Katayoon Kasaian

ORegAnno: an open-access community-driven resource for regulatory annotation

ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regulatory sequences for over 3853 genes and 465 transcription factors from 19 species. A new feature called the ‘publication queue’ allows users to input relevant papers from scientific literature as targets for annotation. The queue contains 4438 gene regulation papers entered by experts and another 54 351 identified by text-mining methods. Users can enter or ‘check out’ papers from the queue for manual curation using a series of user-friendly annotation pages. A typical record entry consists of species, sequence type, sequence, target gene, binding factor, experimental outcome and one or more lines of experimental evidence. An evidence ontology was developed to describe and categorize these experiments. Records are cross-referenced to Ensembl or Entrez gene identifiers, PubMed and dbSNP and can be visualized in the Ensembl or UCSC genome browsers. All data are freely available through search pages, XML data dumps or web services at: http://www.oreganno.org.

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

Complete genomic landscape of a recurring sporadic parathyroid carcinoma

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long‐term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto‐oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto‐oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high‐throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well‐characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient‐matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single‐base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets. Copyright

ORegAnno 3.0: a community-driven resource for curated regulatory annotation

The Open Regulatory Annotation database (ORegAnno) is a resource for curated regulatory annotation. It contains information about regulatory regions, transcription factor binding sites, RNA binding sites, regulatory variants, haplotypes, and other regulatory elements. ORegAnno differentiates itself from other regulatory resources by facilitating crowd-sourced interpretation and annotation of regulatory observations from the literature and highly curated resources. It contains a comprehensive annotation scheme that aims to describe both the elements and outcomes of regulatory events. Moreover, ORegAnno assembles these disparate data sources and annotations into a single, high quality catalogue of curated regulatory information. The current release is an update of the database previously featured in the NAR Database Issue, and now contains 1 948 307 records, across 18 species, with a combined coverage of 334 215 080 bp. Complete records, annotation, and other associated data are available for browsing and download at http://www.oreganno.org/.

Coordinated expression of galectin-3 and caveolin-1 in thyroid cancer†

Galectin‐3 (Gal3) is the single most accurate marker for the diagnosis of differentiated thyroid cancer (DTC). Gal3 overrides the tumour suppressor activity of caveolin‐1 (Cav1) and functions in concert with Cav1 to promote focal adhesion turnover and tumour cell migration and invasion. To study their coordinated role in progression of a human cancer, we investigated the expression of Gal3 and Cav1 in specimens of human benign thyroid lesions, DTC and anaplastic thyroid cancer (ATC). Gal3 and Cav1 expression is significantly associated with DTC and ATC, but not benign nodules. Essentially all Cav1‐positive DTC cancers express Gal3, supporting the synergistic activity of these two proteins in DTC progression. Similarly, coordinated elevated Gal3/Cav1 expression was observed in three DTC‐derived cell lines (papillary TCP1 and KTC1 and follicular FTC133) but only one (ACT1) of five ATC‐derived cell lines. Using siRNA knockdown, Gal3 and Cav1 were shown to be required for RhoA GTPase activation, stabilization of focal adhesion kinase (FAK; a measure of focal adhesion signalling and turnover) and increased migration of the DTC cell lines studied, but not the ATC cell lines, including ACT1, which expresses elevated levels of Gal3 and Cav1. Co‐expression of Gal3 and Cav1 in the T238 anaplastic cell line stabilized FAK‐GFP in focal adhesions. Gal3 and Cav1 therefore function synergistically to promote focal adhesion signalling, migration and progression of DTC. Copyright

A new frontier in personalized cancer therapy: mapping molecular changes

Mutations in the genome of a normal cell can affect the function of its many genes and pathways. These alterations could eventually transform the cell from a normal to a malignant state by allowing an uncontrolled proliferation of the cell and formation of a cancer tumor. Each tumor in an individual patient can have hundreds of mutated genes and perturbed pathways. Cancers clinically presenting as the same type or subtype could potentially be very different at the molecular level and thus behave differently in response to therapy. The challenge is to distinguish the key mutations driving the cancer from the background of mutational noise and find ways to effectively target them. The promise is that such a molecular approach to classifying cancer will lead to better diagnostic, prognostic and personalized treatment strategies. This article provides an overview of advances in the molecular characterization of cancers and their applications in therapy.

Diagnostic Value of Next-Generation Sequencing in an Unusual Sphenoid Tumor

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.

The genomic and transcriptomic landscape of anaplastic thyroid cancer: implications for therapy

BackgroundAnaplastic thyroid carcinoma is the most undifferentiated form of thyroid cancer and one of the deadliest of all adult solid malignancies. Here we report the first genomic and transcriptomic profile of anaplastic thyroid cancer including those of several unique cell lines and outline novel potential drivers of malignancy and targets of therapy.MethodsWe describe whole genomic and transcriptomic profiles of 1 primary anaplastic thyroid tumor and 3 authenticated cell lines. Those profiles augmented by the transcriptomes of 4 additional and unique cell lines were compared to 58 pairs of papillary thyroid carcinoma and matched normal tissue transcriptomes from The Cancer Genome Atlas study.ResultsThe most prevalent mutations were those of TP53 and BRAF; repeated alterations of the epigenetic machinery such as frame-shift deletions of HDAC10 and EP300, loss of SMARCA2 and fusions of MECP2, BCL11A and SS18 were observed. Sequence data displayed aneuploidy and large regions of copy loss and gain in all genomes. Common regions of gain were however evident encompassing chromosomes 5p and 20q. We found novel anaplastic gene fusions including MKRN1-BRAF, FGFR2-OGDH and SS18-SLC5A11, all expressed in-frame fusions involving a known proto-oncogene. Comparison of the anaplastic thyroid cancer expression datasets with the papillary thyroid cancer and normal thyroid tissue transcriptomes suggested several known drug targets such as FGFRs, VEGFRs, KIT and RET to have lower expression levels in anaplastic specimens compared with both papillary thyroid cancers and normal tissues, confirming the observed lack of response to therapies targeting these pathways. Further integrative data analysis identified the mTOR signaling pathway as a potential therapeutic target in this disease.ConclusionsAnaplastic thyroid carcinoma possessed heterogeneous and unique profiles revealing the significance of detailed molecular profiling of individual tumors and the treatment of each as a unique entity; the cell line sequence data promises to facilitate the more accurate and intentional drug screening studies for anaplastic thyroid cancer.

MEN1 Mutations in Hürthle Cell (Oncocytic) Thyroid Carcinoma

CONTEXT AND OBJECTIVE Oncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation, and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management. DESIGN AND PATIENTS Initially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line, and five Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in the multiple endocrine neoplasia I (MEN1) gene. RESULTS Here we report the identification of MEN1 loss-of-function mutations in 4% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumors. CONCLUSION Menin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of MEN1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis, and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

Differential roles of RET isoforms in medullary and papillary thyroid carcinomas

The RET receptor tyrosine kinase mediates cell proliferation, survival and migration in embryogenesis and is implicated in the transformation and tumour progression in multiple cancers. RET is frequently mutated and constitutively activated in familial and sporadic thyroid carcinomas. As a result of alternative splicing, RET is expressed as two protein isoforms, RET9 and RET51, which differ in their unique C-terminal amino acids. These isoforms have distinct intracellular trafficking and associated signalling complexes, but functional differences are not well defined. We used shRNA-mediated knockdown (KD) of individual RET isoforms or of total RET to evaluate their functional contributions in thyroid carcinoma cells. We showed that RET is required for cell survival in medullary (MTC) but not papillary thyroid carcinoma (PTC) cells. In PTC cells, RET depletion reduced cell migration and induced a flattened epithelial-like morphology. RET KD decreased the expression of mesenchymal markers and matrix metalloproteinases and reduced anoikis resistance and invasive potential. Further, we showed that RET51 depletion had significantly greater effects on each of these processes than RET9 depletion in both MTC and PTC cells. Finally, we showed that expression of RET, particularly RET51, was correlated with malignancy in a panel of human thyroid tumour tissues. Together, our data show that RET expression promotes a more mesenchymal phenotype with reduced cell-cell adhesion and increased invasiveness in PTC cell models, but is more important for tumour cell survival, proliferation and anoikis resistance in MTC models. Our data suggest that the RET51 isoform plays a more prominent role in mediating these processes compared to RET9.