Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed ...linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%–87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.
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•Linked-read genome sequencing of mCRPC resolves haplotypes and rearrangements•CDK12 inactivation is associated with a global tandem duplication phenotype•A majority of cases have duplications of an enhancer of the androgen receptor•Progression on androgen pathway inhibitors is associated with gains in AR and AR enhancer
Linked-read genome sequencing data from patients highlight that amplification of an enhancer upstream of the androgen receptor locus is a key feature of metastatic castration-resistant prostate cancer.
Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to ...these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.
In many areas of oncology, we lack sensitive tools to track low-burden disease. Although cell-free DNA (cfDNA) shows promise in detecting cancer mutations, we found that the combination of low tumor ...fraction (TF) and limited number of DNA fragments restricts low-disease-burden monitoring through the prevailing deep targeted sequencing paradigm. We reasoned that breadth may supplant depth of sequencing to overcome the barrier of cfDNA abundance. Whole-genome sequencing (WGS) of cfDNA allowed ultra-sensitive detection, capitalizing on the cumulative signal of thousands of somatic mutations observed in solid malignancies, with TF detection sensitivity as low as 10
. The WGS approach enabled dynamic tumor burden tracking and postoperative residual disease detection, associated with adverse outcome. Thus, we present an orthogonal framework for cfDNA cancer monitoring via genome-wide mutational integration, enabling ultra-sensitive detection, overcoming the limitation of cfDNA abundance and empowering treatment optimization in low-disease-burden oncology care.
Glioblastomas (GBM) with EGFR amplification represent approximately 50% of newly diagnosed cases, and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same ...tumor, which has implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here, we applied a population-based single-cell whole-genome sequencing methodology to characterize genomic heterogeneity in EGFR-amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal LOH and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR carboxyl-terminal deletions) identified in the bulk tumor segregate into nonoverlapping subclonal populations. In vitro and in vivo functional studies show that EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus, the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment.
We developed a novel single-cell sequencing methodology capable of identifying unique, nonoverlapping subclonal alterations from archived frozen clinical specimens. Using GBM as an example, we validated our method to successfully define tumor cell subpopulations containing distinct genetic and treatment resistance profiles and potentially mutually cooperative combinations of alterations in EGFR and other genes.
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
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.
During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient
. Previous case reports and small case ...series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance
. However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.
Abstract
Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA ...duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.
Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and ...accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.
Purpose Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with patient prognosis. Triple-negative ...breast cancer (TNBC) is characterized by few mutations but extensive somatic copy number alterations (SCNAs), yet little is known regarding SCNAs in metastatic TNBC. We sought to evaluate SCNAs in metastatic TNBC exclusively via cfDNA and determine if cfDNA tumor fraction is associated with overall survival in metastatic TNBC. Patients and Methods In this retrospective cohort study, we identified 164 patients with biopsy-proven metastatic TNBC at a single tertiary care institution who received prior chemotherapy in the (neo)adjuvant or metastatic setting. We performed low-coverage genome-wide sequencing of cfDNA from plasma. Results Without prior knowledge of tumor mutations, we determined tumor fraction of cfDNA for 96.3% of patients and SCNAs for 63.9% of patients. Copy number profiles and percent genome altered were remarkably similar between metastatic and primary TNBCs. Certain SCNAs were more frequent in metastatic TNBCs relative to paired primary tumors and primary TNBCs in publicly available data sets The Cancer Genome Atlas and METABRIC, including chromosomal gains in drivers NOTCH2, AKT2, and AKT3. Prespecified cfDNA tumor fraction threshold of ≥ 10% was associated with significantly worse metastatic survival (median, 6.4 v 15.9 months) and remained significant independent of clinicopathologic factors (hazard ratio, 2.14; 95% CI, 1.4 to 3.8; P < .001). Conclusion We present the largest genomic characterization of metastatic TNBC to our knowledge, exclusively from cfDNA. Evaluation of cfDNA tumor fraction was feasible for nearly all patients, and tumor fraction ≥ 10% is associated with significantly worse survival in this large metastatic TNBC cohort. Specific SCNAs are enriched and prognostic in metastatic TNBC, with implications for metastasis, resistance, and novel therapeutic approaches.
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