Chromosome instability drives phenotypic switching to metastasis.

Chromosome instability (CIN) is the most striking feature of human cancers. However, how CIN drives tumor progression to metastasis remains elusive. Here we studied the role of chromosome content changes in generating the phenotypic dynamics that are required for metastasis. We isolated epithelial and mesenchymal clones from human carcinoma cell lines and showed that the epithelial clones were able to generate mesenchymal variants, which had the potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then epithelial phenotypes recapitulated the steps in tumor progression to metastasis. Importantly, the generation of mesenchymal variants from clonal epithelial populations was associated with subtle changes in chromosome content, which altered the chromosome transcriptome and influenced the expression of genes encoding intercellular junction (IJ) proteins, whereas the loss of chromosome 10p, which harbors the ZEB1 gene, was frequently detected in epithelial variants generated from mesenchymal clones. Knocking down these IJ genes in epithelial cells induced a mesenchymal phenotype, whereas knocking down the ZEB1 gene in mesenchymal cells induced an epithelial phenotype, demonstrating a causal role of chromosome content changes in phenotypic determination. Thus, our studies suggest a paradigm of tumor metastasis: primary epithelial carcinoma cells that lose chromosomes harboring IJ genes acquire an invasive mesenchymal phenotype, and subsequent chromosome content changes such as loss of 10p in disseminated mesenchymal cells generate epithelial variants, which can be selected for to generate epithelial tumors during metastatic colonization.

Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events.

Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.

Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma.

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

SWI/SNF Antagonism of PRC2 Mediates Estrogen-Induced Progesterone Receptor Expression.

Endometrial cancer (EC) is characterized by high estrogen levels unopposed by progesterone. Treatment with progestins is standard for early EC, but the response to progestins is dependent on progesterone receptor (PGR) expression. Here, we show that the expression of PGR in endometrial epithelial cells is dependent on ARID1A, a DNA-binding subunit of the SWI/SNF chromatin-remodeling complex that is commonly mutated in EC. In endometrial epithelial cells with estrogen receptor overexpression, we find that ARID1A promotes estrogen signaling and regulates common gene expression programs. Normally, endometrial epithelial cells expressing estrogen receptors respond to estrogen by upregulating the PGR. However, when ARID1A expression is lost, upregulation of PGR expression is significantly reduced. This phenomenon can also occur following the loss of the SWI/SNF subunit BRG1, suggesting a role for ARID1A- and BRG1-containing complexes in PGR regulation. We find that PGR is regulated by a bivalent promoter, which harbors both H3K4me3 and H3K27me3 histone tail modifications. H3K27me3 is deposited by EZH2, and inhibition of EZH2 in the context of ARID1A loss results in restoration of estrogen-induced PGR expression. Our results suggest a role for ARID1A deficiency in the loss of PGR in late-stage EC and a therapeutic utility for EZH2 inhibitors in this disease.

DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse.

We have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. Apc Min/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.

Somatic pairing of chromosome 19 in renal oncocytoma is associated with deregulated EGLN2-mediated [corrected] oxygen-sensing response.

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase EGLN2 [corrected] a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of EGLN2 [corrected] in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.

Evaluation of whole-genome DNA methylation sequencing library preparation protocols.

BACKGROUND: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. RESULTS: An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. RESULTS: The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data reveals MYC activation in high-grade papillary renal cell carcinoma.

Papillary renal cell carcinoma (RCC) represents 10% to 15% of adult renal neoplasms; however, the molecular genetic events that are associated with the development and progression of sporadic papillary RCC remain largely unclear. Papillary RCCs can be divided into two subtypes based on histologic, cytogenetic, and gene expression differences. Type 1 tumors ( approximately 60-70%) are generally low grade with favorable outcome, whereas type 2 tumors ( approximately 30-40%) are associated with increased cytogenetic complexity, high tumor grade, and poor prognosis. In this study, computational analysis of gene expression data derived from papillary RCC revealed that a transcriptional signature indicative of MYC pathway activation is present in high-grade type 2 papillary RCC. The MYC signature is associated with amplification of chromosome 8q and overexpression of MYC that maps to chromosome 8q24. The importance of MYC activation was confirmed by both pharmacologic and short interfering RNA-mediated inhibition of active Myc signaling in a cell line model of type 2 papillary RCC. These results provide both computational and genetic evidence that activation of Myc is associated with the aggressiveness of papillary type 2 RCC. Therefore, it will be useful to consider inhibition of components of the MYC signaling pathway as avenues for therapeutic intervention in high-grade papillary RCC.

Integrated Epigenome, Exome, and Transcriptome Analyses Reveal Molecular Subtypes and Homeotic Transformation in Uterine Fibroids.

Uterine fibroids are benign myometrial smooth muscle tumors of unknown etiology that, when symptomatic, are the most common indication for hysterectomy in the United States. Unsupervised clustering of results from DNA methylation analyses segregates normal myometrium from fibroids and further segregates the fibroids into subtypes characterized by MED12 mutation or activation of either HMGA2 or HMGA1 expression. Upregulation of HMGA2 expression does not always appear to be dependent on translocation but is associated with hypomethylation in the HMGA2 gene body. HOXA13 expression is upregulated in fibroids and correlates with expression of typical uterine fibroid genes. Significant overlap of differentially expressed genes is observed between cervical stroma and uterine fibroids compared with normal myometrium. These analyses show a possible role of DNA methylation in fibroid biology and suggest that homeotic transformation of myometrial cells to a more cervical stroma phenotype could be an important mechanism for etiology of the disease.

ARID1A and PI3-kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion.

ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.

Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

Genomic Status of MET Potentiates Sensitivity to MET and MEK Inhibition in NF1-Related Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+ ; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1ko/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and Nf1ko/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs.Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672-87. ©2018 AACR.

The genomic landscape of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.

Spectral karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro.

The Ink4a/Arf locus is functionally linked to the Rb and p53 pathways through the action of its two gene products. Mouse models null for this locus show rapid onset of cancer with a preponderance of lymphomas and sarcomas. We report on a study of cell lines derived from sarcomas arising in Ink4a/Arf null mice. The cytogenetics of these lines was monitored over the course of serial passage. Results indicate that early passage cells are relatively normal. However, after multiple passages chromosomal instability becomes apparent as evidenced by increasing tetraploidy and aneuploidy, and the concomitant loss of clonality. To further evaluate the effect of Ink4a/Arf-deficiency on chromosomal stability in vitro, we isolated Ink4a/Arf deficient primary murine embryonic fibroblasts (MEFs), serially passaged them, and analyzed their chromosomal stability by spectral karyotyping (a 24-color chromosome paint-FISH technique). We found that chromosomal instability in Ink4a/Arf deficient MEFs developed with the same timing as seen in cell lines derived from Ink4a/Arf deficient sarcomas. Thus, chromosomal instability seen in Ink4a/Arf deficient tumors in vitro may be unrelated to the original phenotype of the tumor in vivo. Therefore, interpretation of cytogenetic data from cell lines derived from Ink4a/Arf deficient tumors should be done on early passage cells.

Human biosample authentication using the high-throughput, cost-effective SNPtrace(TM) system.

Cell lines are the foundation for much of the fundamental research into the mechanisms underlying normal biologic processes and disease mechanisms. It is estimated that 15%-35% of human cell lines are misidentified or contaminated, resulting in a huge waste of resources and publication of false or misleading data. Here we evaluate a panel of 96 single-nucleotide polymorphism (SNP) assays utilizing Fluidigm microfluidics technology for authentication and sex determination of human cell lines. The SNPtrace Panel was tested on 907 human cell lines. Pairwise comparison of these data show the SNPtrace Panel discriminated among identical, related and unrelated pairs of samples with a high degree of confidence, equivalent to short tandem repeat (STR) profiling. We also compared annotated sex calls with those determined by the SNPtrace Panel, STR and Illumina SNP arrays, revealing a high number of male samples are identified as female due to loss of the Y chromosome. Finally we assessed the sensitivity of the SNPtrace Panel to detect intra-human cross-contamination, resulting in detection of as little as 2% contaminating cell population. In conclusion, this study has generated a database of SNP fingerprints for 907 cell lines used in biomedical research and provides a reliable, fast, and economic alternative to STR profiling which can be applied to any human cell line or tissue sample.

Overexpression of HGF Promotes HBV-Induced Hepatocellular Carcinoma Progression and Is an Effective Indicator for Met-Targeting Therapy.

Hepatitis B virus (HBV) is a well-known cause of hepatocellular carcinoma (HCC), but the regulators effectively driving virus production and HCC progression remain unclear. By using genetically engineered mouse models, we show that overexpression of hepatocyte growth factor (HGF) accelerated HCC progression, supporting the genomic analysis that an up-regulated HGF signature is associated with poor prognosis in HBV-positive HCC patients. We show that for both liver regeneration and spontaneous HCC development there is an inclusive requirement for MET expression, and when HGF induces autocrine activation the tumor displays sensitivity to a small-molecule Met inhibitor. Our results demonstrate that HGF is a driver of HBV-induced HCC progression and may serve as an effective biomarker for Met-targeted therapy. MET inhibitors are entering clinical trials against cancer, and our data provide a molecular basis for targeting the Met pathway in hepatitis B-induced HCC.

Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.