Clinical Applications Of Next-generation Sequencing Research Articles

Clinical Applications of Next-Generation Sequencing for Cancer Diagnostics and Targeted Cancer Therapy

Next-generation sequencing (NGS) is a revolutionary technology that has effectively sequence massive quantities of genomic DNA or RNA at a shorter time and a lower cost. The NGS is routinely used in oncology to tailor care for individual patient. In this review, we discuss the role of NGS how it can provide a more personalized approach to targeted cancer therapy. A literature review was conducted via PubMed and articles were screened for publication dates within the past 10 years. Primary literature consisting of clinical trials and review articles were utilized. Search terms included “(next-generation sequencing) AND (cancer)”, “(next-generation sequencing) AND (cancer diagnostics)”, and “(next-generation sequencing) AND “(cancer therapy)”.
 From the literature review, we found that NGS is indicated for daily practice as a diagnostic tool for advanced NSCLC, colon cancer, prostate cancer, cholangiocarcinoma, and some advanced rare cancers. We also found evidence suggesting that NGS is helpful in identifying actionable mutations that will, in turn, allow the patient to be matched to a more individualized cancer treatment.
 As oncology care continues to move in the direction of more personalized care, the potential applications of NGS in the field of oncology will continue to evolve. Our review will help further the understanding of NGS and provide context for its value in patient care and its ability to provide an avenue for targeted therapy.

Clinical applications of next-generation sequencing in the diagnosis of genetic disorders in Korea: a narrative review

Background: Next-generation sequencing (NGS) technologies have revolutionized genetic testing and enabled efficient screening of various genetic conditions in clinical settings. However, the clinical application of genetic test results presents numerous significant challenges. This review aims to provide a comprehensive overview of key concepts for the clinical application of NGS, including (1) technical aspects and limitations, (2) variant classification, (3) clinical interpretation, (4) familial testing and genetic counseling, and (5) ethical considerations.Current Concepts: In short-read-based NGS, several limitations exist in detecting genomic variations, including repetitive sequences or complex structural variations. The variant classification process can be influenced by suspected genomic conditions and the accessibility of genomic databases. Therefore, the final genetic diagnosis depends on the physician’s discretion, which relies on the genotype-phenotype correlations and reverse phenotyping through additional evaluations. Familial testing can help trace the origin of variants and allele segregation and aid in variant interpretation, risk assessment, disease prevention, and family planning. In addition to addressing the clinical impact of genetic results, genetic counselors should also consider potential consequences related to ethical, legal, and social issues, including family dynamics.Discussion and Conclusion: NGS-based genetic testing is a promising diagnostic tool for genetic disorders, but proper variant interpretation and clinical evaluation are crucial for optimal clinical practice. Notably, ethical considerations and regulatory measures are required to prepare for the next era of genomic medicine.

FFPE-Based NGS Approaches into Clinical Practice: The Limits of Glory from a Pathologist Viewpoint.

The introduction of next-generation sequencing (NGS) in the molecular diagnostic armamentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

Clinical Effect of Genetic Alterations in Pediatric Patients with B-Progenitor Acute Lymphoblastic Leukemia

▪BackgroundRecent genetic studies using high-throughput sequencing have substantially revealed genetic alterations in pediatric patients with B-progenitor acute lymphoblastic leukemia (B-ALL). However, their clinical relevance of these alterations has not been fully investigated.MethodsWe performed targeted-capture sequencing of 149 known or putative driver genes for B-ALL in 525 pediatric patients who were uniformly treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol. In addition, we performed whole transcriptome sequencing in 63 of 118 patients who were not categorized into any genetic subgroup defined by RT-PCR status or targeted-capture sequencing results.ResultsIn total, 647 focal deletions (median 1 per patient; range, 0-7) and 993 mutations (median 2 per patient; range, 0-10) were detected in 491 (94%) cases. Some of these alterations were closely associated with disease subtypes. We identified recurrent PHF6 mutations, which were almost exclusively detected in the TCF3 - PBX1- positive ALL genotype. Furthermore, frequent involvement of PAX5 and KMT2D (mutations) and IKZF3 (deletions) were characteristic of TCF3 - PBX1 -positive ALL genotype. To identify genetic alterations associated with a poor prognosis, we analyzed 20 genes that were mutated in at least 20 patients in our study cohort. Univariate analyses revealed genetic alterations in TP53 , KMT2D , and IKZF1 to be associated with a significantly shorter overall survival. Mutations and deletions in IKZF1 were also strongly associated with induction failure (Odds ratio, 12; 95% CI, 2.7-55). This led to death in 80% (n = 4/5) of the patients with IKZF1 alterations. Most patients with TP53 and KMT2D mutations and with disease relapse had fatal disease progression [94% mortality rate (n = 17/18)]. Multivariate analysis, together with the NCI criteria, revealed that two genetic lesions, TP53 and KMT2D , retained prognostic impacts for overall survival. Prediction of 5-year OS improved in each NCI-risk group when patients in each risk group were stratified according to the presence or absence of mutations in TP53 or KMT2D lesions: 69% [with alteration (n = 26)] vs. 85% [without alteration (n = 138)] for the NCI-HR group (P = 0.03) and 85% [with alteration (n = 42)] vs. 97% [without alteration (n = 319)] for the NCI-SR group (P = 0.004). Additionally, the four less frequently mutated genes also had an independent prognostic impact before and after adjustment for risk factors: IKZF2 (n = 6; HR for death, 7.4; 95% CI, 2.3-24), SH2B3 (n = 4; HR for death, 7.0; 95% CI, 1.7-29), JAK2 (n = 7; HR for events, 4.6; 95% CI, 1.7-12), and PTEN (n = 9; HR for death, 3.8; 95% CI, 1.2-12). Notably, mutations in three genes present potentially druggable targets: ruxolitinib for mutations in JAK2 or SH2B3 and poly (ADP-ribose) polymerase inhibitor formutations in PTEN . Finally, based on a gene expression profile, 20 of 63 patients were categorized into discrete subgroups characterized by Ph-like profiles (n = 7), ZNF384 (n = 7), and MEF2D (n = 6) fusions. Ph-like and MEF2D fusion-positive ALLs were associated with a dismal prognosis, whereas all patients with a ZNF384 fusion survived with complete remission. No mutations were associated with a poor prognosis in the 43 remaining patients. Further analysis, such as whole genome sequencing, is warranted for these patients.ConclusionsTargeted-capture sequencing improves the prediction of clinical outcomes and helps detect prognostic and druggable biomarkers. Our results can facilitate the clinical applications of next-generation sequencing to select optimal therapy for pediatric patients with B-ALL. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Abstract 5454: A data driven pipeline to detect somatic mutations in oncology

Abstract The analysis of Next Generation Sequencing (NGS) data relies on workflows that integrate different bioinformatics tools. Designing workflows that are assay agnostic and have a robust computational framework for result generation in a timely manner is particularly important for clinical applications of NGS. These technologies play an important role in classifying a patient's tumor at a molecular level however, there is still a need to develop workflows that generate consistent mutation profiles for oncology. We developed a bioinformatics tool to analyze DNA and RNA sequencing data in a single pipeline and to specifically detect somatic mutations in oncology clinical workflows. Our automated pipeline report contains extensive quality metrics for each sample and generates a robust mutation annotation and filtration module vital for clinical use. We use data driven filters and algorithms that predict the effectiveness of mutations to filter out mutations of poor quality or mutations without clinical significance. We applied our pipeline to standard cell lines and various cancer FFPE samples acquired from the Intermountain Healthcare Biorepository. Our pipeline successfully processes different mutation classes including Single Nucleotide Variants (SNVs), small (<20 bp) and large (>21 bp) insertions and deletions (InDels), gene fusions, and exon skipping mutations. In addition, we established a reference set that allows us to call Copy Number Variants (CNVs) in a gene panel designed to detect clinically relevant mutations in lung cancers. Comparison of this pipeline to a validated external workflow yield high concordance of clinically significant mutations. We conclude that our pipeline offers a sensitive and specific methodology to analyze DNA and RNA sequencing data to clinically relevant results from low input amplicon-based assays for various solid tumor cancer types. Citation Format: Katherine Shortt, Christopher Johnson, Archana Ramesh, Tom Neuwerth, Chris Giauque, Sharanya Raghunath. A data driven pipeline to detect somatic mutations in oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5454.

Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics.

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

Clinical Applications of Next-Generation Sequencing in Precision Oncology.

The ability of next-generation sequencing (NGS) to comprehensively assess the molecular profile of a tumor specimen has transformed the clinical testing landscape in oncology. Accordingly, recent years have seen broad uptake of clinical NGS to inform cancer patient management. However, significant challenges remain. The annotation and clinical interpretation of variants identified by NGS tests often require rigorous review and may vary between laboratories. While a clearer regulatory path has emerged, reimbursement for NGS tests remains a subject of continuing debate. Basket clinical studies such as the National Cancer Institute Molecular Analysis of Therapy Choice are evaluating the degree to which matching of a targeted therapy to tumor molecular profile by NGS can be applied independently of tissue histology. Newer applications of NGS such as for circulating tumor DNA testing and to identify novel RNA fusion driver events continue to expand its clinical utility.

Clinical Applications of Next-Generation Sequencing

The significant drop in sequencing costs boosted by chemistries optimization and sample multiplexation has resulted in an immense growth within the field of next-generation sequencing (NGS) in the last decade. This has allowed a diversification of techniques, promoting a rapid advance in knowledge on the molecular basis of human disease. Due to the applicability and importance of this technology in basic research, it has quickly migrated to the clinical setting. NGS enables clinicians to make improved diagnostic and treatment decisions, which ultimately may influence precision medicine.

Review of Clinical Next-Generation Sequencing.

- Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. - To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. - The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. - The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.

Clinical Applications of Next-Generation Sequencing in Cancer Diagnosis.

With the advancement and improvement of new sequencing technology, next-generation sequencing (NGS) has been applied increasingly in cancer genomics research fields. More recently, NGS has been adopted in clinical oncology to advance personalized treatment of cancer. NGS is utilized to novel diagnostic and rare cancer mutations, detection of translocations, inversions, insertions and deletions, detection of copy number variants, detect familial cancer mutation carriers, provide the molecular rationale for appropriate targeted, therapeutic and prognostic. NGS holds many advantages, such as the ability to fully sequence all types of mutations for a large number of genes (hundreds to thousands) and the sensitivity, speed in a single test at a relatively low cost compared to be other sequencing modalities. Here we described the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead.

Clinical applications of next-generation sequencing in histocompatibility and transplantation.

Next-generation sequencing (NGS) can overcome traditional methodological barriers to facilitate detailed studies of large genomes. Here, we summarize recent NGS-based developments in histocompatibility and transplantation, and highlight the dynamic range of clinical applications achievable on this platform. Multiple NGS-based protocols have been established to achieve unambiguous human leukocyte antigen genotyping. These methods are presently engaged to serve the high-throughput demand of large bone marrow registries; however, the scalable nature of NGS makes it an equally attractive technology for select applications within solid organ transplantation. Recently, the exquisite sensitivity of NGS has been leveraged to perform noninvasive allograft monitoring by tracking the dynamics of donor-derived cell-free DNA. Further, NGS-based T-cell receptor and immunoglobulin heavy chain repertoire profiling appear to be useful in clarifying disease-specific diagnoses in certain complex allograft pathology; detecting/quantifying minimal residual disease following allogeneic stem cell transplantation; and tracking donor-reactive T cells to understand the mechanism of tolerance in kidney transplant recipients. NGS is superior to classical Sanger sequencing in its throughput, sensitivity, and the ability to provide phase-defined sequence data. These unique properties allow its broad application to diverse areas in clinical transplantation.

The translation of cancer genomics: time for a revolution in clinical cancer care.

The introduction of next-generation sequencing technologies has dramatically impacted the life sciences, perhaps most profoundly in the area of cancer genomics. Clinical applications of next-generation sequencing and associated methods are emerging from ongoing large-scale discovery projects that have catalogued hundreds of genes as having a role in cancer susceptibility, onset and progression. For example, discovery cancer genomics has confirmed that many of the same genes are altered by mutation, copy number gain or loss, or structural variation across multiple tumor types, resulting in a gain or loss of function that likely contributes to cancer development in these tissues. Beyond these frequently mutated genes, we now know there is a ‘long tail’ of less frequently mutated, but probably important, genes that play roles in cancer onset or progression. Here, I discuss some of the remaining barriers to clinical translation, and look forward to new applications of these technologies in cancer care.

Clinical Applications of Next-Generation Sequencing: The 2013 Human Genome Variation Society Scientific Meeting

Next-generation sequencing (NGS) has significantly contributed to the transformation of genomic research by providing access to the genome for analysis, by significantly decreasing the sequencing costs and increasing the throughput. The next goal is to exploit this powerful technology in the clinic, namely for diagnostics and therapeutics. The 2013 annual meeting of the Human Genome Variation Society, held in Paris, France, provided a forum to discuss possible clinical applications of NGS, the potential of some of the current NGS systems to transition to the clinic, the identification of causative mutations for rare genetic disorders through whole-genome or targeted genome resequencing, the application of NGS for family genomics, and NGS data analysis tools.

Clinical applications of next-generation sequencing in colorectal cancers

Like other solid tumors, colorectal cancer (CRC) is a genomic disorder in which various types of genomic alterations, such as point mutations, genomic rearrangements, gene fusions, or chromosomal copy number alterations, can contribute to the initiation and progression of the disease. The advent of a new DNA sequencing technology known as next-generation sequencing (NGS) has revolutionized the speed and throughput of cataloguing such cancer-related genomic alterations. Now the challenge is how to exploit this advanced technology to better understand the underlying molecular mechanism of colorectal carcinogenesis and to identify clinically relevant genetic biomarkers for diagnosis and personalized therapeutics. In this review, we will introduce NGS-based cancer genomics studies focusing on those of CRC, including a recent large-scale report from the Cancer Genome Atlas. We will mainly discuss how NGS-based exome-, whole genome- and methylome-sequencing have extended our understanding of colorectal carcinogenesis. We will also introduce the unique genomic features of CRC discovered by NGS technologies, such as the relationship with bacterial pathogens and the massive genomic rearrangements of chromothripsis. Finally, we will discuss the necessary steps prior to development of a clinical application of NGS-related findings for the advanced management of patients with CRC.

Next‐generation sequencing: ready for the clinics?

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing and increasing the throughput. Now, the focus is on using NGS technology for diagnostics and therapeutics. In this review, we discuss the possible clinical applications of NGS and the potential of some of the current systems to transition to the clinic. Clinical use of NGS technologies will enable the identification of causative mutations for rare genetic disorders through whole-genome or targeted genome resequencing, rapid pathogen screening and cancer diagnosis along with the identification of appropriate therapy. Routine clinical use of NGS technologies is appealing, but mandates high accuracy, simple assays, small inexpensive instruments, flexible throughput, short run times and most importantly, easy data analysis as well as interpretation. A number of NGS systems launched recently have least some of these characteristics, namely, small instruments, flexible throughput and short run time, but still face a few challenges. Moreover, simplified data analysis tools will need to be developed to minimize the requirement of sophisticated bioinformatics support in clinics. In summary, for successful transition of NGS to clinic, a sustained collaboration between research labs, clinical practitioners and vendors offering sequencing based genetic tests is required.

Genomics of AML: Clinical Applications of Next-Generation Sequencing

In the past decade, a series of technological advances have revolutionized our ability to interrogate cancer genomes, culminating in whole-genome sequencing, which provides genome-wide coverage at a single base-pair resolution. As sequencing technologies improve and costs decrease, it is likely that whole-genome sequencing of cancer cells will become commonplace in the diagnostic workup of patients with acute myelogenous leukemia (AML) and other cancers. The unprecedented molecular characterization provided by whole-genome sequencing offers the potential for an individualized approach to treatment in AML, bringing us one step closer to personalized medicine. In this chapter, we discuss how next-generation sequencing is being used to study cancer genomes. Recent publications of whole-genome sequencing in AML are reviewed and current limitations of whole-genome sequencing are examined, as well as current and potential future clinical applications of whole-genome sequencing.