ABSTRACT
INTRODUCTION: The 'one biomarker/one drug' scenario is unsustainable because cancer is a complex disorder that involves a number of molecular defects. In the past decade, major technological advances have lowered the overall cost and increased the efficiency of next-generation sequencing (NGS). AREAS COVERED: We review recent regulations on NGS and complementary diagnostics in Japan, mainly focusing on high-quality studies that utilized these new diagnostic modalities and were published within the last 5 years. We highlight significant changes in regulation, and explain the direction of efforts to translate the results of NGS and complementary diagnostics into clinical practice. EXPERT OPINION: NGS holds a number of advantages over conventional companion and complementary diagnostics that enable simultaneous analyzes of multiple cancer genes to detect actionable mutations. Parallel technological developments and regulatory changes have led to the rapid adoption of NGS into clinical practice. NGS-based genomic data have been leveraged to better understand the characteristics of a disease that affects its patient's response to a given therapy. As NGS-based tests become more widespread, however, Japanese authorities will face significant challenges particularly with respect to the complexity of genomic data, which will have to be managed if NGS is to benefit patients.
Subject(s)
High-Throughput Nucleotide Sequencing/trends , Medical Device Legislation , Molecular Diagnostic Techniques/trends , Neoplasms/drug therapy , Precision Medicine/methods , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , DNA Mutational Analysis/economics , DNA Mutational Analysis/methods , DNA Mutational Analysis/trends , Databases, Nucleic Acid , Device Approval/legislation & jurisprudence , Direct-To-Consumer Screening and Testing/economics , Direct-To-Consumer Screening and Testing/legislation & jurisprudence , Drug Resistance, Microbial/genetics , Equipment and Supplies/classification , Genetic Diseases, Inborn/drug therapy , Genetic Diseases, Inborn/genetics , Government Agencies/organization & administration , Health Services Needs and Demand , High-Throughput Nucleotide Sequencing/economics , High-Throughput Nucleotide Sequencing/instrumentation , High-Throughput Nucleotide Sequencing/methods , Humans , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Japan , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Molecular Targeted Therapy , Mutation , National Health Programs , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasms/geneticsABSTRACT
A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection.
Subject(s)
Biotin/chemistry , DNA Mutational Analysis/standards , Mutation , Oligonucleotide Array Sequence Analysis/standards , Oligonucleotides/chemistry , Streptavidin/chemistry , Animals , BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Base Pairing , Biotinylation , Cattle , DNA Mutational Analysis/economics , Humans , Oligonucleotide Array Sequence Analysis/economics , Oligonucleotide Array Sequence Analysis/instrumentation , Polymerase Chain Reaction/standards , Protein Binding , Sensitivity and Specificity , Serum Albumin, Bovine/chemistry , Time FactorsABSTRACT
BACKGROUND: Wilson disease is one of the commonest inherited and potentially fatal yet treatable liver disorders. About 5-27% patients present with acute liver failure and require prompt chelation therapy and life-saving liver transplantation. Diagnosis during acute liver failure is particularly difficult with short time allowance. Direct molecular diagnosis remains the most decisive tool but is often hindered by demanding techniques and numerous mutations. We developed a one-step, 3-h, reproducible, and accurate real-time amplification refractory mutation system which can simultaneously detect 28 ATP7B mutations. METHODS: Primers were designed to complement the mutant sequence at the 3' end. The mutations were p.S105X, p.Q511X, p.R616Q, p.S693P, p.S693C, p.R778L, p.A874V, p.T888P, p.R919G, p.T935M, p.P992L, p.M1025R, p.D1047V, p.I1148T, p.R1156H, p.T1178A, p.V1216M, p.P1273Q, p.G1281C, p.R1320S, p.V1334D, p.V176SfsX28, p.G869EfsX4, IVS3+1G>T, IVS4-1G>C, IVS4-5T>G, IVS6+9A>G, and IVS9+5G>T. Reaction was performed using QuantiTect SYBR Green PCR Master Mix on an Applied Biosystems StepOne thermal cycler. Values of the threshold cycle were compared between normal and mutant alleles. RESULTS: Primers of all mutations were highly specific with absence of wild-type amplification. All the results were validated by direct DNA sequencing. CONCLUSIONS: This rapid and cost-efficient method allows wide mutation coverage, rendering the SYBR-green assay feasible and attractive for large-scale routine application.