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Along with the advances in cancer genomics and the development of targeted therapies, the field of molecular diagnostics has undergone rapid evolution to meet the growing needs associated with patient care. Here, we review the past, present, and possible future of molecular diagnostics, including technologies and testing principles, to provide a comprehensive landscape of molecular diagnostic technologies, testing platforms, and applications. This review is based on the US Food and Drug Administration publications, the National Comprehensive Cancer Network guidelines, and the peer-reviewed English literature published between 2003 and 2021. We conclude that molecular diagnostics has changed dramatically during the past two decades. Next-generation sequencing–based comprehensive genomic profiling has replaced single-gene/single-locus testing for simultaneous detection of mutations, copy number alterations, structural variants, and mutational signatures to facilitate cancer diagnosis, prognosis prediction, targeted therapies, and immunotherapies. Laboratory-developed tests and companion diagnostics approved by the US Food and Drug Administration both play important roles in cancer patient management. Keywords: cancer genomics, molecular diagnostics, past, present, future the reference list of included studies to identify other potentially Introduction useful studies. First, the authors screened the titles and abstracts,  Since the first human genome project was completed in 2003, and then the full texts for keywords, such as “cancer molecu- cancer medicine entered the Genomic Era. Molecular diag- lar diagnostic,” and “molecular technologies” to find those that nostics was incorporated in modern pathology and cancer were potentially suitable. The data extraction process focused work-up to identify genetic alterations associated with diag- on the information about relevant literature. nosis, prognosis, and treatment. Genomic medicine, including targeted therapy and immunotherapies, is becoming important The evolution of molecular diagnostic components of cancer treatment. Here, we review the past, present, and possible future of molecular diagnostics, aiming to methodologies provide a comprehensive summarization of molecular diagnos- Molecular diagnostics is a collection of techniques that can be tic technologies, testing platforms, and applications in cancer used in the context of various diseases to detect genetic alter- management. ations, to assist in diagnosis, classification, and progression pre-  diction, and to monitor treatment response. Retrieval strategy Molecular diagnostics techniques first began to be developed in research laboratories in the middle of last century. Recombinant Literature review was electronically performed using PubMed DNA and cDNA cloning were the technologies used to analyze database. English language and full-text articles published  gene sequences in the early phase of molecular diagnosis. Sanger between 2003 and 2021 were included in this non-systematic sequencing was created in 1977 and became the gold standard review. The authors searched the PubMed database to iden- for gene sequencing in clinical laboratories for several decades. tify relevant publications. The literature search strategy was Subsequently, polymerase chain reaction (PCR) was invented, conducted as follows (1) cancer, (2) molecular diagnostics, (3) and many associated methods were also developed. The first technologies, and (4) future development. The authors screened next-generation sequencing (NGS) instrument was developed in a 2000, while the launch of the Illumina HiSeq and ThermoFisher Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Ion Torrent sequencers in 2010 fully opened the door to large- Cancer Center, New York, NY, Department of Pathology and Laboratory Medicine, Mayo Clinic Hospital, Jacksonville, FL, USA. scale DNA sequencing. Subsequently, innovation focused on *Corresponding author: Jinjuan Yao, Department of Pathology and Laboratory long-read sequencing, also known as third-generation sequenc- Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. E-mail: ing. The Nanopore sequencer is a representative third-genera- Yaoj1@mskcc.org tion sequencer that may reshape the landscape of nucleic acid Copyright © 2022 The Chinese Medical Association, Published by Wolters  sequencing and molecular diagnostics in the near future. Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided Current molecular diagnostic methodologies it is properly cited. The work cannot be changed in any way or used commercially Sanger sequencing without permission from the journal. Journal of Bio-X Research (2022) 5:145–150 Sanger sequencing is also called chain termination sequencing. Received: 5 August 2021; Accepted: 27 October 2022 It was the most popular sequencing method before NGS became http://dx.doi.org/10.1097/JBR.0000000000000136 dominant and is still considered the gold standard. Frederick 145 REVIEW ARTICLE Journal of Bio-X Research Sanger won the 1980 Nobel Prize in Chemistry for developing phase of the PCR reaction, which is a more accurate reflection   this approach to nucleic acid sequencing. The sequencing reac- of the starting amount of template. Quantitative reverse-tran- tion includes primers, DNA polymerase, deoxynucleic acid, and scription PCR (qRT-PCR) is a combination of reverse tran- dideoxynucleic acid. In the process of sequence extension, dide- scription (RT) of RNA to complementary DNA (cDNA) and oxynucleic acid is randomly integrated, resulting in termination quantitative detection that is frequently used in gene expression  of sequence extension. The collection of fragments of various analysis. These methods use probes with fluorescent dyes lengths are then subjected to capillary electrophoresis, and the that are measured by an optical system; then, standard curves bases are read based on the dideoxynucleic acid fluorescence or comparative thresholds are used to calculate the number of  signals. copies in the starting material. qRT-PCR is widely used for min- Sanger sequencing can rapidly detect single-gene or single-lo- imal disease detection in chronic myeloid leukemia to monitor cus mutations, such as EGFR and KRAS mutations in lung cancer, the BCR::ABL1 fusion, in acute myeloid leukemia for NPM1 KIT and PDGFRA mutations in gastrointestinal stromal tumor. mutation, RUNX1::RUNX1T1 and PML::RARA fusions. However, it is time-consuming and low-throughput. In addition, the sensitivity is not optimal; when applied to cancer diagnosis, at Digital PCR least 50% of tumor content are required to be distinguished from the background and achieve a sensitivity of 25%. Digital PCR (dPCR) is a relatively new technique that is used Using locked nucleic acid probes can block sequencing of to directly quantify the amplified nucleic acid. In contrast wild-type fragments, thereby greatly increasing the limit of to traditional PCR, which amplifies the entire sample in a  detection to 1% or even lower. single reaction, dPCR amplification is partitioned such that thousands and millions of partitions are generated from a  single sample. The competitive inhibition that is normally Polymerase chain reaction a factor in a standard PCR reaction is decreased, and the PCR was invented in 1983 by Dr Mullis, the winner of the sensitivity of detection is improved. dPCR is widely used  1993 Nobel Prize in Chemistry. The reaction includes a dou- in absolute quantification, detection of copy number varia- ble-stranded DNA template, primers, nucleotides, and DNA tion, gene expression analysis, and mutation identification. polymerase. After denaturation of the double-stranded tem- Because of its high sensitivity, it is also used to analyze liquid plate, the primers bind and extend from the 5ʹ to the 3ʹ end. biopsy specimens. The newly generated copies of the DNA are used as templates for further replication so that the original template is amplified Next-generation sequencing exponentially in a chain reaction. The amplified product can be visualized directly by gel electrophoresis; subjected to frag- NGS was developed more than a decade ago with the aim of ment analysis and restriction fragment length polymorphism increasing detection capacity and capturing all genetic alter-  (RFLP) analyses based on the size of the PCR fragments ; or ations at the same time. In recent years, it has come to be widely subjected to melting curve analysis based on its dissociation applied in clinical settings, using many different sequencing   properties. The PCR products can also be sequenced by technologies or platforms. Sanger sequencing, pyrosequencing, single-base extension, or The most used platforms are the Illumina and Ion Torrent  NGS. Most of these methods incorporate fluorescent tags into Semiconductor sequencers. These two sequencers have differ- the PCR product that are detected by optical systems to iden- ent chemistries. Illumina detects fluorescence signals, while Ion tify the genomic changes. Torrent detects current change. In Illumina sequencing, the tem- Fragment analysis and RFLP are variations on the same tech- plates are copied on a flow cell using four differently colored flu- nique. Both involve PCR amplification of a template with fluo- orescently tagged deoxyribonucleotide triphosphates (dNTPs). rescently labeled primers followed by capillary electrophoresis During one round of reaction, only the base complementary to sort PCR products by fragment size (length). Fragment anal- to the template is incorporated into the sequencing primer or ysis allows for the rapid detection of small and medium-sized growing chain. The florescent base is excited by a laser, and its insertions and deletions (50 bases to hundreds of bases long), unique emission spectrum is captured by the built-in camera. The some of which could be challenging for other technologies to sequence of the template is determined based on the readout of detect or could be easily missed by massive parallel sequenc- the signals that occur at the same position in sequential pictures. ing. RFLP is a simple, rapid, and cost-effective way to detect In contrast, the Ion Torrent platform is not an optical system, the presence of single-nucleotide variants or methylation at a but rather uses current as a signal. The DNA library fragments given site using sequence-specific restriction enzymes that cut are clonally amplified on the surface of a bead. The sequence of PCR fragments based on the presence of specific palindromic the fragment in each bead is read in a semiconductor chip with sequences. micromachined wells that have an ion sensitive layer and an ion The examples of fragment analysis and RFLP in cancer sensor to detect the hydrogen ions that are released during the molecular diagnostics include the clonality studies of lympho- incorporation of the deoxyribonucleotide triphosphate into the mas, the detection of NPM1 and FLT3 mutations in acute template DNA. myeloid leukemia. In clinical laboratories, enrichment methods are used to select regions of interest for targeted sequencing. Two methods of enrichment that are currently used are hybrid capture and Quantitative PCR amplicon capture. These technologies can enhance the assay Quantitative PCR evolved from PCR. In comparison with PCR, sensitivity, lower cost, shorter turnaround time, and better sup- which detects the end-product of amplification, quantitative port for therapeutic decision-making and patient management,  PCR analyzes the number of DNA copies in the exponential in comparison with single gene assays. 146 Journal of Bio-X Research REVIEW ARTICLE cfDNA can also be obtained from cerebrospinal fluid, urine, and Table 1 other body fluids. A comparison of common molecular techniques The molecular diagnostic technologies described above are Variant types summarized in Table 1. Molecular techniques SNVs Small indels CNV SVs Sensitivity (%) Sanger sequencing ✓ ✓ 25 Future molecular diagnostic technologies fragment analysis and ± ✓ 5 RFLP Nanopore sequencing Allele-specific PCR 1–5 Nanopore sequencing technology was invented in 2014. qPCR ✓ ± ± <1 Currently, it is used in scientific research. Unlike other sequenc- qRT-PCR ✓ ✓ 0.001 dPCR ✓ ± <1 ing technologies that were already available in the clinical NGS-Amplicon capture ✓ ✓ ± ✓ 5–10 laboratories, Nanopore sequencing does not require PCR ampli- NGS-Hybridization capture ✓ ✓ ✓ ✓ 2–5 fication and can generate long reads (10–100kb), reduce cost NGS-liquid biopsy ✓ ✓ ± ✓ <1 and amplification errors, and improve de novo assembly and NGS-RNA sequencing ✓ ± ✓ 5 mapping quality. Nanopore sequencing devices are portable, ±=means may or may not be able to detect those variant types; or with limited ability of detection,  fast, and affordable. In the future, clinical use of this tech- CNV=copy number variation, NGS=next-generation sequencing, RFLP=restriction fragment length nique is expected to yield breakthroughs in germline mutation polymorphism, SNV=single-nucleotide variant, SV=structural variant. detection, virology, and gene fusion testing. In addition, because these devices are portable, it is expected that this technique will Targeted NGS assays are becoming an important integral also be used in the field at point of care. The disadvantages of part of cancer driver mutation detection, as well as the assess- Nanopore are its relatively low accuracy and precision, both of ment of microsatellite instability and tumor mutation burden.  which need to be improved before clinical use. RNA sequencing Single-cell sequencing RNA sequencing (RNA-seq) is a technique that detects gene Single-cell sequencing technology is also increasing in pop-  fusions and analyzes gene expression using NGS. Gene ularity. Single-cell DNA sequencing detects DNA sequences fusions, especially those involving the tyrosine kinase domain and genomic alterations at the level of individual cells, cap- of growth factors, are known driver mutations for many cancer tures information regarding spatial and temporal heterogeneity [18,19] types. These mutations are diagnostic, targetable, or both. within a given tumor, and provides information related to tumor However, clinical use of targeted DNA-seq is limited in that this  evolution, relapse, and metastasis. Single-cell sequencing of technique cannot detect all structural variants, because these RNA or epigenetic modifications further elucidates pheno- events commonly involve introns, which are too long to tile, typic changes by providing information about gene and protein contain unmappable repetitive elements, or have genomic break-  expression. Genomic, transcriptomic, and epigenetic informa- points in alternative introns that are not covered by the panel tion obtained at the level of the individual cell is becoming the design. RNA-seq can capture the junction of exons from the two basis for targeted molecular approaches in cancer therapy. fusion partner genes and offers a direct approach to detecting The disadvantage of single-cell sequencing is the requirement fusions. In addition, fusion genes may have higher expression for fresh or frozen tumor tissue to isolate individual tumor cells.  at the RNA level, which increases the sensitivity of RNA-seq. Its application to hematological diseases, especially myeloid Targeted RNA-seq typically involving using sequence-specific malignancies, is more extensive through the use of flow cytome- primers to known partner genes and universal primers for [25,26] try cell sorting and selection.  unknown genes to further increase the detection sensitivity. Clustered regularly interspaced short palindromic repeat Liquid biopsy/cell-free DNA assay technology Liquid biopsy, also known as a cell-free DNA (cfDNA) assay, Clustered regularly interspaced short palindromic repeat detects circulating tumor DNA (ctDNA) in the blood. In recent (CRISPR) gene editing technology is an emerging tool that is years, this technique has been developed for use in cancer mon- used for cancer mutation detection. Genetic alterations at the itoring, evaluation of drug response, diagnosis, and even early DNA and RNA levels are detected by using CAS12 and CAS13 detection. Liquid biopsy provides a comprehensive analysis [27–29] in combination with the detector and Sherlock technologies. of genomic alterations in both the primary tumor and distant  CRISPR technology can be used to shear wild-type alleles to metastases. It is much less invasive than tissue biopsy, and enrich the mutant allele, which, when followed by amplifica- samples can be collected multiple times through the disease tion and NGS, selectively improves the detection sensitivity for process, providing a dynamic picture of evolutions in genetic low-frequency mutations. cancer alterations. Depending on the design, liquid biopsy can CRISPR will be a valuable addition to clinical molecular be a single-gene assay or can involve a small or large panel of diagnostics because of its clean reads, stability, portability, and genes to detect single-nucleotide variations, small insertions and low cost. deletions, structural variants, and microsatellite instability. In cancer patients, the proportion of ctDNA to total cfDNA varies considerably and depends on tumor type, stage, and size. Some Machine learning and artificial intelligence tumor types are more prone to shed than others. Usually, the later the stage and the larger the size of the tumor, the higher Machine learning (ML) and artificial intelligence (AI) the proportion of ctDNA in the circulation is. Besides blood, approaches have been developed to infer tumor origin based 147 REVIEW ARTICLE Journal of Bio-X Research on large-panel sequencing data, including hotspot mutations, testing approaches cannot capture all relevant biomarkers insertions and deletions, focal or genome-wide copy number within the time frame needed for clinical management and alterations, structural variants, mutational signatures, and from limited tissue samples. Molecular diagnostics laboratories clinical parameters. Computational biologists from Memorial have therefore had to develop more comprehensive platforms Sloan Kettering (MSK) Cancer Center designed and trained an to capture all clinically indicated alterations. Foundation One algorithmic classifier using comprehensive genomic profiling and MSK-IMPACT, representative examples of large, targeted data from 7791 tumors representing 22 cancer types, gener- NGS panels, were launched in 2011 and 2014, respectively. ated during clinical prospective tumor sequencing, using the These two panels apply the pan-cancer strategy, testing all solid MSK-IMPACT (integrated mutation profiling of actionable tumors for a large panel of cancer genes, regardless of tumor cancer targets) targeted panel. They reported that the correct type and whether the tumor has known biomarkers. This type tumor type was predicted in 73.8% of the total training set of strategy has identified many patients with different cancer (5748/7791 patients) and in 74.1% of an independent cohort types who could potentially benefit from basket clinical trials. (8623/11,644 patients). The accurate prediction rate in plasma Since 2017, the FDA has approved three pan-cancer biomarkers cell-free DNA reached 75.0%. These findings indicate that AI and associated drugs based on basket clinical trials, including  technology can further enhance the utility of molecular test- high microsatellite instability and pembrolizumab, NTRK [41,42] ing by providing additional tumor origin information, espe- fusion and larotrectinib, high tumor mutation burden and [43,44] cially in those tumors that have been classified as cancer of pembrolizumab (Fig. 1). In addition, more pan-cancer bio- unknown primary based on histologic and immunophenotypic marker candidates are emerging biomarkers, including BRCA1/ [30,31]  features. BRCA2, PD-L1, RET, FGFR, and so on. Currently, molecular laboratories need to build up a portfo- lio of testing platforms to meet clinical requirements of being The evolution of molecular testing principles both rapid and comprehensive, which is not currently the case Molecular testing principles for solid tumors have evolved with for either single-gene or cancer panel testing. Therefore, an the discovery of cancer biomarkers and the development of cor- algorithm is occasionally needed for testing stratification, espe- responding treatments. cially in tumor types with many targets; for example, in lung In 1998, trastuzumab was approved to treat HER2-positive adenocarcinoma-single gene testing is used to identify EGFR,  metastatic breast cancer, which was the first targeted therapy ALK, or KRAS alterations, followed by NGS testing for the approved by the Food and Drug Administration (FDA). In 2004, detection of mutations, copy number and structural variants in treatment of lung adenocarcinomas with activating mutations other driver genes, as well as the assessment of microsatellite [33–35] in EGFR with gefitinib and erlotinib was reported. Since instability tumor mutation burden to complete the biomarker then, more tumor-specific genetic alterations and associated identification. drugs have been identified and approved, including the KRAS  resistance mutation and cetuximab in colorectal cancer and The role of pathologists in the evolution of  the BRAF V600E mutation and vemurafenib in melanoma. molecular diagnostics In addition, more biomarkers in the same tumor types continue to be identified, such as mutations in ALK, ROS1, KRAS, BRAF, Molecular genetic pathology was created as a joint subspecialty  RET, and many more in lung adenocarcinoma. of the American Board of Medical Genetics and Genomics Molecular diagnostics began as single-gene, single-platform together with the American Board of Pathology in 1999 to pro-  testing >20 years ago. However, with the expansion of cancer vide high-quality training for physicians in the rapidly expand-  biomarkers and targeted therapies, traditional low-throughput ing field of molecular diagnosis. Figure 1. FDA CDx approval and targeted treatment. ABL1=ABL Proto-Oncogene 1, BCR=B-cell receptor, CDK=cyclin dependent kinase, CDx=companion diagnostics, CMS=Centers for Medicare & Medicaid Services, EFGR=epidermal growth factor receptor, FDA=US Food and Drug Administration; FGFR: fibro- blast growth factor receptor, KRAS=Kirsten rat sarcoma virus, MET=mesenchymal epithelial transition factor receptor, MSI=microsatellite instability, MSK- IMPACT=Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets, NGS=next-generation sequencing, NTRK=neurotrophic tyrosine receptor kinase, NYS=New York State, RET=rearranged during transfection. 148 Journal of Bio-X Research REVIEW ARTICLE As an integral part of cancer diagnosis and treatment, pathol- clinically, validation of its accuracy, precision, sensitivity (lower ogy plays a critical role in patient care. Before the genomic era, limit of detection), and specificity are critical. To be approved as surgery, radiation, and chemotherapy were the three main treat- a companion diagnostic (CDx) by the FDA, the clinical validity ment options pursued based on histology and TNM staging. The and utility also need to be verified. Currently, the majority of incorporation of molecular diagnostic technologies and molecular clinical molecular assays are used as laboratory-developed tests, pathology into the field of pathology has brought cancer patients and only a small portion have received clearance or approval the hope of a cure by providing essential information regarding from FDA to serve as a CDx. Selected FDA-cleared or -approved genomic alterations to guide targeted and immune therapies. large-panel NGS platforms are summarized in Table 2. In addition to providing up-to-date training for molecular pathologists, the molecular genetic pathology subspecialty of Limitations pathology has been continuously evolving to incorporate new tests and new platforms that are both rapid and comprehensive The limitations of this review include: possible incomplete to cover all the indicated biomarkers in all tumor types and help retrieval of all relevant publications and bias of NGS-based guide clinical decision-making and selection of targeted thera- technologies due to the authors experience and expertise. pies or immunotherapies (Fig. 2). Based on results from compre- hensive genomic profiling, cancer patients can be grouped based The future of molecular diagnostics on their genomic alterations, rather than their tumor types or tissue origins. These patients will be managed with targeted In the past few years, the field of molecular diagnostics has therapy, immunotherapy, or chemoradiation. undergone rapid, substantial growth, and it will continue to In addition, clinical laboratories are required to meet local grow in the future. Accurate, sensitive, and rapid detection will  and/or federal regulations. For any assay to be applied help facilitate initial diagnosis and disease monitoring. The Figure 2. Comprehensive genomic profiling. MSI=microsatellite instability, TMB=tumor mutation burden. Table 2 FDA-approved large panel NGS testing platforms (selected list) Somatic/ Sample Platform Genes assessed FDA approval Year Mutations germline type MSK- IMPACT (Memorial Sloan Kettering) 505 Authorization 2017 SNVs, Indels and MSI Somatic FFPE FoundationOne CDX (Foundation Medicine) 324 Clearance 2017 SNVs, Indels, CNAs, gene fusions, MSI, and TMB Somatic FFPE Omics Core 468 Authorization 2019 SNVs, Indels, select CNAs and gene fusions, MSI and Somatic FFPE (NantHealth, Inc.) Whole exome TMB (whole exome) PGDx elio™ (Personal Genome Diagnostics) 505 Authorization 2019 SNVs, Indels, CNAs, gene fusions, MSI and TMB Somatic FFPE Guardant360® CDx (Guardant Health, Inc.) 74 Clearance 2020 SNVs (74), CNAs (18), fusion (6) Somatic Plasma FoundationOne® Liquid CDx (Foundation 311 Clearance 2020 SNVs and Indels (311), CNV (3), fusions (4) Somatic Plasma Medicine) Helix The Exome+ Whole exome Authorization 2021 SNVs and small indels Germline Saliva Assay ±=means may or may not be able to detect those variant types; or with limited ability of detection, CNV=copy number variation, FFPE=formalin-fixed, paraffin-embedded, MSI=microsatellite instability, NGS=next-generation sequencing, RFLP=restriction fragment length polymorphism, SNV=single-nucleotide variant, SV=structural variant, TMB=tumor mutation burden. 149 REVIEW ARTICLE Journal of Bio-X Research  Benayed R, Offin M, Mullaney K, et al. 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