Cancer genomics

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations.

The Cancer Genome Atlas (TCGA) Research Network has profiled and analyzed large numbers of human tumors to discover molecular aberrations at the DNA, RNA, protein and epigenetic levels. The resulting rich data provide a major opportunity to develop an integrated picture of commonalities, differences and emergent themes across tumor lineages. The Pan-Cancer initiative compares the first 12 tumor types profiled by TCGA. Analysis of the molecular aberrations and their functional roles across tumor types will teach us how to extend therapies effective in one cancer type to others with a similar genomic profile.

Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis, we identified Sh3gl2 (endophilin A1) as a bladder urothelium-enriched transcript. The gene encoding Sh3gl2 is located on chromosome 9p, a region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to nontumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, which is a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 19 of 20 human UC cell lines but preserved in the low-grade cell line RT4. Stable silencing of Sh3gl2 in RT4 cells by RNA interference 1) enhanced proliferation and colony formation in vitro, 2) inhibited EGF-induced EGFR internalization and increased EGFR activation, 3) stimulated phosphorylation of Src family kinases and STAT3, and 4) promoted growth of RT4 xenografts in subrenal capsule tissue recombination experiments. Conversely, forced re-expression of Sh3gl2 in T24 cells and silenced RT4 clones attenuated oncogenic behaviors, including growth and migration. Together, these findings identify loss of Sh3gl2 as a frequent event in UC development that promotes disease progression.

Increased levels of hypoxia and hypoxia-inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category "Response to hypoxia" was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia-responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF-1α shRNA or Dox at low concentrations blocked HIF-1α induction of VEGF-A by 84-97% and carbonic anhydrase 9 by 83-93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF-1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α target genes that may promote resistance to antiangiogenic and other therapies. HIF-1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.

Identification of somatic rearrangements in cancer genomes has accelerated through analysis of high-throughput sequencing data. However, characterization of complex structural alterations and their underlying mechanisms remains inadequate. Here, applying an algorithm to predict structural variations from short reads, we report a comprehensive catalog of somatic structural variations and the mechanisms generating them, using high-coverage whole-genome sequencing data from 140 patients across ten tumor types. We characterize the relative contributions of different types of rearrangements and their mutational mechanisms, find that ~20% of the somatic deletions are complex deletions formed by replication errors, and describe the differences between the mutational mechanisms in somatic and germline alterations. Importantly, we provide detailed reconstructions of the events responsible for loss of CDKN2A/B and gain of EGFR in glioblastoma, revealing that these alterations can result from multiple mechanisms even in a single genome and that both DNA double-strand breaks and replication errors drive somatic rearrangements.

A large database of copy number profiles from cancer genomes can facilitate the identification of recurrent chromosomal alterations that often contain key cancer-related genes. It can also be used to explore low-prevalence genomic events such as chromothripsis. In this study, we report an analysis of 8227 human cancer copy number profiles obtained from 107 array comparative genomic hybridization (CGH) studies. Our analysis reveals similarity of chromosomal arm-level alterations among developmentally related tumor types as well as a number of co-occurring pairs of arm-level alterations. Recurrent ("pan-lineage") focal alterations identified across diverse tumor types show an enrichment of known cancer-related genes and genes with relevant functions in cancer-associated phenotypes (e.g., kinase and cell cycle). Tumor type-specific ("lineage-restricted") alterations and their enriched functional categories were also identified. Furthermore, we developed an algorithm for detecting regions in which the copy number oscillates rapidly between fixed levels, indicative of chromothripsis. We observed these massive genomic rearrangements in 1%-2% of the samples with variable tumor type-specific incidence rates. Taken together, our comprehensive view of copy number alterations provides a framework for understanding the functional significance of various genomic alterations in cancer genomes.

Microsatellites-simple tandem repeats present at millions of sites in the human genome-can shorten or lengthen due to a defect in DNA mismatch repair. We present here a comprehensive genome-wide analysis of the prevalence, mutational spectrum, and functional consequences of microsatellite instability (MSI) in cancer genomes. We analyzed MSI in 277 colorectal and endometrial cancer genomes (including 57 microsatellite-unstable ones) using exome and whole-genome sequencing data. Recurrent MSI events in coding sequences showed tumor type specificity, elevated frameshift-to-inframe ratios, and lower transcript levels than wild-type alleles. Moreover, genome-wide analysis revealed differences in the distribution of MSI versus point mutations, including overrepresentation of MSI in euchromatic and intronic regions compared to heterochromatic and intergenic regions, respectively, and depletion of MSI at nucleosome-occupied sequences. Our results provide a panoramic view of MSI in cancer genomes, highlighting their tumor type specificity, impact on gene expression, and the role of chromatin organization.

SUMMARY: We have developed Nozzle, an R package that provides an Application Programming Interface to generate HTML reports with dynamic user interface elements. Nozzle was designed to facilitate summarization and rapid browsing of complex results in data analysis pipelines where multiple analyses are performed frequently on big datasets. The package can be applied to any project where user-friendly reports need to be created. AVAILABILITY: The R package is available on CRAN at http://cran.r-project.org/package=Nozzle.R1. Examples and additional materials are available at http://gdac.broadinstitute.org/nozzle. The source code is also available at http://www.github.com/parklab/Nozzle. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers.

To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase ε (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.

We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.

Uterine leiomyomata (UL), the most common neoplasm in reproductive-age women, are classified into distinct genetic subgroups based on recurrent chromosome abnormalities. To develop a molecular signature of UL with t(12;14)(q14-q15;q23-q24), we took advantage of the multiple UL arising as independent clonal lesions within a single uterus. We compared genome-wide expression levels of t(12;14) UL to non-t(12;14) UL from each of nine women in a paired analysis, with each sample weighted for the percentage of t(12;14) cells to adjust for mosaicism with normal cells. This resulted in a transcriptional profile that confirmed HMGA2, known to be overexpressed in t(12;14) UL, as the most significantly altered gene. Pathway analysis of the differentially expressed genes showed significant association with cell proliferation, particularly G1/S checkpoint regulation. This is consistent with the known larger size of t(12;14) UL relative to karyotypically normal UL or to UL in the deletion 7q22 subgroup. Unsupervised hierarchical clustering demonstrated that patient variability is relatively dominant to the distinction of t(12;14) UL compared with non-t(12;14) UL or of t(12;14) UL compared with del(7q) UL. The paired design we employed is therefore important to produce an accurate t(12;14) UL-specific gene list by removing the confounding effects of genotype and environment. Interestingly, myometrium not only clustered away from the tumors, but generally separated based on associated t(12;14) versus del(7q) status. Nine genes were identified whose expression can distinguish the myometrium origin. This suggests an underlying constitutional genetic predisposition to these somatic changes which could potentially lead to improved personalized management and treatment.

OBJECTIVE: Short bowel syndrome remains a condition of high morbidity and mortality, and current therapeutic options carry significant side effects. To identify new treatments we focused on postresection changes in microRNAs--short noncoding RNAs, which suppress target genes--and suggest a previously undiscovered role for microRNA-125a (mir-125a) in intestinal adaptation. METHODS: Rats underwent either 80% massive small bowel resection or transection and were harvested after 48 hours. Jejunum was harvested for microRNA microarrays, laser capture microdissection, and RNA and protein analysis. Mir-125a was overexpressed in intestinal epithelium-6 (crypt-derived) cells (IEC-6) and effects on proliferation and apoptosis determined using MTS and flow cytometry. Expression of potential targets of mir-125a in rat jejunum and IEC-6 cells was determined using quantitative real-time polymerase chain reaction (RNA) and Western blotting (protein). RESULTS: Resection upregulated mir-125a and mir-214 by 2.4-folds and 3.2-folds, respectively. Highest levels of expression were noted in the crypt fraction. Mir-125a overexpression induced apoptosis and resultant growth arrest in IEC-6 cells. The expression of the prosurvival Bcl-2 family member Mcl-1 was downregulated in both mir-125a-overexpressing IEC-6 cells and in jejunum of resected rats, confirming Mcl-1 as a previously undiscovered target of mir-125a. CONCLUSIONS: Upregulation of mir-125a suppresses the prosurvival protein Mcl1, producing the increase in apoptosis known to accompany the proliferative changes characteristic of intestinal adaptation. Our data highlight a potential role for microRNAs as mediators of the adaptive process and may facilitate the development of new therapeutic options for short bowel syndrome.

Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas, and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnormalities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions, suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion increases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associated development of aggressive biologic behavior in meningiomas.

Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.

Glioblastoma contains a hierarchy of stem-like cancer cells, but how this hierarchy is established is unclear. Here, we show that asymmetric Numb localization specifies glioblastoma stem-like cell (GSC) fate in a manner that does not require Notch inhibition. Numb is asymmetrically localized to CD133-hi GSCs. The predominant Numb isoform, Numb4, decreases Notch and promotes a CD133-hi, radial glial-like phenotype. However, upregulation of a novel Numb isoform, Numb4 delta 7 (Numb4d7), increases Notch and AKT activation while nevertheless maintaining CD133-hi fate specification. Numb knockdown increases Notch and promotes growth while favoring a CD133-lo, glial progenitor-like phenotype. We report the novel finding that Numb4 (but not Numb4d7) promotes SCF(Fbw7) ubiquitin ligase assembly and activation to increase Notch degradation. However, both Numb isoforms decrease epidermal growth factor receptor (EGFR) expression, thereby regulating GSC fate. Small molecule inhibition of EGFR activity phenocopies the effect of Numb on CD133 and Pax6. Clinically, homozygous NUMB deletions and low Numb mRNA expression occur primarily in a subgroup of proneural glioblastomas. Higher Numb expression is found in classical and mesenchymal glioblastomas and correlates with decreased survival. Thus, decreased Numb promotes glioblastoma growth, but the remaining Numb establishes a phenotypically diverse stem-like cell hierarchy that increases tumor aggressiveness and therapeutic resistance.

mRNA expression profiling has suggested the existence of multiple glioblastoma subclasses, but their number and characteristics vary among studies and the etiology underlying their development is unclear. In this study, we analyzed 261 microRNA expression profiles from The Cancer Genome Atlas (TCGA), identifying five clinically and genetically distinct subclasses of glioblastoma that each related to a different neural precursor cell type. These microRNA-based glioblastoma subclasses displayed microRNA and mRNA expression signatures resembling those of radial glia, oligoneuronal precursors, neuronal precursors, neuroepithelial/neural crest precursors, or astrocyte precursors. Each subclass was determined to be genetically distinct, based on the significant differences they displayed in terms of patient race, age, treatment response, and survival. We also identified several microRNAs as potent regulators of subclass-specific gene expression networks in glioblastoma. Foremost among these is miR-9, which suppresses mesenchymal differentiation in glioblastoma by downregulating expression of JAK kinases and inhibiting activation of STAT3. Our findings suggest that microRNAs are important determinants of glioblastoma subclasses through their ability to regulate developmental growth and differentiation programs in several transformed neural precursor cell types. Taken together, our results define developmental microRNA expression signatures that both characterize and contribute to the phenotypic diversity of glioblastoma subclasses, thereby providing an expanded framework for understanding the pathogenesis of glioblastoma in a human neurodevelopmental context.

PURPOSE: Numerous preclinical studies have demonstrated that angiogenesis inhibitors can increase the efficacy of radiotherapy (RT). We sought to examine the safety and efficacy of bevacizumab (BV) and RT in soft tissue sarcomas and explore biomarkers to help determine the treatment response. METHODS AND MATERIALS: Patients with ≥5 cm, intermediate- or high-grade soft tissue sarcomas at significant risk of local recurrence received neoadjuvant BV alone followed by BV plus RT before surgical resection. Correlative science studies included analysis of the serial blood and tumor samples and serial perfusion computed tomography scans. RESULTS: The 20 patients had a median tumor size of 8.25 cm, with 13 extremity, 1 trunk, and 6 retroperitoneal/pelvis tumors. The neoadjuvant treatment was well tolerated, with only 4 patients having Grade 3 toxicities (hypertension, liver function test elevation). BV plus RT resulted in ≥80% pathologic necrosis in 9 (45%) of 20 tumors, more than double the historical rate seen with RT alone. Three patients had a complete pathologic response. The median microvessel density decreased 53% after BV alone (p <.05). After combination therapy, the median tumor cell proliferation decreased by 73%, apoptosis increased 10.4-fold, and the blood flow, blood volume, and permeability surface area decreased by 62-72% (p <.05). Analysis of gene expression microarrays of untreated tumors identified a 24-gene signature for treatment response. The microvessel density and circulating progenitor cells at baseline and the reduction in microvessel density and plasma soluble c-KIT with BV therapy also correlated with a good pathologic response (p <.05). After a median follow-up of 20 months, only 1 patient had developed local recurrence. CONCLUSIONS: The results from the present exploratory study indicated that BV increases the efficacy of RT against soft tissue sarcomas and might reduce the incidence of local recurrence. Thus, this regimen warrants additional investigation. Gene expression profiles and other tissue and circulating biomarkers showed promising correlations with treatment response.

PURPOSE: Meningiomas are the most common central nervous system tumors in the population of age 35 and older. WHO defines three grades predictive of the risk of recurrence. Clinical data supporting histologic malignant progression of meningiomas are sparse and underlying molecular mechanisms are not clearly depicted. EXPERIMENTAL DESIGN: We identified genetic alterations associated with histologic progression of 36 paired meningioma samples in 18 patients using 500K SNP genotyping arrays and NF2 gene sequencing. RESULTS: The most frequent chromosome alterations observed in progressing meningioma samples are early alterations (i.e., present both in lower- and higher-grade samples of a single patient). In our series, NF2 gene inactivation was an early and frequent event in progressing meningioma samples (73%). Chromosome alterations acquired during progression from grade I to grade II meningioma were not recurrent. Progression to grade III was characterized by recurrent genomic alterations, the most frequent being CDKN2A/CDKN2B locus loss on 9p. CONCLUSION: Meningiomas displayed different patterns of genetic alterations during progression according to their NF2 status: NF2-mutated meningiomas showed higher chromosome instability during progression than NF2-nonmutated meningiomas, which had very few imbalanced chromosome segments. This pattern of alterations could thus be used as markers in clinical practice to identify tumors prone to progress among grade I meningiomas.

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.