features of CRC, and improved recovery of associations between gene expression and protein abundance of MLH1, FASN, CDX2, MGMT, and SIRT1 in CRC tumors.Conclusions: These methods for large-scale QC of FFPE expression profiles enable study of the cancer transcriptome in relation to extensive clinicopathological information, tumor molecular biomarkers, and long-term lifestyle and outcome data. Clin Cancer Res; 18(22); 6136–46. ©2012 AACR.

PDF file - 31K, Supplemental Table S1: Correlation between mRNA expression and status of protein expression, promoter CpG island methylation, or other tumor biomarker status.

Validation of tumor fraction confidence interval and effects of biological variability on tumor fraction inference. (A) Confidence intervals inferred from simulated data were aggregated into coverage probability for each condition and compared against theoretical coverage probability of 95% (see Methods). (B) Biological variability of clonal evolution loss was simulated by removing tumor evidence from a fraction of the fingerprint panel. Tumor fraction was calculated for simulated and original samples for comparison.

Patients who experienced local recurrence only after treatment for early stage breast cancer. cfDNA time points for all patients who experienced local recurrence only. End points represent time of last follow up or death.

PDF file - 1787K, Supplemental Figure S1: Immunohistochemistry for selected proteins. Supplemental Figure S2: Pairwise scatterplots of technical variables for 1,003 DASL arrays from the NHS/HPFS study, including median value of Illumina control probes, sample age, RNA concentration, fraction of probes called present (p

Supplementary Methods

Supplementary Tables

Application of MRD testing to healthy donor cfDNA samples. Patient specific panels designed for 14 different patients were pooled together and applied to cfDNA collected from four different healthy donors. Reported are the number of sites where ctDNA was detected. We required at least two sites to call a sample MRD+.

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Digital droplet PCR (ddPCR) testing for single mutation in dilution series. (A) The observed VAFs of HapMap serial dilutions measured by using a ddPCR assay for a single mutation (with three replicates) vs. the expected VAFs; (B) The representative ddPCR raw data and fluorescence intensity thresholds for determining positive or negative droplets.