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Open AccessArticle

Aberrant MUC1-TRIM46-KRTCAP2 Chimeric RNAs in High-Grade Serous Ovarian Carcinoma

by Kalpana Kannan, Gona Karimi Kordestani, Anika Galagoda, Cristian Coarfa and Laising Yen

Cancers 2015, 7(4), 2083-2093; https://doi.org/10.3390/cancers7040878 - 19 Oct 2015

Cited by 10 | Viewed by 5153

Abstract

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. By analyzing the mRNA-seq reads from The Cancer Genome Atlas (TCGA), we uncovered a novel cancer-enriched chimeric RNA as the result of splicing between MUC1, a highly glycosylated [...] Read more.

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. By analyzing the mRNA-seq reads from The Cancer Genome Atlas (TCGA), we uncovered a novel cancer-enriched chimeric RNA as the result of splicing between MUC1, a highly glycosylated transmembrane mucin, TRIM46, a tripartite motif containing protein, and KRTCAP2, a keratinocyte associated protein. Experimental analyses by RT-PCR (reverse transcription PCR) and Sanger sequencing using an in-house cohort of 59 HGSC patient tumors revealed a total of six MUC1-TRIM46-KRTCAP2 isoforms joined by different annotated splice sites between these genes. These chimeric isoforms are not detected in non-cancerous ovaries, yet are present in three out of every four HGSC patient tumors, a significant frequency given the exceedingly heterogeneous nature of this disease. Transfection of the cDNA of MUC1-TRIM46-KRTCAP2 isoforms in mammalian cells led to the translation of mutant MUC1 fusion proteins that are unglycosylated and cytoplasmically localized as opposed to the cell membrane, a feature resembling the tumor-associated MUC1. Because the parental MUC1 is overexpressed in 90% of HGSC tumors and has been proposed as a clinical biomarker and therapeutic target, the chimeric MUC1-TRIM46-KRTCAP2 isoforms identified in this report could represent significantly better MUC1 variants for the same clinical utilities. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Review

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Open AccessReview

Revealing the Complexity of Breast Cancer by Next Generation Sequencing

by John Verigos and Angeliki Magklara

Cancers 2015, 7(4), 2183-2200; https://doi.org/10.3390/cancers7040885 - 06 Nov 2015

Cited by 25 | Viewed by 6003

Abstract

Over the last few years the increasing usage of “-omic” platforms, supported by next-generation sequencing, in the analysis of breast cancer samples has tremendously advanced our understanding of the disease. New driver and passenger mutations, rare chromosomal rearrangements and other genomic aberrations identified [...] Read more.

Over the last few years the increasing usage of “-omic” platforms, supported by next-generation sequencing, in the analysis of breast cancer samples has tremendously advanced our understanding of the disease. New driver and passenger mutations, rare chromosomal rearrangements and other genomic aberrations identified by whole genome and exome sequencing are providing missing pieces of the genomic architecture of breast cancer. High resolution maps of breast cancer methylomes and sequencing of the miRNA microworld are beginning to paint the epigenomic landscape of the disease. Transcriptomic profiling is giving us a glimpse into the gene regulatory networks that govern the fate of the breast cancer cell. At the same time, integrative analysis of sequencing data confirms an extensive intertumor and intratumor heterogeneity and plasticity in breast cancer arguing for a new approach to the problem. In this review, we report on the latest findings on the molecular characterization of breast cancer using NGS technologies, and we discuss their potential implications for the improvement of existing therapies. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

Next-Generation Sequencing in Clinical Molecular Diagnostics of Cancer: Advantages and Challenges

by Rajyalakshmi Luthra, Hui Chen, Sinchita Roy-Chowdhuri and R. Rajesh Singh

Cancers 2015, 7(4), 2023-2036; https://doi.org/10.3390/cancers7040874 - 14 Oct 2015

Cited by 103 | Viewed by 13822

Abstract

The application of next-generation sequencing (NGS) to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has [...] Read more.

The application of next-generation sequencing (NGS) to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has been difficult to manage by traditional low- and/or medium-throughput sequencing platforms. Massively parallel sequencing capabilities of NGS provide a much-needed alternative for mutation screening in multiple genes with a single low investment of DNA. However, implementation of NGS technologies, most of which are for research use only (RUO), in a diagnostic laboratory, needs extensive validation in order to establish Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP)-compliant performance characteristics. Here, we have reviewed approaches for validation of NGS technology for routine screening of tumors. We discuss the criteria for selecting gene markers to include in the NGS panel and the deciding factors for selecting target capture approaches and sequencing platforms. We also discuss challenges in result reporting, storage and retrieval of the voluminous sequencing data and the future potential of clinical NGS. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

Next-Generation Sequencing Approaches in Cancer: Where Have They Brought Us and Where Will They Take Us?

by Veronique G. LeBlanc and Marco A. Marra

Cancers 2015, 7(3), 1925-1958; https://doi.org/10.3390/cancers7030869 - 23 Sep 2015

Cited by 47 | Viewed by 6297

Abstract

Next-generation sequencing (NGS) technologies and data have revolutionized cancer research and are increasingly being deployed to guide clinicians in treatment decision-making. NGS technologies have allowed us to take an “omics” approach to cancer in order to reveal genomic, transcriptomic, and epigenomic landscapes of [...] Read more.

Next-generation sequencing (NGS) technologies and data have revolutionized cancer research and are increasingly being deployed to guide clinicians in treatment decision-making. NGS technologies have allowed us to take an “omics” approach to cancer in order to reveal genomic, transcriptomic, and epigenomic landscapes of individual malignancies. Integrative multi-platform analyses are increasingly used in large-scale projects that aim to fully characterize individual tumours as well as general cancer types and subtypes. In this review, we examine how NGS technologies in particular have contributed to “omics” approaches in cancer research, allowing for large-scale integrative analyses that consider hundreds of tumour samples. These types of studies have provided us with an unprecedented wealth of information, providing the background knowledge needed to make small-scale (including “N of 1”) studies informative and relevant. We also take a look at emerging opportunities provided by NGS and state-of-the-art third-generation sequencing technologies, particularly in the context of translational research. Cancer research and care are currently poised to experience significant progress catalyzed by accessible sequencing technologies that will benefit both clinical- and research-based efforts. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

Analysis of Pre-Analytic Factors Affecting the Success of Clinical Next-Generation Sequencing of Solid Organ Malignancies

by Hui Chen, Rajyalakshmi Luthra, Rashmi S. Goswami, Rajesh R. Singh and Sinchita Roy-Chowdhuri

Cancers 2015, 7(3), 1699-1715; https://doi.org/10.3390/cancers7030859 - 28 Aug 2015

Cited by 97 | Viewed by 6711

Abstract

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to [...] Read more.

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection

by Phillip N. Gray, Charles L.M. Dunlop and Aaron M. Elliott

Cancers 2015, 7(3), 1313-1332; https://doi.org/10.3390/cancers7030837 - 17 Jul 2015

Cited by 47 | Viewed by 10456

Abstract

The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to [...] Read more.

The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

Exploring the Mechanisms of Gastrointestinal Cancer Development Using Deep Sequencing Analysis

by Tomonori Matsumoto, Takahiro Shimizu, Atsushi Takai and Hiroyuki Marusawa

Cancers 2015, 7(2), 1037-1051; https://doi.org/10.3390/cancers7020823 - 15 Jun 2015

Cited by 7 | Viewed by 6346

Abstract

Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic [...] Read more.

Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic mutations in tumor cells, NGS technologies enable us to approach the subject of carcinogenic mechanisms from new perspectives. Deep sequencing, a method of optimizing the high throughput capacity of NGS technologies, allows for the detection of genetic aberrations in small subsets of premalignant and/or tumor cells in noncancerous chronically inflamed tissues. Genome-wide NGS data also make it possible to clarify the mutational signatures of each cancer tissue by identifying the precise pattern of nucleotide alterations in the cancer genome, providing new information regarding the mechanisms of tumorigenesis. In this review, we highlight these new methods taking advantage of NGS technologies, and discuss our current understanding of carcinogenic mechanisms elucidated from such approaches. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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433 KiB

Open AccessReview

Next-Generation Sequencing in Clinical Oncology: Next Steps Towards Clinical Validation

by Nigel C. Bennett and Camile S. Farah

Cancers 2014, 6(4), 2296-2312; https://doi.org/10.3390/cancers6042296 - 18 Nov 2014

Cited by 44 | Viewed by 10086

Abstract

Compelling evidence supports the transition of next generation sequencing (NGS) technology from a research environment into clinical practice. Before NGS technologies are fully adopted in the clinic, they should be thoroughly scrutinised for their potential as powerful diagnostic and prognostic tools. The importance [...] Read more.

Compelling evidence supports the transition of next generation sequencing (NGS) technology from a research environment into clinical practice. Before NGS technologies are fully adopted in the clinic, they should be thoroughly scrutinised for their potential as powerful diagnostic and prognostic tools. The importance placed on generating accurate NGS data, and consequently appropriate clinical interpretation, has stimulated much international discussion regarding the creation and implementation of strict guidelines and regulations for NGS clinical use. In the context of clinical oncology, NGS technologies are currently transitioning from a clinical research background into a setting where they will contribute significantly to individual patient cancer management. This paper explores the steps that have been taken, and those still required, for the transition of NGS into the clinical area, with particular emphasis placed on validation in the setting of clinical oncology. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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