Challenges and opportunities for next-generation sequencing in companion diagnostics.

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SPECIAL FOCUS y In vitro companion diagnostics

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Challenges and opportunities for next-generation sequencing in companion diagnostics Expert Rev. Mol. Diagn. Early online, 1–17 (2014)

Erick Lin1, Jeremy Chien2, Frank S Ong1 and Jian-Bing Fan*1 1 Illumina, Inc. 5200 Illumina Way, San Diego, CA 92122, USA 2 University of Kansas Cancer Center, 3901 Rainbow Blvd, Kansas City, KS 66160, USA *Author for correspondence: [email protected]

The rapid decline in sequencing costs has allowed next-generation sequencing (NGS) assays, previously ubiquitous only in research laboratories, to begin making inroads into molecular diagnostics. Genotypic assays – DNA sequencing – include whole genome sequencing, whole exome sequencing, focused assays that target only a handful of genes. Phenotypic assays comprise a broader spectrum of options and can query a variety of epigenetic modifications of DNA (such as ChIP-seq, bisulfite sequencing, DNase-I hypersensitivity site-sequencing, Formaldehyde-Assisted Isolation of Regulatory Elements-sequencing, etc.) that regulate gene expression-related processes or gene expression (RNA-sequencing) itself. To date, the US FDA has only cleared 12 DNA-based companion diagnostic tests, all in cancer. Although challenges exist for NGS in companion diagnostics, the wide-ranging capabilities of NGS offer extraordinary opportunities for the development and implementation of NGS-based companion diagnostics to probe oncogenes, tumor suppressor genes and cancer-enabling genes. KEYWORDS: bioinformatics • companion diagnostics • CTC • ctDNA • next-generation sequencing • NGS • precision medicine • precision oncology • tumor heterogeneity

Introduction to next-generation sequencing technology

When the first human genome was sequenced in 2001, the cost to sequence a single human genome was approximately US$3 billion and required years of dedicated and collaborative efforts from laboratories across the globe [1]. With the advent of next-generation sequencing (NGS) technology in 2005 and subsequent entry into the sequencing market in 2007, the cost to sequence a full human genome at 30X coverage has dropped substantially from US $1 million in 2008 to US$10,000 in 2011 (FIGURE 1). The throughput and cost of NGS has reached the point where a whole human genome can be sequenced for less than US $1000 [2]. The rapid decline in sequencing costs has allowed genomic sequencing assays, previously ubiquitous only in research laboratories, to begin making inroads into molecular diagnostics laboratories. A standard NGS workflow informahealthcare.com

10.1586/14737159.2015.961916

involves four major steps: sample preparation, amplification, sequencing and data analysis (FIGURE 2). Once the original DNA is extracted from a sample, enrichment strategies may be utilized for a subset of applications such as targeted gene panels or exome sequencing in order to focus only on a subset of genomic targets. NGS platform-specific adapters are added to the original DNA molecules to allow for sequencing to occur on the associated NGS platform. The amplification step clonally amplifies individual molecules and allows subsequent sequencing reactions to be spatiallyarranged or spatially-separated. Next, the detection of individual sequencing reactions may occur via digital imaging or measurement of pH changes. Once the data from the sequencing reactions are acquired, a series of data processing steps is required to convert the raw output from the NGS platform in DNA sequencing information. The final step in the process involves processing the sequencing reads through a bioinformatics pipeline

Ó 2014 Informa UK Ltd

ISSN 1473-7159

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Review

Lin, Chien, Ong & Fan

datasets raises concerns over information access, data security and subject/patient 1000 privacy [4]. In the USA, electronic health Moore’s law record (EHR) systems are subject to fed$1M genome 100 eral regulatory legislation and oversight with the Health Insurance Portability and 10 Accountability Act of 1996. In 2003, the $10K genome 2001 1 IHGSC reports the security regulations requiring appropriate 2005 2007 $1,000 sequence of the first Start of Entry of NGS administrative, physical and technical genome 0.10 human genome NGS into the market safeguards to ensure the confidentiality, integrity and security of stored or in0.01 transit to electronic protected health information were published by the US Department of Health and Services as Figure 1. Cost per raw megabase of DNA sequence in US dollars (2000–2014). The Security Rule [5]. As the sheer volData taken from National Human Genome Research Institute and [68]. ume of cumulative NGS data grows, providing information access, data security designed to detect DNA variants. The ability of NGS to and subject/patient privacy is a constant challenge, especially sequencing hundreds of thousands to millions of DNA mole- for genomic sequencing assays that target the whole genome or cules in parallel has fundamentally shifted genomic sequencing whole exome. assays away from automated Sanger sequencing [3]. As previously illustrated in FIGURE 1, the growth in the raw Applications of NGS technology sequencing data output NGS platforms has outstripped NGS assays can be broadly separated based on what genotype Moore’s Law relating to advances in information technology or phenotype they are seeking to decipher. Genotypic and storage capacity. As a result, molecular diagnostic laborato- assays – DNA sequencing – differ as to the level of comprehenries face the daunting challenge of storing and protecting the siveness they seek, ranging from whole genome sequencing sequence data (i.e., FASTQ, BAM or VCF file formats). The (WGS), to whole exome sequencing (WES), to focused assays availability of both deeply sequenced and large genomic that target only a handful of genes, from germline polymorphism analysis to somatic mutation detection. Phenotypic assays comprise a Original DNA is prepared into a DNA library based broader spectrum of options; reflective of on the NGS platform-specific sequencing adapters the fact that phenotype can manifest itself 1 Sample preparation Target enrichment steps may be utilized for in many ways. Phenotypic changes can certain applications (i.e., enrichment for targeted also be queried with NGS for a variety of gene panels, whole exome sequencing) epigenetic modifications of DNA (such as ChIP-seq, bisulfite sequencing, Individual DNA library molecules are clonally DNase-I hypersensitivity site-sequencing, amplified Formaldehyde-Assisted Isolation of Regu2 Amplification Based on NGS-platform,amplification can occur latory Elements-sequencing, etc.) that regvia clonal bridge amplification, emulsion PCR or ulate gene expression-related processes or gridded-DNA nanoballs gene expression (RNA-sequencing [RNASeq]) itself. During amplification,the clonally amplified DNA Although WGS can be used for de molecules arespatially arranged (i.e., clonal clusters, beads, or balls) novo sequencing, application of WGS, 3 Sequencing WES and targeted sequencing in human Millions to billions of individual sequencing reactions occur in parallel genomics broadly fall into the category of (massively parallel sequencing) DNA re-sequencing, meaning that a reference genome is available and that DNA Data acquired for each individual sequencing from specific individual is sequenced to chemical reactions test for known mutations (genotyping) or Each seqencing reaction is distinctly detected 4 Data analysis to scan for somatic and germline via digital imaging or pH change variations in entire genome, exome or Data formations: FASTQ file Æ BAM file Æ VCF file smaller sub-regions. The sequencing assay remains the same irrespective of the Figure 2. Introduction to next-generation sequencing. targeted area; the targeting process is 20 00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14


Cost per raw megabase of DNA sequence (USD)

10,000

doi: 10.1586/14737159.2015.961916

Expert Rev. Mol. Diagn.


NGS in companion diagnostics

Review

addressed during sample preparation. The Table 1. Comparison of next-generation sequencing-based DNA key factors in deciding which approaches sequencing assays. to take include: budget, the degree of WGS • Hypothesis-free approach exploration one wishes to engage in, and • Provides most complete snapshot (i.e., exons, introns, regulatory regions) sample availability (i.e., quality and quan• Lowest depth of coverage tity). The primary reason for performing • Most difficult data interpretation WGS is comprehensiveness. For WES, • Targets coding regions of genome Whole the purpose is to interrogate all of pro• Increased sequencing depth over WGS exome tein-coding regions within the genome. • Easier data interpretation compared with WGS sequencing Of the three genotypic assays, targeted sequencing uses the least machine time, Targeted • Hypothesis-driven approach reagents, computer time, data storage and sequencing • Provides highest depth of sequencing expert analysis; it is less expensive per • Easiest data interpretation sample than either WGS or WES. Given WGS: Whole genome sequencing. this, one of the chief benefits of targeted sequencing is that it is inexpensive enough to permit sequencing with great depth, that is, Nanopore DNA sequencing is method based on feeding a with many independent reads through a given region. strand of DNA through a biological pore. The nucleotides are By deeply sampling the regions of interest, one can discover identified by measuring the difference in their electrical conmutations present even in the face of the two key sources of ductivity as they pass through the pore [7]. Current NGS techsignal dilution in a cancer sample: contamination of the tumor nologies are based on short-read lengths, while the SMRT specimen with normal cells and clonal heterogeneity. TABLE 1 sequencing and nanopore sequencing are long-read length techshows a summary comparing WGS, WES and targeted nologies. The combination of current NGS and emerging sequencing technologies provides powerful complementary tools sequencing. The most common functional genomic assay, RNA-Seq ana- for genomic analysis. lyzes RNA as opposed to DNA. RNA-Seq experiments are used to study the transcriptome of an organism, which, as a Unlocking the power of the genome: building the necessary intermediate of protein expression, can be closely companion diagnostics knowledge base linked to phenotype, including disease state. Bisulfite sequenc- One of the major challenges for clinical utilization of NGS ing is an application used to study the patterns of cytosine genetic data is building a knowledge base for variant calling methylation in the genome that control the transcription of and annotation for clinical interpretation and results reporting. nearby genes to RNA. Similarly, NGS can be used to probe Identification of variants beyond the well-studied single nucleoother aspects of epigenetic state, including chromatin-bound tide variants and small indels remains a major obstacle for stanportions of DNA and those bound by particular transcription dardization of the data analysis pipelines. Complex variant factors. Thus, like RNA-Seq, bisulfite sequencing can be used types such as copy number variants (CNVs; loss or gain of one as a readout of biological states linked to disease. Other DNA or more copies of a region of the genome), structural variants sequencing, such as DNase-I hypersensitivity site-sequencing (SVs; including duplications, inversions and translocations) and and Formaldehyde-Assisted Isolation of Regulatory Elements- epigenetic variations all pose difficulties in detection and are sequencing provide readouts of regulatory regions that are cell therefore associated with higher error rates [8–12]. Both CNVs type- and disease state-dependent. All of these experimental and SVs are more difficult to detect using the current generatechniques may be applied toward the understanding of human tion of variant callers. In the case of CNVs, the variant will diseases, with the goal of ultimately improving patient manifest as slightly higher or lower coverage on average in the particular CNV region compared with the remainder of the outcomes. genome [9]. In the case of SVs, one read of a paired-end sequencing experiment will align to one part of the genome, Emerging sequencing technologies While multiple NGS platforms are commercially available, two and the other read will align to a different part of the emerging sequencing technologies have been or are in the pro- genome [11]. However, due to the presence of repeat elements cess of being commercialized. Single-molecule real-time in human genome, homology with alternate genomic regions, (SMRT) sequencing works by sequencing strands of DNA on the presence of regions with unresolved sequence order and chips containing thousands of zero-mode wave-guides, small structure and the complexity of SVs and heterogeneity in spewell-like containers with a DNA polymerase and capturing and cific diseases like human cancers, sensitive and specific SV calldetection tools located at the bottom of the well. According to ing algorithms need to be refined in order to further build on the SMRT technology developer, this methodology allows the ever-growing knowledge base. As the previously described detection of nucleotide modifications (such as cytosine methyl- emerging sequencing technologies continue to develop, some of ation) through observations of polymerase kinetics [6]. the current difficulties with SVs may be addressed with the informahealthcare.com

doi: 10.1586/14737159.2015.961916


Review


long-read technologies by providing a broader genomic context for alignments. Regardless of the variant type, once a set of variants is identified from a cancer sample, a further challenge is to classify each as driver versus passenger [13]. Current methods that are utilized in the clinical research setting for such classification are generally based on calculation of risk scores, such as Sorting Intolerant from Tolerant or Polymorphism Phenotyping [14,15]; determination of the protein coding effect of each mutation, either sense, missense or nonsense or identification within a relevant database, such as dbSNP, COSMIC, OMIM or HapMap [16–19]. All of these methods are largely qualitative in nature, and the result of such methods is not a final classification of each variant, but rather a ranking of the identified variants according to likelihood of pathogenicity. Some pipelines endeavor to identify drivers versus passengers at the gene level rather than at the variant level, with the rationale that many variants within a single gene are more likely to affect its function and are thus related to cancer progression [20–23]. This gap in knowledge limits rapid translation of genomic information into clinically actionable genetic data, but analyses of genomic data on an individual and population level are helping to bridge this gap and accelerating the development of NGS-based tests. Many studies have been published across cancer types that identify risk genes rather than specific variants; however, more recently, prominent groups have raised concern at this method, since as sample size increases, the significant results balloons and is dominated by false positives [20]. After noting many published studies that implausibly identified olfactory receptors as risk genes for a wide range of cancers, Lawrence et al. developed MutSigCV, which accounts for the mutational heterogeneity of cancer when evaluating candidate genes [20]. Related tools to identify driver variants and genes have been developed by other groups and are well-summarized by Gonzalez-Perez et al. [21]. Even as bioinformatics tools for alignment and variant calling continue to be refined and/or developed, the downstream variant annotation process will remain as an additional bioinformatics hurdle for clinical interpretation and results reporting. Challenges in analyzing & aggregating complex genomic data

Using NGS sequencing to guide clinical decision-making is bioinformatically complex, as it involves building connections between genetic information in the biological realm, and treatment information in the clinical realm. Public and private initiatives are currently underway to assist in the identification of and the development of a consensus on medically relevant genomic variants. One public initiative for analyzing and aggregating variant data is ClinVar [24], a freely available archive of reports of relationships among medically important variants and phenotypes. ClinVar is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human genome assemblies. The database contains doi: 10.1586/14737159.2015.961916

accessioned submissions reporting human variation, interpretations of the relationship of that variation to human health/ disease and the evidence supporting each interpretation. The phenotypic descriptions associated with ClinVar are maintained in MedGen [25]. The information on ClinVar requires expert review and analysis by a genetics professional prior to applying the genetics data for direct diagnostic use or in the medical decision-making process. One important note is that the submissions to ClinVar are not independently verified by NIH. Beyond the variant databases, initiatives are currently underway that take a patient-oriented or population-based analysis of genomic data. The flagship 100,000 Genomes Project led by Genomics England, a company owned by the Department of Health in the UK, plans to sequence 100,000 whole genomes by 2017 with a focus on patients with rare diseases, and their families, as well as patients with common cancers. For cancer-specific initiatives, The Cancer Genome Atlas (TCGA) launched in 2006 and the International Cancer Genome Consortium [26] launched in 2008 both have the common goals to coordinate and organize a large number of research projects among different groups to comprehensively characterize and catalog the genomic alterations present in many forms of cancer. The TCGA pilot project demonstrated that an atlas of changes could be created for specific cancer types by utilizing a national network of research and technology teams working on distinct but related projects. TCGA pilot project showed that results from multiple projects could be pooled and developed into an infrastructure for making the data publicly accessible, thereby enabling researchers anywhere around the world to validate clinically significant discoveries. On the international stage, the International Cancer Genome Consortium has received commitments from funding organizations in Asia, Australia, Europe, North America and South America for 71 project teams in 17 jurisdictions to study over 25,000 tumor genomes [27]. Ultimately, public initiatives aid in the comprehensive analysis and characterization of cancer genomes by providing a public information exchange to share genomic results. As these large-scale studies are beginning to create and accumulate a wealth of genomic information, general insights can be gleaned from these datasets. In general, recurrent point mutations are frequently observed in proto-oncogenes, and the resulting oncogenes are prime targets for cancer therapies. Examples include V600 mutations in BRAF in cancer that lead to constitutive activation of BRAF, thereby allowing one to target the BRAF activity by several drugs. Other types of alterations in proto-oncogenes include amplification, translocation and overexpression, exemplified by human epidermal growth factor receptor 2 (HER2), BCR-ABL and c-Myc. HER2 and BCR-ABL alterations can be targeted with various inhibitors (such as Herceptin for HER2 amplified breast cancer and Gleevec for BCR-ABL translocation in chronic myeloid leukemia), and therefore these alterations and related companion diagnostics are actionable in the management of specific cancers. One of the most recent major developments in molecular diagnostics Expert Rev. Mol. Diagn.



and clinical oncology is the discovery of the EML4-ALK fusion oncogene in a subset of non-small-cell lung cancer (NSCLC), which arises from an inversion on the short arm of chromosome 2, inv(2)(p21p23) that joins exons 1–13 of EML4 to exons 20–29 of anaplastic lymphoma kinase (ALK) [28]. From the time of publication of the EML4-ALK fusion oncogene in 2007 to the conditional approval by the US FDA of the EML4-ALK inhibitor, crizotinib, in August 2011, only a total of 4 years was required to bring this single discovery from the bench to the bedside. In this particular case, the rapid development and validation of the companion diagnostic test Vysis ALK Break Apart FISH Probe Kit (*CE-marked – from Abbott Molecular; approved in September 2010) was a key factor to quickly bring crizotinib to patients and to improve patient outcomes. In contrast, although c-Myc overexpression is common in a variety of cancers, therapeutic agents that target c-Myc are not fully developed for clinical use, and developing potential companion diagnostics for c-Myc is a challenge. Similarly, many tumor suppressor genes that are frequently mutated in cancers are difficult to target for therapeutic benefits. Therefore, mutations in tumor suppressor genes represent another challenge in companion diagnostics for treatment of cancers. Footnote:*CE marking indicates the compliance with EU legislation of a product and enables its free movement within the European Economic Area and is intended for national market surveillance and enforcement authorities for the EU. Expert commentary Opportunities for NGS in companion diagnostics

The translational utility of genomic sequencing is clear, from understanding of human genetic variation and its association with disease risk and individual response to treatment, to the interpretation and translation of the data for clinical decisionmaking. As NGS technology becomes more widely adopted in the molecular diagnostic laboratories, one can expect NGSbased diagnostic assays and NGS companion diagnostics to become a central piece of routine healthcare management and genomic medicine. The roadmap for the development of NGSbased companion diagnostics gained some clarity after Illumina’s MiSeqDx instrument received CE markings in June 2013 and FDA-clearance as a class II device in November 2013 (510(k) number K123989). Additionally, the FDA cleared three NGS-based assays, two assays for cystic fibrosis carrier screening (Illumina’s 139-Variant Assay and Clinical Sequencing Assay) and the MiSeqDx Universal Kit that allows molecular diagnostic laboratories to design their own assays using the MiSeqDx instrument. The FDA has approved a multitude of nucleic acid-based tests over the years (TABLE 2) [29]. However, as of July 2014, the FDA has only cleared 12 DNA-based companion diagnostic tests. Of the 12 FDA-cleared DNA-based companion diagnostic tests, 7 are FISH/CISH and probe for either HER2 or ALK (TABLE 3) [30]. The five remaining companion diagnostic DNAbased tests are all real-time qualitative PCR assays that are designed to detect specific mutations in either Kirsten rat informahealthcare.com

Review

sarcoma 2 viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) or v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) (TABLE 4) [30]. No FDA-cleared NGS-based companion diagnostic test exists on the market today. The current FDA guidance mandates that if an in vitro diagnostic test is necessary for the safe and effective use of a therapeutic product then a companion diagnostic test is required. The first such effort for developing a NGS-based companion diagnostic comes from the announced partnership between Illumina and Amgen to develop a multigene NGS-based test, which will be used as a companion diagnostic for Amgen’s cancer treatment, panitumumab (VectibixÒ) [31]. In Amgen’s prospective-retrospective study of the completed Panitumumab Randomized Trial In Combination With Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy study [32], additional RAS mutations predicted a lack of response in patients who received panitumumab–FOLFOX4. In patients who had metastatic colorectal cancer without RAS mutations, improvements in overall survival were observed with panitumumab–FOLFOX4 therapy [33]. The Illumina–Amgen partnership will seek to develop an NGS-based test to identify RAS mutation status of patients to determine eligibility for treatment with Vectibix with the ultimate goal of improving patient outcomes. With the emergence of personalized medicine and precision oncology, the partnership between Illumina and Amgen could conceivably be the first of many NGS-based companion diagnostic tests. Opportunities also exist for NGS in companion diagnostics due to the ability of NGS to target a wide spectrum of mutations across a large number of genes/pathways. For the current 12 FDA-approved DNA-based companion diagnostic tests outlined in TABLE 3, each test is only approved for a specific cancer type (i.e., HER2 for breast cancer, KRAS for colorectal cancer, EGFR and ALK for lung cancer, BRAF for melanoma). Although different cancer types may also exhibit HER2 amplification or KRAS, EGFR or BRAF mutations, the efficacy of the approved therapy has only been demonstrated with the specific companion diagnostic test with the specific therapy. The use of NGS to target multiple genes/pathways could provide details regarding the underlying genomic alterations for specific cancer types that provide susceptibility or resistance to specific therapies. Studies into various cancer genomes demonstrate that cancer genomes evolve over time or in response to treatment. In the case of HER2-positive breast cancers, 24% of patients with HER2-positive primary breast tumors were found to have HER2-negative metastatic tumors [34]. There are also several well-characterized acquired resistance genes by certain cancers due to treatment; the T315I ‘gatekeeper mutation’ leads to acquired BCR-ABL tyrosine kinase inhibitor resistance [35] and the T790M mutation leads to acquired EGFR tyrosine kinase inhibitor resistance. The ability for NGS to target a wide spectrum of mutations across a large number of genes/pathways could provide diagnostic information to identify focal amplifications responsible for drug resistance and corresponding targeted therapeutic agent(s) likely to be effective in treating patients as well as providing monitoring for acquired resistance to treatment. doi: 10.1586/14737159.2015.961916

Vysis Vysis

Vysis CLL FISH Probe Kit

CEP 12 SpectrumOrange Direct Labeled Chromosome Enumeration DNA Probe

doi: 10.1586/14737159.2015.961916 Nanosphere, Inc. Third Wave Technology, Inc. Celera Diagnostics

Verigene CFTR and Verigene CFTR PolyT Nucleic Acid Tests

InPlex CF Molecular Test

Cystic Fibrosis Genotyping Assay

FFPE: Formalin-fixed paraffin-embedded; MTHFR: Methylenetetrahyrofolate reductase; PCA3: Prostate cancer gene 3.

Clinical Micro Sensors, Inc.

Luminex Molecular Diagnostics Inc.

xTAG Cystic Fibrosis 71 Kit v2

eSensor Cystic Fibrosis Carrier Detection System



Tm Bioscience Corporation



Tag-It Cystic Fibrosis Kit

Osmetech Molecular Diagnostics

eSensor CF Genotyping Test

BRACAnalysis

Illumina, Inc.

Myriad

Dako TOP2A FISH PharmDx Kit

Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay

Dako Denmark A/S

GeneSearch Breast Lymph Node (BLN) Test Kit

Illumina, Inc.

Veridex, LLC

MammaPrint

Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

Agendia BV

Prosigna Breast Cancer Prognostic Gene Signature Assay

Breast cancer

Cystic fibrosis

Nanostring Technologies

Vysis UroVysion Bladder Cancer Recurrence Kit

Bladder cancer

Vysis

Abbott Molecular Inc.

Vysis EGR1 FISH Probe Kit

B-cell chronic lymphocytic leukemia


Vysis D7S486/CEP 7 FISH Probe Kit

Acute myeloid leukemia

Manufacturer

Trade name

Disease

Table 2. FDA-approved nucleic acid-based tests.

Genotyping of CFTR mutations for cystic fibrosis








Genotyping of 23 ACOG/ACMG recommended mutations for cystic fibrosis

Next-generation sequence analysis of 139 CFTR variants

Next-generation sequence analysis of CFTR gene

BRCA1 and BRCA2 mutations

Copy number of TOP2A gene to indicate an elevated risk of post-surgical recurrence of the breast cancer or decreased long-term survival

Intra-operative assay for qualitative test for metastases in lymph node

Identification of patents with high risk of distant recurrence for breast cancer

Prognosis of distant recurrence-free survival at 10 years

Aneuploidy for Chr 3, 7, 17 and 9p21 loss

chr-12 enumeration

(13q-, +12) vs (11q- or 17p-) groups

EGR1 deletion

Monosomy 7 and 7q deletion

Intended use


Review Lin, Chien, Ong & Fan


informahealthcare.com

AutoGenomics, Inc. Nanosphere, Inc.

INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin

Verigene Warfarin Metabolism Nucleic Acid Test and Verigene System

Nanosphere, Inc.

Verigene F5 Nucleic Acid Test


Verigene MTHFR Nucleic Acid Test

Verigene F2 Nucleic Acid Test

Cepheid

Xpert HemosIL FII & FV

Genotyping of MTHFR

Genotyping of Factor II

Genotyping of Factor V

Genotyping of Factor II (20210G>A) and Factor V (1691G>A) mutations for thrombophilia

Genotyping of 20210G>A mutation in Factor II for thrombophilia

Genotyping of the Factor V G1691A Leiden mutation to identify patients with hereditary risk factor for venous thrombosis

Genotyping of CYP2C9 *2 and *3 polymorphisms and VKORC1 (1173C>T) to identify patients at risk for warfarin-related adverse events

Genotyping of CYP2C9 *2 and *3 polymorphisms and VKORC1 (-1639G>A) to identify patients at risk for warfarin-related adverse events

Genotyping of CYP2C9 *2 and *3 polymorphisms and VKORC1 (-1639G>A and 1173C>T) to identify patients at risk for warfarin-related adverse events

Genotyping of Factor II, Factor V and 5, 10 MTHFR for thrombophilia

ParagonDx, LLC

Gentris Rapid Genotyping Assay – CYP2C9 & VKORCI




eSensor Warfarin Sensitivity Test and XT-8 Instrument


eSensor Thrombophilia Risk Test, eSensor FII-FV Genotyping Test, eSensor Fll Genotyping Test, eSensor FV Genotyping Test, eSensor MTHFR Genotyping Test

TrimGen Corporation

eQ-PCR LC Warfarin Genotyping kit


Genotyping of Factor V Leiden G1691A and Factor II G20210A mutations for thrombophilia

GenMark Diagnostics

eSensor Warfarin Sensitivity Saliva Test

Genotyping of 33 CYP2D6 and 3 CYP2C19 alleles and CYP2D6 copy number alterations

Illumina, Inc.

Roche Molecular Systems, Inc.

Roche AmpliChip CYP450 microarray

Genotyping of UGT1A1 *28 for the chemotherapeutic drug irinotecan

Hologic, Inc.

Third Wave Technologies Inc.

Invader UGT1A1 Molecular Assay

Genotyping of the *2, *3 and *17 alleles of CYP2C19

Illumina VeraCode Genotyping Test for Factor V and Factor II

AutoGenomics, Inc.

INFINITI CYP2C19 Assay


Invader Factor II

Nanosphere, Inc.

Verigene CYP2C 19 Nucleic Acid Test


Hologic, Inc.

Spartan Bioscience, Inc.

Spartan RX CYP2C19 Test System

Genotyping and copy number analysis of CYP2D6

Intended use

Invader Factor V

Luminex Molecular Diagnostics, Inc.

xTAG CYP2D6 Kit v3

Drug metabolizing enzymes

Coagulation factors

Manufacturer

Trade name

Disease

Table 2. FDA-approved nucleic acid-based tests (cont.).



Review

doi: 10.1586/14737159.2015.961916

doi: 10.1586/14737159.2015.961916 Myriad

Pathwork Diagnostics Inc.

Pathwork Tissue of Origin Test

PREZEON

Pathwork Diagnostics Inc.

Myriad

CCP Assay

Pathwork Tissue of Origin Test Kit – FFPE

Gen-Probe, Inc.

PROGENSA PCA3 Assay


Cancer

Tissue of origin

Iris Molecular Diagnostics

Vysis

CEP X SpectrumOrange/Y SpectrumGreen DNA Probe Kit

NADiA ProsVue

Vysis

CEP 8 Spectrumorange DNA Probe Kit

Prostate cancer

Vysis

AneuVysion

Hologic, Inc.

Invader MTHFR 1298

Affymetrix, Inc.

Hologic, Inc.

Invader MTHFR 677

Affymetrix CytoScan Dx Assay

Roche Diagnostics Corporation

Factor V Leiden Kit

Chromosome abnormalities

Roche Diagnostics Corporation

Factor II (Prothrombin) G20210A Kit

xDx

Autogenomics, Inc.

INFINITI System

AlloMap Molecular Expression Testing

Manufacturer

Trade name

Heart transplant

Disease

Table 2. FDA-approved nucleic acid-based tests (cont.).

An in vitro diagnostic assay to determine the loss of PTEN function for use in clinical trials for the targeted cancer drugs such as PI3K, EGFR, MEK, AKT and mTOR inhibitors

An in vitro diagnostic assay to measure the degree of similarity between RNA expression patterns in a patient’s tumor and that from a reference database consisting of 15 tumor types

An in vitro diagnostic assay to measure the degree of similarity between RNA expression patterns in a patient’s formalin-fixed paraffin-embedded tumor and that from a reference database consisting of 15 tumor types

An in vitro diagnostic assay to assess the aggressiveness of prostate cancer by measuring the expression level of genes involved with cell cycle progression in tumor specimens to predict disease outcome

An in vitro diagnostic assay for the prostate-specific antigen RNA and PCA3 RNA molecules to determine the need for repeat prostate biopsies

An in vitro diagnostic assay for determining rate of change of serum total prostate specific antigen over a period of time (slope, pg/ml per month)

Enumeration of Chr X and Y by FISH for patients receiving opposite-sex bone marrow transplantation for hematologic disorders

Enumeration of Chr 8 by FISH for chronic myelogenous leukemia, acute myeloid leukemia, myeloproliferative disorder, myelodysplastic syndrome and hematological disorders

Enumeration of Chr 13, 18, 21, X and Y by FISH for prenatal diagnosis

Detect chromosomal variations for developmental delay or intellectual disability in children

Gene expression profiling of RNA isolated from peripheral blood mononuclear cells for the identification of heart transplant recipients with stable allograft function

Genotyping of MTHFR

Genotyping of MTHFR

Genotyping of Factor V

Genotyping of Factor II

Genotyping of Factor II, Factor V

Intended use


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PATHVYSION HER2 DNA Probe Kit

SPOT-LIGHT HER2 CISH Kit

HER2 CISH PharmDx Kit

Herceptin (trastuzumab)



HER2 CISH PharmDx kit is intended for dual-color chromogenic visualization of signals achieved with directly labeled in situ hybridization probes targeting the HER2 gene and centromeric region of chromosome 17. The kit is designed to quantitatively determine HER2 gene status in FFPE breast cancer tissue specimens. Red and blue chromogenic signals are generated on the same tissue section for evaluation under bright field microscopy. The CISH procedure is automated using Dako Autostainer instruments HER2 CISH pharmDx Kit is indicated as an aid in the assessment of patients for whom Herceptin (trastuzumab) treatment is being considered. Results from the HER2 CISH pharmDx Kit are intended for use as an adjunct to the clinicopathologic information currently used for estimating prognosis in stage II, node-positive breast cancer patients This kit is for IVD use only The INFORM HER2 Dual ISH DNA Probe Cocktail is intended for use in determining HER2 gene status by enumeration of the ratio of the HER2 gene to chromosome 17. The HER2 and chromosome 17 probes are detected using two color chromogenic ISH in FFPE human breast cancer tissue specimens following staining on Ventana BenchMark XT automated slide stainers (using NexES software), by light microscopy. The INFORM HER2 Dual ISH DNA Probe Cocktail is indicated as an aid in the assessment of patients for whom Herceptin (trastuzumab) treatment is being considered This product should be interpreted by a qualified reader in conjunction with histological examination, relevant clinical information and proper controls This reagent is intended for IVD use

Dako Denmark A/S

Ventana Medical Systems, Inc.

For in vitro diagnostic use The SPOT-Light HER2 CISH Kit is intended to quantitatively determine HER2 gene amplification in FFPE breast carcinoma tissue sections using CISH and brightfield microscopy This test should be performed in a histopathology laboratory The SPOT-Light HER2 CISH Kit is indicated as an aid in the assessment of patients for whom Herceptin (trastuzumab) treatment is being considered. The assay results are intended for use as an adjunct to the clinicopathological information currently being used as part of the management of breast cancer patients. Interpretation of test results must be made within the context of the patient’s clinical history by a qualified pathologist

The PathVysion HER2 DNA Probe Kit (PathVysion Kit) is designed to detect amplification of the HER2/neu gene via FISH in FFPE human breast cancer tissue specimens. Results from the PathVysion Kit are intended for use as an adjunct to existing clinical and pathologic information currently used as prognostic factors in stage II, node-positive breast cancer patients. The PathVysion Kit is further indicated as an aid to predict disease-free and overall survival in patients with stage II, node positive breast cancer treated with adjuvant cyclophosphamide, doxorubicin, and 5-fluorouracil chemotherapy. The Pathvysion Kit is indicated as an aid in the assessment of patients for whom herceptin (trastuzumab) treatment is being considered (see herceptin package insert)


Life Technologies, Inc.

The Inform Her2/Neu gene detection system is a FISH DNA probe assay that determines the qualitative presence of Her2/Neu gene amplification on FFPE human breast tissue as an aid to stratify breast cancer patients according to risk for recurrence or disease-related death. It is indicated for use as an adjunct to existing clinical and pathologic information currently used as prognostic indicators in the risk stratification of breast cancer in patients who have had a priori invasive, localized breast carcinoma and who are lymph node-negative

Intended use/indications for use

Ventana Medical Systems, Inc.

Device manufacturer

ALK: Anaplastic lymphoma kinase; CISH: Chromogenic in situ hybridization; FFPE: Formalin-fixed paraffin-embedded; ISH: In situ hybridization; IVD: In vitro diagnostic.

INFORM HER2 DUAL ISH DNA Probe Cocktail

INFORM HER2/ NEU



Device trade name

Drug trade name (generic name)

Table 3. FDA-approved companion diagnostic tests for cancer (FISH/CISH-based assays).



Review

doi: 10.1586/14737159.2015.961916

The Vysis ALK Break Apart FISH Probe Kit is a qualitative test to detect rearrangements involving the ALK gene via FISH in FFPE non-small-cell lung cancer tissue specimens to aid in identifying patients eligible for treatment with Xalkori (crizotinib). This is for prescription use only Abbott Molecular Inc. VYSIS ALK Break Apart FISH Probe Kit Xalkori (crizotinib)

doi: 10.1586/14737159.2015.961916

ALK: Anaplastic lymphoma kinase; CISH: Chromogenic in situ hybridization; FFPE: Formalin-fixed paraffin-embedded; ISH: In situ hybridization; IVD: In vitro diagnostic.

HER2 IQFISH pharmDx is a direct FISH assay designed to quantitatively determine HER2 gene amplification in FFPE breast cancer tissue specimens and FFPE specimens from patients with metastatic gastric or gastroesophageal junction adenocarcinoma HER2 IQFISH pharmDx is indicated as an aid in the assessment of breast and gastric cancer patients for whom Herceptin (trastuzumab) treatment is being considered and for breast cancer patients for whom Perjeta (pertuzumab) or Kadcyla (ado-trastuzumab emtansine) treatment is being considered (see Herceptin, Perjeta and Kadcyla package inserts) For breast cancer patients, results from the HER2 IQFISH pharmDx are intended for use as an adjunct to the clinicopathologic information currently used for estimating prognosis in stage II, node-positive breast cancer patients Dako Denmark A/S HER2 FISH PharmDx Kit Herceptin (trastuzumab); Perjeta (pertuzumab); Kadcyla (adotrastuzumab emtansine)

Intended use/indications for use Device manufacturer Device trade name



Table 3. FDA-approved companion diagnostic tests for cancer (FISH/CISH-based assays) (cont.).


Review

There are already instances of NGS technology being utilized to probe for acquired resistance mutations due to mutations. Utilizing NGS to deep sequence, the entire ALK kinase domain of tumor DNA from NSCLC patients who have relapsed after crizotinib treatment revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A [36]. For patients who demonstrate early or acquired resistance to targeted therapy, NGS has been used to elucidate the genetic landscape of clinical resistance. The use of RAF inhibitors for BRAFV600-mutant metastatic melanoma improves patient outcomes, but most patients demonstrate early or acquired resistance to this targeted therapy [37]. In the study by Van Allen et al., WES was performed on patients to experimentally assess new observed mechanisms to define potential subsequent treatment strategies. In addition to probing for acquired resistance mutations and identify subsequent targets for therapy post-resistance acquisition, NGS has also been utilized to understand and learn about ‘exceptional responders’, rare patients with unexpected exquisite sensitivity or durable responses to therapy. The study of exceptional responders represents a promising approach to better understand the mechanisms that underlie sensitivity to targeted anticancer therapies [38]. In the study by Wagle et al., two activating mTOR mutations were identified in a patient with exquisite sensitivity to everolimus and pazopanib, suggesting an approach to identifying patients who might benefit most from mTOR inhibitors. Challenges for NGS in companion diagnostics Tumor heterogeneity

The promises of early detection and molecularly precise diagnosis for many cancers have given patients and clinicians new hope for targeted, personalized treatment and improved outcomes. NGS has been used to identify promising biomarker candidates for early diagnosis, to detect the causative mutations in clinical cases and to guide targeted treatment decisions [39–43]. Though cancer is a genetic disease, the heterogeneity among the cells in a single tumor is astounding, making detection of low-level mutations difficult [44,45]. However, the ability of NGS to provide deep sequencing of tumors, that is, at depths of 1000 or greater is sufficiently robust to allow for sensitive and specific single nucleotide variant mutation analysis that can be incorporated easily into the clinical laboratory for routine testing [46]. Tumors are known to vary in terms of their mutational load, that is, secondary tumors are not genetically identical to the primary tumor from which they originate [45]. This fact is one possible explanation why the rate of relapse is so high in patients whose cancer has metastasized to a secondary location. Traditional treatments involve surgery to remove the primary tumor followed by one or more rounds of chemotherapy and/or radiation in order to destroy any traces of the cancer missed by the surgical extraction. Ideally, the preferred chemotherapy should be selected based on the specific genetic characteristics of that patient’s cancer. For example, Expert Rev. Mol. Diagn.

The Cobas 4800 BRAF V600 Mutation Test is an in vitro diagnostic device intended for the qualitative detection of the BRAF V600E mutation in DNA extracted from FFPE human melanoma tissue. The Cobas 4800 BRAF V600 Mutation Test is a real-time PCR test on the Cobas 4800 system, and is intended to be used as an aid in selecting melanoma patients whose tumors carry the BRAF V600E mutation for treatment with vemurafenib The THxID BRAF kit is an In Vitro Diagnostic device intended for the qualitative detection of the BRAF V600E and V600K mutations in DNA samples extracted from FFPE human melanoma tissue. The THxIDTM BRAF kit is a real-time PCR test on the ABI 7500 Fast Dx system and is intended to be used as an aid in selecting melanoma patients whose tumors carry the BRAF V600E mutation for treatment with dabrafenib [Tafinlar] and as an aid in selecting melanoma patients whose tumors carry the BRAF V600E or V600K mutation for treatment with trametinib [Mekinist]


bioMe´rieux Inc.

therascreen EGFR RGQ PCR Kit


cobas EGFR Mutation Test

COBAS 4800 BRAF V600 Mutation Test

THxIDTM BRAF Kit

Tarceva (erlotinib)

Zelboraf (vemurafenib)

Mekinist (tramatenib); Tafinlar (dabrafenib)

The cobasÒ EGFR Mutation Test is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the EGFR gene in DNA derived from FFPET human NSCLC tumor tissue. The test is intended to be used as an aid in selecting patients with metastatic NSCLC for whom TarcevaÒ (erlotinib), an EGFR TKI, is indicated

The therascreen EGFR RGQ PCR Kit is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the EGFR gene in DNA derived from FFPE NSCLC tumor tissue. The test is intended to be used to select patients with NSCLC for whom GILOTRIF (afatinib), an EGFR TKI, is indicated. Safety and efficacy of GILOTRIF (afatinib) have not been established in patients whose tumors have L861Q, G719X, S768I, exon 20 insertions and T790M mutations, which are also detected by the therascreen EGFR RGQ PCR Kit. Specimens are processed using the QIAamp DSP DNA FFPE Tissue Kit for manual sample preparation and the Rotor-Gene Q MDx instrument for automated amplification and detection

CRC: Colorectal cancer; EGFR: Epidermal growth factor receptor; FFPE: Formalin-fixed paraffin-embedded; NSCLC: Non-small-cell lung cancer; TKI: Tyrosine kinase inhibitor.


Qiagen Manchester, Ltd.

The therascreen KRAS RGQ PCR Kit is a real-time qualitative PCR assay used on the Rotor-Gene Q MDx instrument for the detection of seven somatic mutations in the human KRAS oncogene, using DNA extracted from formalin FFPE CRC tissue. The therascreen KRAS RGQ PCR Kit is intended to aid in the identification of CRC patients for treatment with Erbitux (cetuximab) based on a KRAS no mutation detected test result

Gilotrif (afatinib)

Qiagen Manchester, Ltd.

therascreenKRAS RGQ PCR Kit

Intended use/indications for use

Erbitux (cetuximab)

Device manufacturer

Device trade name


Table 4. FDA-approved companion diagnostic tests for cancer (quantitative PCR -based assays).



Review

doi: 10.1586/14737159.2015.961916


Review


if one patient’s cancer is driven by a mutation in PTEN or BRCA1/2, the selected treatment should not be the same as if his/her cancer were driven by a mutation in RAS. In the first case, PTEN acts as a tumor suppressor, and for cancers where mutations in PTEN lead to pathway disinhibition, the resulting activated downstream targets such as AKT or mTOR may be treated using inhibitors. For the second case, treatment with poly (ADP-ribose) polymerase inhibitors for tumors harboring germline BRCA1/2 mutations has been demonstrated to effectively kill cancer cells through the concept of ‘synthetic lethality’ [47]. Although the exact mechanism for BRCA1/2 and poly (ADP-ribose) polymerase inhibitor ‘synthetic lethality’ remains up for debate [48], the observed effect provides an additional target for treatment of cancers harboring BRCA1/2 mutations. In the last case, RAS acts as an oncogene, and the goal of treatment would thus be to de-activate the pathway that was upregulated in the cancer cells. While this makes intuitive sense, tumor heterogeneity makes such one-size-fit-all ideas impractical. A primary tumor, driven by a mutation in a tumor suppressor gene, may give rise to secondary tumors, undetectable at the time of surgery, which additionally have mutations in one or more oncogenes [45]. Thus, adjuvant chemotherapy will confer temporary remission, but only until the secondary population is detectable by clinical tests. Such circumstances are unfortunately all too common in cancer treatment. Other technical difficulties persist as well. Because of tumor heterogeneity, some authors have recommended sequencing both primary and secondary tumors in order to understand the range of somatic mutations present [49–51]. Even in the case of a single primary tumor, some authors have proposed sequencing from multiple areas [52,53]. In many cancers, but especially in the case of lung cancer, this requirement may be technically challenging to perform for interventional radiologists based on tumor location(s) [44]. Additionally, requiring continuous biopsies in order to monitor the effect of treatment is not a feasible long-term approach. Another issue relating to the clinical presentation of cancer is the fact that there are many disease subtypes that are not currently differentiable to the pathologist or oncologist; in fact, each presentation of cancer in an individual is effectively unique. For example, in colorectal cancer, two subtypes exist: hypermutated and non-hypermutated. In hypermutated cases, the genetic signature is marked by microsatellite instability; in non-hypermutated cases, the genetic signature was differentiated by mutation of the TP53 and APC genes. Interestingly, it was observed that for non-hypermutated colorectal cancers, 60% of patients have a mutation in TP53, while only 20% of patients with the hypermutated form have such mutations [54]. One can envision future work, further classifying nonhypermutated cancers into finer subtypes to guide treatment decisions. Ultimately, however, this goal of increased granularity must give way to the view of cancer as an individual disease rather than as a disease to be treated based on population-level patterns and outcomes statistics. doi: 10.1586/14737159.2015.961916

In order to eventually reach this goal, and also because of genetic heterogeneity, researchers are required to sequence a large number of samples in order to identify mutations that are of lower frequency in the general population [55]. The requisite number is magnified when one considers that the manifestation of subtypes may vary significantly based on population. For example, activating mutations of EGFR in NSCLC tumors have been found to be approximately 10-fold higher in a Japanese NSCLC population compared with a US population, which was consistent with previously observed population differences in response to EGFR inhibitors [56]. Thus, when performing genomic analysis for the purpose of biomarker discovery, it can be important to sample different populations to ensure that findings are broadly applicable. Accessing a large number of samples for research purposes is also hampered by basic sample collection difficulties, especially in the case of lung cancer, where the tumor is not easily accessible for biopsy, and the biopsy process itself is difficult and painful for the patient. It is clearly preferable to avoid multiple biopsies, but for the purposes of research, it is of extreme importance to understand the manner in which a tumor population changes over time and is based on treatment. Although in their early stages, NGS has been applied to study both circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Analyzing CTCs and/or ctDNA are approaches to studying metasynchronous or synchronous metastases in which limited tumor samples are available for genomic profiling. The development of single-cell sequencing technologies has made CTCs an attractive target for assessing prognosis, monitoring response to therapy, pharmacodynamic studies and rational selection of therapies in cancer patients. In a recent study, WES of circulating tumors in patients with metastatic prostate cancer revealed that 51 of 73 (70%) of CTC mutations were found in matched tissue [57] providing support for the use of CTC sequencing for genomic profiling. Analysis of ctDNA for detecting and monitoring cancers has been an active area of clinical research. In the area of metastatic breast cancer, ctDNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified and provided the earliest measure of treatment response in 10 of 19 women (53%) [58]. For lung cancer, ctDNA was used to detect the cancer and assess response to treatment by utilizing a method called cancer personalized profiling by deep sequencing, which identifies recurrent mutations in tumors that serve as specific targets for quantitating ctDNA in patients [59]. All of the outstanding challenges listed above represent facets of the presentation of cancer that make its study difficult from a practical point of view; however, they also represent hurdles for the analysis of genetic data in general, specifically from the standpoint of downstream bioinformatics. When reviewing NGS data, tumor heterogeneity manifests itself as low-level somatic variants that are seen in only a small number of sequencing reads. For rare variants, identification may be difficult or impossible because it may not be differentiable Expert Rev. Mol. Diagn.



from errors due to instrument noise [60]. For example, if the genome is sequenced to a mean depth of 100, then a variant present at a level of 1% would be expected to appear in only one read. Currently, sequencing accuracy is very high, generally reported at 99.9% or above for the majority of bases [61]. However, this implies that one base per thousand sequenced will be in error. At a depth of 100, this means that 300 billion bases will be sequenced across the genome, and therefore, that 1 in 10 positions (or 300 million bases across the genome) will have a single read reporting an alternate base. Clearly, a cancer signature does not encompass 300 million sites, so a 1% somatic frequency is effectively impossible to differentiate from noise. By contrast, a 5% somatic frequency is much more unique, and setting such a threshold (or increasing sequencing depth) would reduce the number of false-positive calls substantially. NGS in molecular diagnostic laboratory workflows & reimbursements

Similar to the current FDA-cleared real-time PCR companion diagnostic tests, NGS-based companion diagnostic tests will also have to rely on formalin-fixed paraffin-embedded (FFPE) tissue as the starting material to ensure that sufficient amounts of neoplastic cells are present. DNA isolated from FFPE tissue poses technical challenges since FFPE processing both fragments the DNA and also causes base modifications such as cytosine deamination. One current approach for mitigating base modification artifacts is using amplicon-based sequencing strategy that assesses both strands of DNA. In conjunction with pretreatment using uracil-DNA glycosylase, the amplicon-based sequencing strategy instead of the enrichment or captured-based strategies has been shown to reduce these C:G>T:A single nucleotide changes and facilitate accurate discrimination of mutations in FFPE samples [62]. The poor DNA quality makes it challenging to extract sufficient amounts of amplifiable DNA, as assessed by a quantitative PCR, for subsequent NGS-based testing, especially in the context of certain cancers such as lung cancer where tumor tissue is typically more difficult to obtain in larger quantities. The current FDA-cleared real-time PCR companion diagnostic tests for KRAS, EGFR and BRAF have rapid turnaround times (TATs), on the order of several days after specimen acquisition. In comparison, NGS-based assays currently require 7–14 days TAT due to laboratory steps including library preparation, sequencing machine run-time and subsequent bioinformatics analyses. The advantage for NGS-based companion diagnostics for a targeted re-sequencing will have the ability to identify all mutations present in the targeted regions compared with current companion diagnostic tests that only probe for specific mutations. As NGS become more routine in the molecular diagnostic laboratory setting, and more automation becomes available, the TAT for NGS diagnostic tests would likely shorten and fall within a range that is acceptable for most clinicians. One clinical research study that illustrates the informahealthcare.com

Review

potential of NGS with rapid TAT is the application of rapid WGS for genetic disease diagnosis in neonatal intensive care units. The study demonstrates a 50-h differential diagnosis of genetic disorders by WGS that features automated bioinformatics analysis and is intended to be a prototype for use in neonatal intensive care units [63]. Perhaps some of the most serious challenges to the realization of NGS in companion diagnostics and personalized medicine are not technical, but rather those related to institutional adoption and barriers related to the workflow in the practice of medicine. Until NGS-based assays such as WGS, WES and targeted gene panels are reimbursed by insurance carriers, the hurdle to widespread adoption is very high. Even after a majority of carriers and providers adopt this technology, incorporation of NGS results into a patient’s EHR is a challenge [64–66]. In a review of six Clinical Sequencing Exploratory Research sites that are incorporating WGS and WES into the EHR, TarczyHornoch et al. reported that development across sites was independent and non-standardized [67]. Workflows varied widely, and in all cases, the final output was a human-readable, PDF report that was attached to the EHR, with only three sites additionally providing a machine-readable version of identified variants. Progress in institutional implementation is generally much slower than the pace of development, so it is important that this issue is addressed quickly and collaboratively by healthcare networks and EHR vendors [64], ideally with input from the NGS community. Five-year view

Conceptually, future efforts in the development of companion diagnostics in cancer should focus on three areas: oncogenes, tumor suppressor genes and cancer-enabling genes. In the oncogene-related companion diagnostics, these efforts should focus not only on developing diagnostics that provide clinically actionable information on mutations and available drugs that target these mutations, but also on developing knowledge base of additional mutations that contribute to resistance to targeted therapies. As discussed previously, although therapies that target activated oncogenes are effective, durable response is not often achieved with the exception of imatinib therapy [68], due to compensatory activation of parallel signaling pathways, secondary mutations in the target genes or activation of downstream component of the targeted pathway [69]. As such, it is important to identify downstream regulators and modulators that crosstalk with the targeted pathway so that multiple pathways may be simultaneously targeted to achieve durable response. Moreover, with increasing appreciation of clonal heterogeneity in several types of cancer, there is an increasing need to combine multiple targeted therapies to tailor treatment for each individual cancer patient. This approach presents new challenges in understanding drug and pharmacogenomics interactions and a comprehensive companion diagnostics that cover multiple drug targets as well as host factors to minimize adverse drug interactions. Although co-development of doi: 10.1586/14737159.2015.961916


Review


drugs and companion diagnostics are underway by pharmaceutical industry, it is important that these efforts are integrated with the development of other targeted agents so that a comprehensive knowledge base is created to understand which targeted agents can be combined or sequenced to achieve synergistic, durable response. Companion diagnostics will also be important in the development of adaptive clinical trials [70]. One clinical trial study design that utilizes the adaptive model is the so-called ‘basket’ clinical trial in which various cancer types with the same genetic drivers are treated as a disease. This emerging paradigm in the treatment of cancers not based on the tissue of origin but based on shared genetic factors that drive cancer progression in various tissues requires robust companion diagnostics that can group patients into separate trial arms for therapeutic purposes. Another clinical trial study design with an adaptive model is the so-called ‘umbrella’ clinical trial, which tests the impact of different drugs on different mutations in a single type of cancer. For this emerging paradigm, the treatment of cancers is based on the unique genetic factors that drive cancer progression for an individual patient and requires comprehensive mutational profiling from a robust companion diagnostic test designed to test a large number of genomic targets in order to match the possible treatments with the patient’s unique tumor profile. These clinical trials represent opportunities to co-develop companion diagnostics and targeted therapies for precision cancer medicine. On the research and development front, companion diagnostics should also focus on co-developing assays and drugs that target tumor suppressor genes. The most frequently mutated gene in human cancer is the tumor suppressor gene TP53 [71]. At least 50% of human tumors harbor mutations in TP53 [72], and these mutations are recorded throughout the coding region with some recurrent mutations in a few hotspots. TP53 mutations are expected to produce three distinct yet not mutually exclusive phenotypes: loss-of-function (amorphic), dominantnegative and gain-of-function (neomorphic) phenotypes. Lossof-function phenotype is expected from all somatic missense and nonsense mutations that result in protein structural alterations and truncation. Dominant-negative mutations are expected in cases where heterozygous mutations are detected in tumor samples [73], and these mutations result in mixed tetramers with loss of DNA binding or transactivation activity [74]. Gain-of-function mutations are expected to be neomorphic and more difficult to characterize by specific features. However, these gain-of-function mutations may be more amenable to therapeutic targeting because they may produce oncogene addiction or non-oncogenic addiction, such as metabolic dependency.

doi: 10.1586/14737159.2015.961916

Despite the high prevalence of somatic mutations and relevance of these mutations in tumor development and progression, not much is known about which TP53 mutations are targetable and which drugs can be used to target them. Because of the high prevalence of somatic mutations in TP53, development of actionable knowledge on TP53 mutations and companion diagnostics could have broader impact on the treatment of cancers. Toward this goal, effort should focus on generating mutant library of TP53 and development of functional assays to assist in classification of mutants with loss-of-function, gain-of-function and dominant-negative phenotype. In addition, functional assays should be developed to screen a large library of drug-like compounds to determine which drugs can be used to restore the function of mutant TP53 or inhibit the gain-of-function or dominantnegative phenotype of mutant TP53. These efforts will ultimately produce a companion diagnostics that provide oncologists with actionable knowledge on specific drugs that can be used to target specific TP53 mutations. Finally, the third category of genes for which companion diagnostics should be developed is those involved in neomorphic effects and non-oncogene addiction [75,76]. Mutations in IDH1 and IDH2 produce gain-of-function phenotype that produces ‘oncometabolite’ 2-hydroxyglutarate from isocitrate metabolic pathway [77]. Genes that are involved in counteracting various types of stress (DNA damage and replication stress, proteotoxic stress, mitotic stress, metabolic stress and oxidative stress) by themselves are not oncogenic but enable oncogenes to promote cancer progression by mitigating oncogenic stress [76]. Therefore, these co-dependencies between oncogenes and those mitigate oncogenic stresses are being actively investigated and targeted therapies are being developed. In conclusion, companion diagnostics that facilitate the identification of the type of oncogenes and oncogenic stresses that can be jointly targeted would become essential armamentarium in clinical oncology. Financial & competing interests disclosure

E Lin, J-B Fan and FS Ong are employees of Illumina, Inc. J Chien is funded by the University of Kansas Endowment Association and the Department of Defense Ovarian Cancer Research Program under award number W81XWH-10-1-0386. Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



Review

Key issues • The rapid decline in sequencing costs has allowed next-generation sequencing (NGS) platforms to be utilized in molecular diagnostics laboratories. As NGS technology becomes more widely adopted in the molecular diagnostic laboratories, one can expect NGS-based diagnostic assays and NGS companion diagnostics to become a central piece of routine healthcare management and genomic medicine. • Development of a knowledge base for clinical utilization of NGS genetic data for variant calling and annotation for clinical interpretation and results reporting is occurring on multiple fronts and includes refinement of current bioinformatics algorithms and pipelines as well as public and private initiatives to assist in the identification of and the development of a consensus on medically relevant genomic variants. Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.

• NGS is being utilized to understand and learn about ‘exceptional responders’, rare patients with unexpected exquisite sensitivity or durable responses to therapy. The study of exceptional responders represents a promising approach to better understand the mechanisms that underlie sensitivity to targeted anticancer therapies and adds to the knowledge base for clinical utilization of NGS data. • The ability for NGS to target a wide spectrum of mutations across a large number of genes/pathways could provide diagnostic information to identify focal amplifications responsible for drug resistance and corresponding targeted therapeutic agent(s) likely to be effective in treating patients as well as provide monitoring for acquired resistance to treatment. • NGS has been applied to studying circulating tumor cells and circulating tumor DNA and may be useful for future efforts in determining prognosis, monitoring response to therapy and rational selection of therapies in cancer patients. • The advent of adaptive model clinical trial models that include the ‘basket’ and ‘umbrella’ clinical trials provides an opportunity for co-development companion diagnostics and targeted therapies for precision cancer medicine. • Future effort in the development of companion diagnostics in cancer should focus on three areas: oncogenes, tumor suppressor genes and cancer-enabling genes. In the oncogene-related companion diagnostics, these efforts should focus not only on developing diagnostics that provide clinically actionable information on mutations and available drugs that target these mutations, but also on developing knowledge base of additional mutations that contribute to resistance to targeted therapies.

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