Next-generation sequencing: from conventional applications to breakthrough genomic analyses and precision oncology

KEYWORDS:

• Next-generation sequencing
• conventional and breakthrough methods
• intratumor heterogeneity
• circulating cell-free DNA sequencing
• common diseases
• cancer
• precision oncology

1. Introduction

The validity, accuracy, and decreasing cost of genome analyses in health and disease [1,2] and their successful integration in the ENCODE project [3] have revolutionized life sciences. Progress in the research of major complex diseases, including neurodevelopmental and degenerative disorders, psychiatric syndromes, and particularly cancer has been slow. Rapid advances in next-generation sequencing (NGS) applications from static, conventional, single-biopsy NGS to dynamic, breakthrough, multiple-biopsy analyses, assessing genomic clone evolution in time and space, have laid the foundation of precision medicine [4]. Conventional NGS refers to single-biopsy analyses at a single time point, while breakthrough NGS includes multi-regional analyses of the primary tumor for the identification of intratumor heterogeneity (ITH), as well as serial liquid biopsy analyses, using circulating cell-free DNA (cfDNA) followed by NGS (cfDNA-NGS). Here, we summarize the potential and challenges of NGS integration into appropriately designed studies, to achieve predictive, preventive, and therapeutic clinical implications.

Common complex diseases remain an unresolved health problem. In some cases, such as cardiovascular disease, there has been substantial progress in prevention, although the management of these patients requires lifelong treatment and intensive follow-up [5]. Other diseases, such as neurodevelopmental and neurodegenerative disorders, schizophrenia, and cancer, remain incurable, lacking means of early and accurate diagnosis, as well as effective therapies, and featuring poor quality of life.

Subsequently, it has become clear that, without understanding the germline and/or somatic mutational landscape underlying these complex diseases and how structural genome variants affect molecular networks, gene-expression profiles, transcription and translation, significant progress toward cure will never occur.

The rapid evolution of NGS platforms during the past decade and their unprecedented potential to identify genetic and genomic aberrations in tissue and liquid biopsies have rendered these genomic analyses into the most accurate tool for precise diagnosis and individualized treatment.

2. NGS technologies and the revolution of life sciences

The integration of NGS systems into basic translational and clinical research within the past decade and, recently, the clinical utilization of targeted NGS (tNGS) pose as the most promising strategy to achieve precision cancer medicine [4,6] with subsequent dramatic improvement of public health and individualized patient management.

The revolution in biomedical research brought by the introduction of Sanger sequencing 40 years ago has reached new heights of progress with the development of NGS methods. Continuously dropping costs, especially in comparison to the Sanger method, as well as high speed and accuracy have led to the widespread use of next-generation sequencers for research, or even clinical purposes. Several competing companies currently offer their respective NGS technologies, such as Roche, Illumina, and Thermo Fisher Scientific. Some sequencers, including the HiSeq series and particularly the HiSeq 2500, 3000, and 4000 (Illumina), Ion Proton (Ion Torrent), SeqCap EZ (Roche, and PromethION (Oxford Nanopore Technologies) are capable of scanning whole exomes or genomes very swiftly and reliably, while others, such as Ion PGM (Ion Torrent) and MiSeq (Illumina) are only capable of targeted sequencing of a known gene panel. Furthermore, some sequencing machines allow for sequencing of parts or the whole transcriptome, for instance the NextSeq, HiSeq and NovaSeq series from Illumina and the Ion Proton system [7,8]. More recently, serial cfDNA-NGS provides the opportunity for patient monitoring and identification of circulating subclones probably responsible for tumor relapse.

3. Genomic medicine cancer genome analysis with NGS

Precision medicine offers the potential to dramatically improve clinical care and genetics are already being increasingly used to guide clinical decision-making. The widespread market availability and accuracy of NGS platforms has facilitated the implementation of sequencing into patient care. Although the clinical implications of NGS include a variety of major common complex diseases, including neurodevelopmental and neurodegenerative diseases, cardiovascular disease, the metabolic syndrome, immune disorders, and psychotic syndromes [9], the research interest of NGS studies is currently shifted toward cancer genome analyses. The availability of tumor specimens allows for the analysis of paired cancer-normal tissue, including the widespread use of conventional, single-biopsy, static NGS methods, as well as breakthrough, dynamic, multiple-biopsy genomic analyses [10].

Especially in solid tumors, the availability and the potential for thorough molecular exploration of the primary tumor, coupled with repeated DNA analyses of cfDNA for the identification of circulating genomic subclones (cGSs) at different time points, provide unprecedented clinical implications. Two major cancer genome projects explore cancer driver genes, aiming at the completion of the respective catalogue of genes and mutations, for different cancer types [11,12]. In specific, NGS analyses of multiregional biopsies and serial cfDNA samples, to identify ITH and cGSs, respectively, shape a new era of combinational therapy, targeting distinct tumor cell subpopulations in an individual patient.

For the first time, NGS technologies were implemented into the ENCODE project [3], early after their market availability in 2006. The utilization of sequencing systems for basic research has revolutionized life sciences, establishing the concept of non-coding genome functionality and negating the term ‘junk DNA’, previously used to describe the non-coding genome, accounting for 98% of the whole genome. Moreover, the integration of protein coding gene expression profiling to delineate regulatory, molecular and gene–gene interaction networks paves new avenues toward the realization of precision medicine [13,14].

The successful integration of NGS into the ENCODE international, multicenter consortium has led to the publication, at first, of small NGS studies in 2011, analyzing patient-derived clinical samples, aiming at personalized medicine [15]. Since then, progress has been dramatic, especially regarding the validity with which NGS can translate basic research findings into clinical implications. Although tNGS was met with widespread acclaim for its ability to scan known genes involved in cancer and its clinical utility, the discovery of new genes and novel therapeutic targets, on the basis of gene mutations, amplifications, gene fusions, and other aberrations, requires the conduction of large-scale clinico-genomic studies for valid results [10]. Recently, the valid identification of novel cancer driver genes and druggable mutations has been achieved by large scale WES [10] and whole-genome sequencing (WGS) studies, especially on breast [16], liver [17], and pancreatic cancers [18].

Beyond the completion of the list of genes involved in tumorigenesis and the discovery of novel oncotargets, understanding and overcoming intrinsic and acquired resistance, as well as the prediction and potentially the prevention of disease recurrence, obviously remain an unrealistic goal, based on the conventional, single-biopsy strategy for NGS analysis.

The greatest challenge at the present is to overcome primary and acquired therapeutic resistance. Recent intensive research has focused on two major entities to explain tumor non-responsibility. The first is the assessment of ITH of the primary tumor. The new method of multi-regional biopsies followed by NGS aims to the therapeutic targeting of distinct genomic subclones within the primary tumor with a combination of targeted drugs [19]. The second is the genomic analysis of liquid biopsies. The technological revolution brought by non-invasive genome analyses from individual patients gives rise to high expectations in the fields of both personalized treatment to improve therapeutic response, as well as patient monitoring after the completion of the therapeutic approaches.

4. Future perspectives and conclusions

Overcoming therapeutic resistance, increasing the time to relapse, or even preventing recurrence in the adjuvant setting, in order to substantially prolong overall survival still remain unresolved problems. Researchers and clinicians share a common dream to achieve these goals in the war against cancer and the implementation of innovative NGS methods poses as a compelling and realistic future direction toward the realization of precision cancer medicine.

The extensive genomic heterogeneity among patients sharing the same disease highlights the necessity for large-scale clinical studies to achieve strong statistical significance (p < 0.01) [10]. Indeed, the rapid progress of NGS technologies and bioinformatics has enabled the conduction of large genomic studies, including WGS studies with 560 breast and 300 liver cancer patients by Nik-Zainal [16] and Fujimoto et al. [17] respectively, as well as two large studies on pancreatic cancer by Bailey [18] and Humphris and colleagues [20], applying WES or WGS on a total of 383 and 385 participants respectively, with the first implementing RNA sequencing as well. The aforementioned studies returned highly promising results, showcasing significant advances in the discovery of novel mutations and oncotargets, as well as the molecular classification of the studied diseases. Lately, three exciting projects, in conjunction with Illumina, have been designed to accelerate cancer research with clinical implications in the fields of diagnosis and therapy of cancer. Genomics England ultimately aims to sequence 100,000 samples, with more than 16,000 samples already analyzed in the latest report. Furthermore, two even larger collaborative projects from the United States, the Cancer Moonshot program by the National Cancer Institute and the Precision Medicine Initiative by the National Institute of Health, intend to sequence samples from as many as 1 million volunteers, in order to identify novel biomarkers, study the environmental impact on health and disease, and develop new targeted drugs [21].

Recent evidence on the genomic dynamics in the evolution of cancer shapes the new era of the spatiotemporal emergence of tumor heterogeneity and the dissemination to distant organs through cGSs [19]. This proposed fundamental principle of cancer metastasis can now be systematically explored with two innovative methods. First, using tNGS, WES, or WGS for multiregional analysis of the primary tumor can identify subclones within it based on known tNGS or novel genes WES, WGS, through appropriately designed, large studies. The assessment of ITH could enable the development of new combinational therapies, targeting the comprehensive tumor diversity [22].

Second, the establishment of DNA analysis on plasma cfDNA in individual patients before, during, and after the completion of therapy allows for the assessment of genomic diversity in both the tumor and the circulation [23]. Innovative designs of emerging and future studies combining the concepts of ITH and serial plasma sampling for cGS identification raise now, for the first time, the potential to predict and reduce the rates of primary and secondary therapeutic resistance, as well as predict and, presumably, prevent disease relapse.

In summary, both the conventional and breakthrough methodologies of clinical NGS studies provide unprecedented potential of clinical implications. Conventional, static, single–biopsy-based NGS analyses could broaden the list of approved targeted drugs, through the valid identification of novel druggable mutations. On the other hand, the assessment of the spatiotemporal emergence of resistant subclones through breakthrough NGS applications could revolutionize current research strategies toward overcoming therapeutic resistance, predicting relapse and substantially prolonging time to recurrence in individual patients.

Declaration of interest

The authors have no 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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Funding

This paper is not funded.