RESUMO
Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.
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Variação Estrutural do Genoma/genética , Genômica/métodos , Neoplasias/genética , Inversão Cromossômica/genética , Cromotripsia , Variações do Número de Cópias de DNA/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Humanos , Mutação/genética , Sequenciamento Completo do Genoma/métodosRESUMO
SUMMARY: We present a new version of the popular somatic variant caller, Lancet, that supports the analysis of linked-reads sequencing data. By seamlessly integrating barcodes and haplotype read assignments within the colored De Bruijn graph local-assembly framework, Lancet computes a barcode-aware coverage and identifies variants that disagree with the local haplotype structure. AVAILABILITY AND IMPLEMENTATION: Lancet is implemented in C++ and available for academic and non-commercial research purposes as an open-source package at https://github.com/nygenome/lancet. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Sequenciamento de Nucleotídeos em Larga Escala , Software , Algoritmos , Diploide , Análise de Sequência de DNARESUMO
Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.
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Neoplasias Cerebelares , Meduloblastoma , Humanos , Criança , Animais , Camundongos , Meduloblastoma/genética , Transposases/genética , Transposases/metabolismo , Proteínas Hedgehog/metabolismo , Fatores de Transcrição/genética , Mutagênese , Neoplasias Cerebelares/genéticaRESUMO
In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.
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DNA Tumoral Circulante , Variações do Número de Cópias de DNA , Aprendizado de Máquina , Neoplasia Residual , Carga Tumoral , Humanos , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Neoplasia Residual/genética , Sequenciamento Completo do Genoma , Neoplasias/genética , Neoplasias/sangue , Neoplasias/terapia , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologiaRESUMO
BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , Dosagem de Genes , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Intracranial metastases in prostate cancer are uncommon but clinically aggressive. A detailed molecular characterization of prostate cancer intracranial metastases would improve our understanding of their pathogenesis and the search for new treatment strategies. We evaluated the clinical and molecular characteristics of 36 patients with metastatic prostate cancer to either the dura or brain parenchyma. We performed whole genome sequencing (WGS) of 10 intracranial prostate cancer metastases, as well as WGS of primary prostate tumors from men who later developed metastatic disease (n = 6) and nonbrain prostate cancer metastases (n = 36). This first whole genome sequencing study of prostate intracranial metastases led to several new insights. First, there was a higher diversity of complex structural alterations in prostate cancer intracranial metastases compared to primary tumor tissues. Chromothripsis and chromoplexy events seemed to dominate, yet there were few enrichments of specific categories of structural variants compared with non-brain metastases. Second, aberrations involving the AR gene, including AR enhancer gain were observed in 7/10 (70%) of intracranial metastases, as well as recurrent loss of function aberrations involving TP53 in 8/10 (80%), RB1 in 2/10 (20%), BRCA2 in 2/10 (20%), and activation of the PI3K/AKT/PTEN pathway in 8/10 (80%). These alterations were frequently present in tumor tissues from other sites of disease obtained concurrently or sequentially from the same individuals. Third, clonality analysis points to genomic factors and evolutionary bottlenecks that contribute to metastatic spread in patients with prostate cancer. These results describe the aggressive molecular features underlying intracranial metastasis that may inform future diagnostic and treatment approaches.
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While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett's esophagus compared to 40 Barrett's patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett's tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett's versus those who progress to cancer is acquisition and expansion of TP53-/- cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett's esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies.
Assuntos
Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Adenocarcinoma/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Progressão da Doença , Neoplasias Esofágicas/patologia , HumanosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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 10-5. 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.
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Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA de Neoplasias/genética , Neoplasias/sangue , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Variações do Número de Cópias de DNA/genética , DNA de Neoplasias/sangue , Intervalo Livre de Doença , Feminino , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Masculino , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Carga Tumoral/genética , Sequenciamento Completo do GenomaRESUMO
To test the performance of a new sequencing platform, develop an updated somatic calling pipeline and establish a reference for future benchmarking experiments, we performed whole-genome sequencing of 3 common cancer cell lines (COLO-829, HCC-1143 and HCC-1187) along with their matched normal cell lines to great sequencing depths (up to 278x coverage) on both Illumina HiSeqX and NovaSeq sequencing instruments. Somatic calling was generally consistent between the two platforms despite minor differences at the read level. We designed and implemented a novel pipeline for the analysis of tumor-normal samples, using multiple variant callers. We show that coupled with a high-confidence filtering strategy, the use of combination of tools improves the accuracy of somatic variant calling. We also demonstrate the utility of the dataset by creating an artificial purity ladder to evaluate the somatic pipeline and benchmark methods for estimating purity and ploidy from tumor-normal pairs. The data and results of the pipeline are made accessible to the cancer genomics community.
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Perfilação da Expressão Gênica , Neoplasias/genética , Sequenciamento Completo do Genoma/métodos , Algoritmos , Alelos , Calibragem , Linhagem Celular Tumoral , Biologia Computacional , Reações Falso-Positivas , Variação Genética , Genoma Humano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
The tumor genome of a patient with advanced pancreatic cancer was sequenced to identify potential therapeutic targetable mutations after standard of care failed to produce any significant overall response. Matched tumor-normal whole-genome sequencing revealed somatic mutations in BRAF, TP53, CDKN2A, and a focal deletion of SMAD4 The BRAF variant was an in-frame deletion mutation (ΔN486_P490), which had been previously demonstrated to be a kinase-activating alteration in the BRAF kinase domain. Working with the Novartis patient assistance program allowed us to treat the patient with the BRAF inhibitor, dabrafenib. The patient's overall clinical condition improved dramatically with dabrafenib. Levels of serum tumor marker dropped immediately after treatment, and a subsequent CT scan revealed a significant decrease in the size of both primary and metastatic lesions. The dabrafenib-induced remission lasted for 6 mo. Preclinical studies published concurrently with the patient's treatment showed that the BRAF in-frame mutation (ΔNVTAP) induces oncogenic activation by a mechanism distinct from that induced by V600E, and that this difference dictates the responsiveness to different BRAF inhibitors. This study describes a dramatic instance of how high-level genomic technology and analysis was necessary and sufficient to identify a clinically logical treatment option that was then utilized and shown to be of clinical value for this individual.
Assuntos
Imidazóis/uso terapêutico , Oximas/uso terapêutico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/genética , Idoso , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Neoplasias Pulmonares/genética , Masculino , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Indução de Remissão , Sequenciamento Completo do Genoma/métodos , Neoplasias PancreáticasRESUMO
Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy-resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.
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Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Criança , Resistencia a Medicamentos Antineoplásicos , Genes do Tumor de Wilms , Genes p53 , Humanos , Leucemia Mieloide Aguda/genéticaRESUMO
BACKGROUND: Prompted by the revolution in high-throughput sequencing and its potential impact for treating cancer patients, we initiated a clinical research study to compare the ability of different sequencing assays and analysis methods to analyze glioblastoma tumors and generate real-time potential treatment options for physicians. METHODS: A consortium of seven institutions in New York City enrolled 30 patients with glioblastoma and performed tumor whole genome sequencing (WGS) and RNA sequencing (RNA-seq; collectively WGS/RNA-seq); 20 of these patients were also analyzed with independent targeted panel sequencing. We also compared results of expert manual annotations with those from an automated annotation system, Watson Genomic Analysis (WGA), to assess the reliability and time required to identify potentially relevant pharmacologic interventions. RESULTS: WGS/RNAseq identified more potentially actionable clinical results than targeted panels in 90% of cases, with an average of 16-fold more unique potentially actionable variants identified per individual; 84 clinically actionable calls were made using WGS/RNA-seq that were not identified by panels. Expert annotation and WGA had good agreement on identifying variants [mean sensitivity = 0.71, SD = 0.18 and positive predictive value (PPV) = 0.80, SD = 0.20] and drug targets when the same variants were called (mean sensitivity = 0.74, SD = 0.34 and PPV = 0.79, SD = 0.23) across patients. Clinicians used the information to modify their treatment plan 10% of the time. CONCLUSION: These results present the first comprehensive comparison of technical and machine augmented analysis of targeted panel and WGS/RNA-seq to identify potential cancer treatments.
Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Sequenciamento Completo do Genoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Ploidias , Reprodutibilidade dos TestesRESUMO
Following publication of the original article [1], it was reported that the given name of the fourteenth author was incorrectly published. The incorrect and the correct names are given below.
RESUMO
Reliable detection of somatic variations is of critical importance in cancer research. Here we present Lancet, an accurate and sensitive somatic variant caller, which detects SNVs and indels by jointly analyzing reads from tumor and matched normal samples using colored de Bruijn graphs. We demonstrate, through extensive experimental comparison on synthetic and real whole-genome sequencing datasets, that Lancet has better accuracy, especially for indel detection, than widely used somatic callers, such as MuTect, MuTect2, LoFreq, Strelka, and Strelka2. Lancet features a reliable variant scoring system, which is essential for variant prioritization, and detects low-frequency mutations without sacrificing the sensitivity to call longer insertions and deletions empowered by the local-assembly engine. In addition to genome-wide analysis, Lancet allows inspection of somatic variants in graph space, which augments the traditional read alignment visualization to help confirm a variant of interest. Lancet is available as an open-source program at https://github.com/nygenome/lancet.
RESUMO
Next-generation DNA sequencing (NGS) technologies are currently being applied in both research and clinical settings for the understanding and management of disease. The goal is to use high-throughput sequencing to identify specific variants that drive tumorigenesis within each individual's tumor genomic profile. The significance of copy number and structural variants in glioblastoma makes it essential to broaden the search beyond oncogenic single nucleotide variants toward whole genome profiles of genetic aberrations that may contribute to disease progression. The heterogeneity of glioblastoma and its variability of cancer driver mutations necessitate a more robust examination of a patient's tumor genome. Here, we present patient whole genome sequencing (WGS) information to identify oncogenic structural variants that may contribute to glioblastoma pathogenesis. We provide WGS protocols and bioinformatics approaches to identify copy number and structural variations in 41 glioblastoma patient samples. We present how WGS can identify structural diversity within glioblastoma samples. We specifically show how to apply current bioinformatics tools to detect EGFR variants and other structural aberrations from DNA whole genome sequencing and how to validate those variants within the laboratory. These comprehensive WGS protocols can provide additional information directing more precise therapeutic options in the treatment of glioblastoma.
Assuntos
Variação Genética , Genoma Humano , Glioblastoma/genética , Sequenciamento Completo do Genoma , Biomarcadores Tumorais , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Receptores ErbB/genética , Expressão Gênica , Glioblastoma/patologia , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Medicina de PrecisãoRESUMO
We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.
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Variação Genética , Neoplasias/genética , Relatório de Pesquisa , Transcriptoma/genética , Sequenciamento Completo do Genoma/métodos , Variações do Número de Cópias de DNA/genética , Frequência do Gene/genética , Humanos , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
NUTM1-rearranged tumors are defined by the presence of a gene fusion between NUTM1 and various gene partners and typically follow a clinically aggressive disease course with poor outcomes despite conventional multimodality therapy. NUTM1-rearranged tumors display histologic features of a poorly differentiated carcinoma with areas of focal squamous differentiation and typically express the BRD4-NUTM1 fusion gene defining a distinct clinicopathologic entity-NUT carcinoma (NC). NCs with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high-grade spindle cell sarcoma harboring a novel MGA-NUTM1 fusion. Whole-genome sequencing identified the presence of complex rearrangements resulting in a MGA-NUTM1 fusion gene in the absence of other significant somatic mutations. Genetic rearrangement was confirmed by fluorescence in situ hybridization, and expression of the fusion gene product was confirmed by transcriptomic analysis. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1-22) and NUTM1 (exons 3-8). Histopathologically, both cases were high-grade spindle cell sarcomas without specific differentiation markers. In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA-NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high-grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Sarcoma/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Criança , Feminino , Fusão Gênica/genética , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Recombinação Genética/genética , Sarcoma/metabolismo , Sarcoma Sinovial/genética , Fatores de Transcrição/genética , Translocação Genética/genética , Sequenciamento Completo do Genoma/métodosRESUMO
Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection.Significance: New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. Cancer Discov; 8(9); 1112-29. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.