Implementation Of Next-generation Sequencing Research Articles

Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests.

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.

Current use of chromosomal microarray by Australian paediatricians and implications for the implementation of next generation sequencing.

Chromosomal microarray (CMA) is an important diagnostic test for children with multiple congenital anomalies or certain developmental behavioural problems suggestive of an underlying genetic diagnosis. However, there are medical and ethical complexities to its use and few Australian policies to guide practice. We aimed to describe the current practice of Australian paediatricians in relation to CMA testing. We hypothesised that there are knowledge gaps in their use of CMA. Online survey completed between September 2015 and January 2016 by paediatricians in secondary care settings. Participants were members of the Australian Paediatric Research Network. One hundred and sixty five (43%) of 383 active members responded. Our main outcome measures comprised: (i) the indications for which paediatricians request CMA; (ii) their approach to consent; (iii) their interpretation of results; and (iv) their understanding of the impact on patient management. A significant proportion of paediatricians (21-52%) did not regularly use CMA for conditions with established evidence of diagnostic yield. Paediatricians under-estimated the potential for CMA findings to alter patient management. There was wide variability in paediatricians' approach to consent, and low use of consent forms and fact sheets. Paediatricians reported difficulties interpreting CMA results, with high rates of referral to clinical genetics services. The reported practice of Australian paediatricians is not consistent with international standards on CMA. Australian practice could be improved by a standardised approach to ordering CMA, consenting patients and interpreting results. We provide resources for CMA ordering and make recommendations about preparation for next generation sequencing.

Are we ready to detect nematode diversity by next generation sequencing?

In a Technical Advance article, Porazinska et al. (2009, Molecular Ecology Resources, 9, 1439–1450) assessed next generation sequencing (NGS) as a method for metagenomic analysis of nematode diversity. We agree that NGS has great potential here. However, it is not an easy path to the successful implementation of NGS for environmental DNA analysis of nematodes. Here, we describe the method's limitations and discuss prospective research questions. For instance, only a few direct extraction kits are suitable for nematode DNA extraction from bulk samples without adaptation. They enable the analysis of extracellular nematode DNA. The most crucial and unresolved issue remains the limited availability of suitable primers.

Changing the Therapeutic Landscape in Non-small Cell Lung Cancers: the Evolution of Comprehensive Molecular Profiling Improves Access to Therapy.

Targeting genomic alterations has led to a paradigm shift in the treatment of patients with lung cancer. In an effort to better identify potentially actionable alterations that may predict response to FDA-approved and or investigational therapies, many centers have migrated towards performing targeted exome sequencing in patients with stage IV disease. The implementation of next-generation sequencing (NGS) in the evaluation of tumor tissue from patients with NSCLC has led to the discovery of targetable alterations in tumors that previously had no known actionable targets by less comprehensive profiling. An improved understanding of the molecular pathways that drive oncogenesis in NSCLC and a revolution in the technological advances in NGS have led to the development of new therapies through biomarker-driven clinical trials. This review will focus on the advances in molecular profiling that continue to fuel the revolution of precision medicine, identifying targets such as MET exon 14 skipping alterations and select recurrent gene alterations with increasing frequency.

MA12.01 Next Generation Sequencing Based Clinical Framework for Analyses of Treatment Predictive Mutations and Gene Fusions in Lung Cancer

The use of new, emerging techniques in the search of tailored patient therapies is rapidly becoming a reality. Here we describe the optimization and implementation of next generation sequencing for treatment predictive mutation screening in parallel with gene fusion status of ALK, RET and ROS1 in non-small cell lung cancer (NSCLC) patients. The Illumina TruSight tumor 26-gene NGS panel was validated in 81 clinical routine FFPE or cytology specimens and implemented in 533 diagnostic NSCLCs during one year of clinical analysis. In parallel, a RNA-based NanoString method was evaluated in 169 cases for gene fusion status of ALK, RET and ROS1. We have successfully established a streamlined workflow with a 5-day turnaround time from specimen arrival to mutation report. The concordance in the validation cohort was 99% for comparable variants. In the 533 diagnostic samples, 1-2 variants were detected in 79% of the cases. Most frequently mutated genes included TP53, KRAS, EGFR, STK11, and BRAF, all with differences in mutational patterns between histological subgroups. The RNA-based NanoString assay was successfully established and validated. The success rate in the 169 cases was 80% and 10 gene fusions were found (five ALK fusions, three RET fusions and two ROS1 fusions) all in adenocarcinomas. Integration of mutation and gene fusion status revealed that 68% of adenocarcinomas, 13% of SqCCs and 56% of NSCLC-NOS harbored ≥1 actionable alteration ALK, RET, ROS1, EGFR, KRAS, PIK3CA, BRAF, NRAS, MAP2K1, ERBB2 or AKT1. Specifically, in 13.2% of the adenocarcinomas where no EGFR or ALK alteration was detected emerging targeted therapy may be considered in addition to the 15.3% of patients that was eligible for EGFR or ALK inhibitors. The corresponding proportions for SqCCs were 5.5% in addition to the 2.2%, and for NSCLC-NOS 2.5% in addition to the 11.2% eligible for EGFR or ALK inhibitors. Next generation sequencing in combination with the NanoString technology is time- and cost efficient in the diagnostic routine for treatment predictive mutation screening and gene fusion status detection. The techniques represent valuable tools for pinpointing patients eligible to standard targeted therapies in addition to new emerging therapies.

Developing a framework to assess the costeffectiveness of COMPARE - a global platform for the exchange of sequence-based pathogen data.

Analysing the genomic data of pathogens with the help of next-generation sequencing (NGS) is an increasingly important part of disease outbreak investigations and helps guide responses. While this technology has already been successfully employed to elucidate and control disease outbreaks, wider implementation of NGS also depends on its cost-effectiveness. COMPARE - short for 'Collaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks' - is a major project, funded by the European Union, to develop a global platform for sharing and analysing NGS data and thereby improve the rapid identification, containment and mitigation of emerging infectious diseases and foodborne outbreaks. This article introduces the project and presents the results of a review of the literature, composed of previous relevant cost-benefit and cost-effectiveness analyses. The authors also outline the implications for a methodological framework to assess the costeffectiveness of COMPARE and similar systems.

P2.03b-028 Improved Overall Survival Following Implementation of NGS in Routine Diagnostics of Advanced Lung Cancer in Germany: Results of the NGM: Topic: Biomarkers

Broad implementation of molecular diagnostics and personalized cancer care is hampered by insufficient molecular screening, missing reimbursement for comprehensive molecular testing and lack of access to appropriate drugs. The Network Genomic Medicine (NGM) Lung Cancer is a health care provider network offering comprehensive next generation sequencing (NGS)-based multiplex genotyping on a central diagnostics platform in Cologne for all inoperable lung cancer patients (pts) in Germany. The NGS panel used in NGM consists of 14 genes and 102 amplicons to cover potentially targetable aberrations. Mutation analyses were run on an Illumina (MySeq) platform, while FISH analyses were performed separately. In 2015, we have started the second outcome evaluation for all NGM pts who had received NGS-based molecular diagnostics. In particular, we have focused on molecular subgroups of EGFR, ALK, BRAF-V600E, HER2 and ROS1 positive pts and especially on NGM pts treated in clinical trials. From 2013-2015 6210 lung cancer pts (n=4244 non-squamous NSCLC) were genotyped. Preliminary data show the overall survival (OS) of 934 NSCLC pts including of 110 NSCLC pts treated in clinical trials. For 108 EGFR+ pts, the OS of clinical trials pts treated with so called 3rd generation EGFR-TKIs was 55 months (n=25) vs 22 months in control group (n=83) (p=0,002; mean OS: 29 months; 95% CI: 36-83 months). For 85 ALK+ pts, the OS of pts treated in clinical trials was 35 months (n=19) compared to OS of 23 months for 45 pts treated with one ALK inhibitor and 8 months for 19 pts treated with no ALK inhibitors (P<0,0001; mean OS: 22 months; 95% CI: 22-33 months). While the first NGM evaluation in 2013 already showed a survival benefit of 2 years in EGFR-TKI treated EGFR+ pts compared to chemotherapy, our current evaluation in pts treated with 3rd generation EGFR-TKIs after acquired resistance to 1st gen. EGFR-TKIs shows the significant increasing of the OS. Similarly, we show a significant longer OS for ALK+ pts treated with 2 ALK inhibitors compared to treatment with one or no ALK inhibitor. Further results of this ongoing NGM evaluation will be provided.

Clinical Next Generation Sequencing to Identify Novel Platelet Disorders

Inherited platelet disorders (IPDs) comprise a heterogeneous group of disorders with a complex genetic etiology, characterized by impairments in platelet formation, morphology and function. Since the implementation of next generation sequencing (NGS) in 2009, the gene list for diagnosis of IPDs rapidly expanded from 39 to 53 genes. A diagnostic high-throughput targeted NGS platform (referred to as ThromboGenomics; www.thrombogenomics.org.uk) was very recently described as an affordable DNA-based test of 76 genes to diagnose patients ’suspected of having a known inherited platelet, thrombotic or bleeding disorder’ (BPD). When the phenotype is strongly indicative of the presence of a particular disease etiology but the variants are unknown, sensitivity remains high (>90% based on 61 samples) while patients included with an uncertain disease such as delta storage pool disease, mostly receive no genetic diagnosis (only 10% a genetic diagnosis was obtained). Such IPDs should be included in gene discovery NGS programs such as the BRIDGE-BPD2 study. For this study, whole genome sequencing results of the DNA samples of nearly 1000 probands with uncharacterized IPDs, analyzed using assigned Human Phenotype Ontology (HPO) terms have helped to identify pathogenic variants in almost 20% of cases. New clustering algorithms to group cases with similar phenotypes have been used to identify two novel IPD genes (DIAPH1 and SRC2) and several putative ones. Still many IPD patients don’t receive a genetic diagnosis. A majority of cases either harbor pathogenic variants in unknown genes or in regulatory regions or are the result of a digenic mode of inheritance. NGS combined with data from RNA-seq, ChIP-seq, gene regulatory network analysis, epigenome, proteomics and mouse knock-out studies amongst others will also help explore the non-coding regulatory space and gene-gene interactions. Given the existence of many non-pathogenic variants in any individual’s genome, the main challenge faced by researchers when interpreting NGS data of an IPD case is determining which variants are causing the disorder.3Interpreting the functional consequences of novel rare variants is not easy and it is extremely important to apply rigorous standards when assigning pathogenicity. Clinical genomic data are the same as other complex medical data and should be interpreted by a multidisciplinary team comprising typically a statistical geneticist, clinical geneticist, and genetic counselors, who have the skills to interpret these results in the context of the test methodology, the theoretical background of genetics, Bayesian reasoning, and a myriad of other factors.1. Simeoni I, Stephens JC, Hu F, et al. A comprehensive high-throughput sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders. Blood. 2016; 127: 279.2. Turro E, Greene D, Wijgaerts A, et al. A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. Sci Transl Med. 2016;8:328.3. Lentaigne C, Freson K, Laffan MA, et al. Inherited platelet disorders: towards DNA-based diagnosis. Blood. 2016; 127: 2814. DisclosuresNo relevant conflicts of interest to declare.

Next generation sequencing-based exploration of genomes and transcriptomes of medicinal plants

Medicinal plants are known for their therapeutic potential and have been associated with human history for their use in traditional medicine systems in different countries. Recent advances in next generation sequencing (NGS) technologies have accelerated research on medicinal plants with reduced cost and efforts. NGS technologies not only provide opportunity for high throughput whole genome sequencing, they also facilitate direct RNA sequencing. The sequence data-sets generated can further be explored for application in various areas of research such as comparative genomics, data mining for small and long non-coding RNAs, mining of molecular markers, gene discovery, etc. Continuous efforts are being made by commercial sequencing service providers in improving technology to overcome bioinformatics challenges in NGS data analysis. In recent past, genome sequence projects on various medicinal plants have been successfully accomplished and few others are in pipeline. Similarly, enormous NGS-based transcriptome data have been generated in a number of medicinal plants. In the present review, we have briefly attempted to address advancement in NGS technology, genomic and transcriptomics studies on medicinal plants with special emphasis on seabuckthorn (Hippophae sp.), a medicinally important plant of Indian Himalayas. Moreover, the scope of implementation of NGS based research on medicinal plants have been explored for the selection of candidate genes involved in particular biosynthesis pathways. The identified genes can be exploited for engineering medicinal plants for producing improved quality biologically active phytocompounds.

Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls

We live in an era of genomic medicine. The past five years brought about many significant achievements in the field of cancer genetics, driven by rapidly evolving technologies and plummeting costs of next-generation sequencing (NGS). The official completion of the Cancer Genome Project in 2014 led many to envision the clinical implementation of cancer genomic data as the next logical step in cancer therapy. Stemming from this vision, the term ‘precision oncology’ was coined to illustrate the novelty of this individualised approach. The basic assumption of precision oncology is that molecular markers detected by NGS will predict response to targeted therapies independently from tumour histology. However, along with a ubiquitous availability of NGS, the complexity and heterogeneity at the individual patient level had to be acknowledged. Not only does the latter present challenges to clinical decision-making based on sequencing data, it is also an obstacle to the rational design of clinical trials. Novel tissue-agnostic trial designs were quickly developed to overcome these challenges. Results from some of these trials have recently demonstrated the feasibility and efficacy of this approach. On the other hand, there is an increasing amount of whole-exome and whole-genome NGS data which allows us to assess ever smaller differences between individual patients with cancer. In this review, we highlight different tumour sequencing strategies currently used for precision oncology, describe their individual strengths and weaknesses, and emphasise their feasibility in different clinical settings. Further, we evaluate the possibility of NGS implementation in current and future clinical trials, and point to the significance of NGS for translational research.

Erratum: Guidelines for diagnostic next-generation sequencing

We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a 'rating system' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.

The Role of Next-Generation Sequencing in Castration-Resistant Prostate Cancer Treatment.

Molecular biomarkers play little role in the current treatment of metastatic castration-resistant prostate cancer (CRPC). The advent of next-generation sequencing (NGS) has enabled the comprehensive molecular characterization of the genomic and transcriptomic landscape of both untreated primary prostate cancer and CRPC. Recent studies demonstrating the feasibility of interinstitution studies obtaining and NGS profiling of metastatic biopsies, targeted NGS approaches applicable to routine formalin-fixed, paraffin-embedded specimens, and NGS approaches applicable to circulating DNA and circulating tumor cells portend near-term adoption of NGS approaches in the management and treatment of CRPC. Important considerations in the clinical implementation of NGS include interpatient and intrapatient heterogeneity, disease progression to neuroendocrine/small cell prostate carcinoma, and incorporation into clinical trial design to demonstrate clinical utility. We review the recent progress in NGS-based characterization of CRPC to understand disease biology and inform on barriers to widespread clinical adoption.

Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway.

Holoprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high heterogeneity, both in clinics and genetics. We have developed a novel targeted next-generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling.

Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories

Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials.

From HLA typing to anti-HLA antibody detection and beyond: The road ahead.

The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA antibodies have led to continuously develop novel practical and feasible technologies in the field of organ and tissue transplantation. Technologies to identify HLA molecules have evolved from the serological to the molecular methods and a true innovation in the DNA sequencing has taken place with the development of next generation sequencing. An interesting field to explore is how the information resulting from the HLA-DNA sequencing can be applied in the clinical setting by including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce the number of mismatched antigens. Furthermore, high resolution HLA allele typing could help to understand the epitopes against which antibodies are developed. However, the improvements should not only concern the diagnostic tools in the pre-transplantation phase, but also the monitoring of post transplantation outcome. There is a growing interest in developing new non-invasive biomarkers to monitor the rejection. Currently, increasing evidence has focused on miRNAs, epigenetic markers emerged as regulators of molecular events that are differently expressed in biopsies and blood as well as in urinary samples of transplanted recipients. The implementation of next generation sequencing and genome-wide expression analysis together with functional assays may provide useful tools to evaluate the epigenetic modulation in transplantation biology but many efforts are requested for translating in the clinical arena the results obtained in experimental models.

Classifying Germline Sequence Variants in the Era of Next-Generation Sequencing.

Genetic medicine has taken tremendous strides in recent years due in large part to the clinical implementation of next-generation sequencing (NGS).10 Laboratories are now able to sequence more genes for less cost, resulting in advances in diagnostics, prognostics, therapeutic decision-making, and risk assessment. New germline and somatic gene/variant panel NGS tests are continually emerging, and exome/genome sequencing for rare inherited disorders is becoming more widely available. The increasing adoption of NGS testing has already led to a dramatic increase in the number of variants that need to be interpreted, ranging from completely novel variants to well-characterized variants with multiple publications describing their functional consequence. Catalyzed by the adoption of exome sequencing, an increasing need to evaluate the validity and strength of gene–disease association claims has emerged and adds yet another dimension to the already complex process of genomic data curation and interpretation. Depending on the amount of data available for a given variant (and its associated gene), clinical variant assessment can be a very challenging and time-consuming process. In addition, variants whose impact on the protein may be less obvious (e.g., missense variants) or variants in genes which are less well characterized may remain variants of uncertain clinical significance for quite some time, leading to an abundance of inconclusive results and possible confusion for ordering clinicians as well as patients. Clinical variant classification typically incorporates many data elements such as disease prevalence, variant frequency in patient and control populations, and gene function. However, these variables are often incomplete or poorly understood, further exacerbating the challenges for the overall medical community. Updated standards and guidelines for germline sequence variant interpretation were recently published as a joint consensus recommendation involving stakeholders from the American College of Medical Genetics and Genomics (ACMG), the Association for Molecular Pathology (AMP), and the College of …

Survival following implementation of next-generation sequencing in routine diagnostics of advanced lung cancer: Results of the German Network Genomic Medicine.

9085Background: The Network Genomic Medicine (NGM) Lung Cancer is a health care provider network offering comprehensive next generation sequencing (NGS)-based multiplex genotyping on a central diag...

The Changing Landscape of Molecular Diagnostic Testing: Implications for Academic Medical Centers.

Over the last decade, the field of molecular diagnostics has undergone tremendous transformation, catalyzed by the clinical implementation of next generation sequencing (NGS). As technical capabilities are enhanced and current limitations are addressed, NGS is increasingly capable of detecting most variant types and will therefore continue to consolidate and simplify diagnostic testing. It is likely that genome sequencing will eventually serve as a universal first line test for disorders with a suspected genetic origin. Academic Medical Centers (AMCs), which have been at the forefront of this paradigm shift are now presented with challenges to keep up with increasing technical, bioinformatic and interpretive complexity of NGS-based tests in a highly competitive market. Additional complexity may arise from altered regulatory oversight, also triggered by the unprecedented scope of NGS-based testing, which requires new approaches. However, these challenges are balanced by unique opportunities, particularly at the interface between clinical and research operations, where AMCs can capitalize on access to cutting edge research environments and establish collaborations to facilitate rapid diagnostic innovation. This article reviews present and future challenges and opportunities for AMC associated molecular diagnostic laboratories from the perspective of the Partners HealthCare Laboratory for Molecular Medicine (LMM).

Report from the Standards for Pathogen Identification via Next-Generation Sequencing (SPIN) Workshop

Next-generation sequencing (NGS) is not routinely used in applied settings due to lack of confidence in results. This workshop convened experts to identify measurement challenges impeding NGS implementation and potential standards-based solutions to address these challenges.

Challenges and opportunities of next-generation sequencing: a cytopathologist's perspective.

Molecular cytopathology has gene sequencing as its core technology. Until recently, cytological samples were only tested by sequential single-gene mutational tests. Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more informative than a single biomarker. Next-generation sequencing (NGS), matched with the multiplex capture of targeted gene regions and analysed by sophisticated bioinformatics tools, enables the simultaneous detection of multiple mutations in multiple genes. With the development of miniaturised technology and benchtop sequencers, it is not unlikely that NGS will soon be adopted for routine molecular diagnostics, including cytological samples. This review addresses (1) the most relevant methodological and technical aspects of the NGS analysis workflow and the diverse platforms available; (2) the issues related to daily practice implementation, namely, the cytological sample requirement and the validation procedures; and (3) the opportunities that NGS offers in different fields of cytopathology, to increase mutation detection sensitivity in paucicellular smears and to extend the analysis to a larger number of gene regions. Cytopathologists involvement and coordination in this rapidly evolving field is crucial for the effective implementation of NGS in the present and future cytological practice.