Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

Oncogenic Signaling Pathways in The Cancer Genome Atlas.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFß signaling, p53 and ß-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.

Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.

Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin.

Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All data sets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies.

The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Genetic and epigenetic evolution as a contributor to WT1-mutant leukemogenesis.

Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms' tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.

Regional differences in gallbladder cancer pathogenesis: Insights from a multi-institutional comparison of tumor mutations.

BACKGROUND: Although rare in the United States, gallbladder cancer (GBCA) is a common cause of cancer death in some parts of the world. To investigate regional differences in pathogenesis and outcomes for GBCA, tumor mutations were analyzed from a sampling of specimens. METHODS: Primary tumors from patients with GBCA who were treated in Chile, Japan, and the United States between 1999 and 2016 underwent targeted sequencing of known cancer-associated genes. Fisher exact and Kruskal-Wallis tests assessed differences in clinicopathologic and genetic factors. Kaplan-Meier methods evaluated differences in overall survival from the time of surgery between mutations. RESULTS: A total of 81 patients were included. Japanese patients (11 patients) were older (median age, 72 years [range, 54-81 years]) compared with patients from Chile (21 patients; median age, 59 years [range, 32-73 years]) and the United States (49 patients; median age, 66 years [range, 46-87 years]) (P = .002) and had more well-differentiated tumors (46% vs 0% for Chile/United States; P < .001) and fewer gallstone-associated cancers (36% vs 67% for Chile and 69% for the United States; P = .13). Japanese patients had a median mutation burden of 6 (range, 1-23) compared with Chile (median mutation burden, 7 [range, 3-20]) and the United States (median mutation burden, 4 [range, 0-27]) (P = .006). Tumors from Japanese patients lacked AT-rich interaction domain 1A (ARID1A) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations, whereas Chilean tumors lacked Erb-B2 receptor tyrosine kinase 3 (ERBB3) and AT-rich interaction domain 2 (ARID2) mutations. SMAD family member 4 (SMAD4) was found to be mutated similarly across centers (38% in Chile, 36% in Japan, and 27% in the United States; P = .68) and was univariately associated with worse overall survival (median, 10 months vs 25 months; P = .039). At least one potentially actionable gene was found to be altered in 80% of tumors. CONCLUSIONS: Differences in clinicopathologic variables suggest the possibility of distinct GBCA pathogenesis in Japanese patients, which may be supported by differences in mutation pattern. Among all centers, SMAD4 mutations were detected in approximately one-third of patients and may represent a converging factor associated with worse survival. The majority of patients carried mutations in actionable gene targets, which may inform the design of future trials.

GNAQ Mutations in Diffuse and Solitary Choroidal Hemangiomas.

PURPOSE: GNAQ mutations have been identified in port wine stains (both syndromic and nonsyndromic) and melanocytic ocular neoplasms. This study investigates the presence of GNAQ mutations in diffuse (those associated with Sturge-Weber syndrome [SWS]) and solitary choroidal hemangiomas. PARTICIPANTS: Tissue from 11 patients with the following diagnoses: port wine stain (n = 3), diffuse choroidal hemangioma (n = 1), solitary choroidal hemangioma (n = 6), and choroidal nevus (n = 1). METHODS: Ten specimens were interrogated with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets, a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 468 key cancer genes in formalin-fixed, paraffin-embedded tumors. Digital polymerase chain reaction was used to detect GNAQ Q209 mutation in 1 specimen. MAIN OUTCOME MEASURES: Detection of GNAQ codon-specific mutation. RESULTS: Activating somatic GNAQ mutations (c.547C > T; p.Arg183Cys) were found in 100% (3 of 3) of the port wine stain and in the diffuse choroidal hemangioma. Somatic GNAQ mutations (c.626A > T; p.Gln209Leu) were found in 100% (6 of 6) of the solitary choroidal hemangiomas and (c.626A > C; p.Gln209Pro) in the choroidal nevus. CONCLUSIONS: GNAQ mutations occur in both diffuse and solitary hemangiomas, although at distinct codons. An R183 codon is mutant in diffuse choroidal hemangiomas, consistent with other Sturge-Weber vascular malformations. By contrast, solitary choroidal hemangiomas have mutations in the Q209 codon, similar to other intraocular melanocytic neoplasms.

Integrated genomic characterization of endometrial carcinoma.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ∼25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.

Mutational landscape and significance across 12 major cancer types.

The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/ß-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.

Whole-genome analysis informs breast cancer response to aromatase inhibition.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.

BACKGROUND: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma.

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%-60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.

Genome Modeling System: A Knowledge Management Platform for Genomics.

In this work, we present the Genome Modeling System (GMS), an analysis information management system capable of executing automated genome analysis pipelines at a massive scale. The GMS framework provides detailed tracking of samples and data coupled with reliable and repeatable analysis pipelines. The GMS also serves as a platform for bioinformatics development, allowing a large team to collaborate on data analysis, or an individual researcher to leverage the work of others effectively within its data management system. Rather than separating ad-hoc analysis from rigorous, reproducible pipelines, the GMS promotes systematic integration between the two. As a demonstration of the GMS, we performed an integrated analysis of whole genome, exome and transcriptome sequencing data from a breast cancer cell line (HCC1395) and matched lymphoblastoid line (HCC1395BL). These data are available for users to test the software, complete tutorials and develop novel GMS pipeline configurations. The GMS is available at https://github.com/genome/gms.

MuSiC: identifying mutational significance in cancer genomes.

Massively parallel sequencing technology and the associated rapidly decreasing sequencing costs have enabled systemic analyses of somatic mutations in large cohorts of cancer cases. Here we introduce a comprehensive mutational analysis pipeline that uses standardized sequence-based inputs along with multiple types of clinical data to establish correlations among mutation sites, affected genes and pathways, and to ultimately separate the commonly abundant passenger mutations from the truly significant events. In other words, we aim to determine the Mutational Significance in Cancer (MuSiC) for these large data sets. The integration of analytical operations in the MuSiC framework is widely applicable to a broad set of tumor types and offers the benefits of automation as well as standardization. Herein, we describe the computational structure and statistical underpinnings of the MuSiC pipeline and demonstrate its performance using 316 ovarian cancer samples from the TCGA ovarian cancer project. MuSiC correctly confirms many expected results, and identifies several potentially novel avenues for discovery.

Metapipeline-DNA: A Comprehensive Germline & Somatic Genomics Nextflow Pipeline.

Summary: DNA sequencing is becoming more affordable and faster through advances in high-throughput technologies. This rise in data availability has contributed to the development of novel algorithms to elucidate previously obscure features and led to an increased reliance on complex workflows to integrate such tools into analyses pipelines. To facilitate the analysis of DNA sequencing data, we created metapipeline-DNA, a highly configurable and extensible pipeline. It encompasses a broad range of processing including raw sequencing read alignment and recalibration, variant calling, quality control and subclonal reconstruction. Metapipeline-DNA also contains configuration options to select and tune analyses while being robust to failures. This standardizes and simplifies the ability to analyze large DNA sequencing in both clinical and research settings. Availability: Metapipeline-DNA is an open-source Nextflow pipeline under the GPLv2 license and is freely available at https://github.com/uclahs-cds/metapipeline-DNA.

DNMT3A mutations in acute myeloid leukemia.

BACKGROUND: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS: Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS: A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS: DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

BreakFusion: targeted assembly-based identification of gene fusions in whole transcriptome paired-end sequencing data.

UNLABELLED: Despite recent progress, computational tools that identify gene fusions from next-generation whole transcriptome sequencing data are often limited in accuracy and scalability. Here, we present a software package, BreakFusion that combines the strength of reference alignment followed by read-pair analysis and de novo assembly to achieve a good balance in sensitivity, specificity and computational efficiency. AVAILABILITY: http://bioinformatics.mdanderson.org/main/BreakFusion

PathScan: a tool for discerning mutational significance in groups of putative cancer genes.

MOTIVATION: The expansion of cancer genome sequencing continues to stimulate development of analytical tools for inferring relationships between somatic changes and tumor development. Pathway associations are especially consequential, but existing algorithms are demonstrably inadequate. METHODS: Here, we propose the PathScan significance test for the scenario where pathway mutations collectively contribute to tumor development. Its design addresses two aspects that established methods neglect. First, we account for variations in gene length and the consequent differences in their mutation probabilities under the standard null hypothesis of random mutation. The associated spike in computational effort is mitigated by accurate convolution-based approximation. Second, we combine individual probabilities into a multiple-sample value using Fisher-Lancaster theory, thereby improving differentiation between a few highly mutated genes and many genes having only a few mutations apiece. We investigate accuracy, computational effort and power, reporting acceptable performance for each. RESULTS: As an example calculation, we re-analyze KEGG-based lung adenocarcinoma pathway mutations from the Tumor Sequencing Project. Our test recapitulates the most significant pathways and finds that others for which the original test battery was inconclusive are not actually significant. It also identifies the focal adhesion pathway as being significantly mutated, a finding consistent with earlier studies. We also expand this analysis to other databases: Reactome, BioCarta, Pfam, PID and SMART, finding additional hits in ErbB and EPHA signaling pathways and regulation of telomerase. All have implications and plausible mechanistic roles in cancer. Finally, we discuss aspects of extending the method to integrate gene-specific background rates and other types of genetic anomalies. AVAILABILITY: PathScan is implemented in Perl and is available from the Genome Institute at: http://genome.wustl.edu/software/pathscan.

Genome Nexus: A Comprehensive Resource for the Annotation and Interpretation of Genomic Variants in Cancer.

PURPOSE: Interpretation of genomic variants in tumor samples still presents a challenge in research and the clinical setting. A major issue is that information for variant interpretation is fragmented across disparate databases, and aggregation of information from these requires building extensive infrastructure. To this end, we have developed Genome Nexus, a one-stop shop for variant annotation with a user-friendly interface for cancer researchers and clinicians. METHODS: Genome Nexus (1) aggregates variant information from sources that are relevant to cancer research and clinical applications, (2) allows high-performance programmatic access to the aggregated data via a unified application programming interface, (3) provides a reference page for individual cancer variants, (4) provides user-friendly tools for annotating variants in patients, and (5) is freely available under an open source license and can be installed in a private cloud or local environment and integrated with local institutional resources. RESULTS: Genome Nexus is available at https://www.genomenexus.org. It displays annotations from more than a dozen resources including those that provide variant effect information (variant effect predictor), protein sequence annotation (Uniprot, Pfam, and dbPTM), functional consequence prediction (Polyphen-2, Mutation Assessor, and SIFT), population prevalences (gnomAD, dbSNP, and ExAC), cancer population prevalences (Cancer hotspots and SignalDB), and clinical actionability (OncoKB, CIViC, and ClinVar). We describe several use cases that demonstrate the utility of Genome Nexus to clinicians, researchers, and bioinformaticians. We cover single-variant annotation, cohort analysis, and programmatic use of the application programming interface. Genome Nexus is unique in providing a user-friendly interface specific to cancer that allows high-performance annotation of any variant including unknown ones. CONCLUSION: Interpretation of cancer genomic variants is improved tremendously by having an integrated resource for annotations. Genome Nexus is freely available under an open source license.