Application of next-generation sequencing technology to precision medicine in cancer: joint consensus of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology

Introduction

Precision medicine, also referred to as personalized medicine, is an emerging concept in healthcare that adapts therapy based on the genome, physiology, and lifestyle of the patient. This method of disease management requires the incorporation of clinical and genetic data, enabling the clinicians to provide an efficient treatment course to attain a significant outcome^1,2. In the present era of personalized oncology therapy, a comprehensive analysis of cancer driver genes and their mutations underlying the pathophysiology of cancer development is crucial for designing the most appropriate treatment strategy for patients using targeted therapies³. Although Sanger sequencing⁴ is still being used as a gold standard in clinical applications, the more recently developed second-generation technology, namely the next-generation sequencing (NGS) technology, has superseded it owing to simpler usage and functional advantage^5–7.

Over the last decade, economic growth and changing lifestyle in China has led to an increase in the incidence of cancer and cancer-related mortality⁸. Continuous progressive research is being conducted in the areas of molecular targeted therapy, which is currently considered as a promising treatment. China has supported research initiatives directed towards progress in precision medicine, considering it as a new phase in health care. On September 17^th, 2015, the Chinese Society of Clinical Oncology (CSCO) convened a group of renowned oncologists, pathologists, biologists, and NGS experts in Xiamen to set up the China Actionable Genome Consortium (CAGC). The CAGC announced the start of the precision oncology initiative (CAGC-POI), and the framework and goals of this organization were laid out. According to the 2015 Chinese cancer report, the most commonly diagnosed cancers in China are carcinomas of the lung and bronchus, stomach, esophagus, liver, colorectum, and breast in the order listed. The overall national incidence of all cancer types in 2015 was approximately 4.29 million, and cancer-related mortality was 2.81 million people⁸. Therefore, there is a significant medical need for precision oncology for diagnosis and treatment in China.

Under the preliminary consensus on the clinical application of NGS, the CAGC-POI project stage I aims to utilize NGS technology to profile cancer driver genes for five malignant solid tumors, including lung cancer, breast cancer, hepatocellular carcinoma, gastric cancer, and colorectal cancer. On September 27th 2015, hematopoietic malignancy was included as the 6th tumor type after discussions between CSCO Chairman Professor Yi-long Wu, Academician Zhu Chen (President of Chinese Medical Association), and Academician Saijuan Chen, aiming to perform a more comprehensive mutational atlas study on leukemia. Under the consensus, the CAGC-POI will determine a set of optimized standard operating procedures (SOPs) and mutational atlases of driver gene mutations across these six cancers. This will guide the recruitment of patients with such mutations into precision medicine-related clinical trials. In the final version of the consensus, standards and compliance rules suitable for clinical oncology in China regarding NGS techniques, analysis tools, along with diagnosis and treatment models will be put forward, which will further fill the gap between NGS laboratory and clinical application, ultimately improving the health of cancer patients. The goal of CAGC-POI stage I is to establish a consensus on the application of NGS technology in clinical oncology, based on the advice and opinions from the expert group, which would serve as a guideline for oncology-related clinical and personal testing in the near future. Currently, there is an urgent need for a consensus based on the discussions from expert professionals in all related fields to guide the application of NGS in cancer diagnosis and precision medicine.

Overview of NGS technology

NGS technology, also referred to as massively parallel sequencing (MPS), is a parallel sequencing technology applied to specific samples obtained from patients with cancer, which has the ability to sequence billions of DNA base pairs in a single run.

NGS for cancer samples can range from targeted gene panels analyzing few thousand base cells to whole-exome sequencing (WES) and whole-genome sequencing (WGS) analyzing 40 to 50 million bases and 3.3 billion bases, respectively. The target panels in NGS offer a larger exon coverage, greater than that with WES and WGS applications. The targeted panel sequencing detects only the specific carcinogenic genes, while WES and WGS can identify unknown variants along with known mutations. WGS provides the most unbiased examination of the cancer genome, thereby paving the way for the discovery of previously unrecognizable mutations⁹.

Presently, NGS of targeted gene panels are very common in clinical practice for the purpose of finding targetable genomic alterations. Whole exome or genome sequencing are mainly used for research purposes. NGS of gene panels longer than one million megabases are under extensive validation for TMB (tumor mutational burden) estimation, aiming to serve as surrogate test for whole exome sequencing based TMB analysis. In the future, whole exome sequencing might be validated for the use of identifying functional neoantigens or other clinical biomarkers.

Although NGS technology has the advantage of a high-throughput workflow and can discover hereto unknown mutations^10,11, it also confronts many challenges such as technology, data management, data analysis, interpretation, reporting, and genetic consulting. Many publications and international consensus have reported the application of NGS in single-gene inheritance diseases and prenatal diagnostics^12–15; however, there has been no consensus on the application of NGS technology in clinical oncological practice, especially when used in cancer precision diagnosis and treatment to date¹⁶. With the widespread scope of tumor gene testing, NGS for precision diagnostics has gradually progressed from single-gene analysis to the profiling of several hundreds of genes. It is possible in the future that integrated information from the exome, transcriptome, whole genome, and epigenome will be adopted into clinical practice. Furthermore, as biotechnology is also evolving, it is foreseeable that the application of high-throughput NGS in clinical diagnosis and precision treatment will change perpetually in terms of testing technology, analysis tools, variant interpretation, etc., in the future, which will also bring various challenges to the practical application.

This technical consensus describes quality requirements, clinical tumor-related NGS testing content, sample processing, sequencing procedures, data management, informatics analysis, interpretation of reports, and consulting. This consensus also describes patient-informed consent and quality control of NGS tests, and adds a note on the differences in research and diagnostic NGS.

Quality requirements of NGS in cancer diagnosis

With evolving technology, it is of crucial importance to understand the current status of NGS technology in order to ensure a superior survival outcome and guarantee the well-being of the patient. A variety of NGS platforms are being used in different clinical laboratories. All laboratories, regardless of the platform used, should conduct NGS tests in accordance with the recommendations in this consensus and use them to validate the platform and associated analytical tools for clinical use.

Quality management (QM) plays a pivotal role in the standardization of NGS workflow by providing basic guidelines to ensure an advanced reproducibility of data and high turnover with reduced cost. Quality documentation containing procedural instructions and verified documents is the preliminary requirement to a good standardization method¹⁷, which improves the transparency and reliability of the results. One of the most important criteria for QM is quality assurance (QA). The QA program provides quality control (QC) methods for the predetermined checkpoints, such as contamination identification including initial sample check, fragmentation, library evaluation, error rate monitoring, and data analysis. These methods assist to confirm the formerly established performance status of a sample and indicate an error in case of any change in the status. The aforementioned QC features ensure that no sequence or sample data is used in the testing without meeting the established laboratory quality standards, and the QA procedure minimizes the risk of errors due to contamination. This is of central importance, especially in the case of a high or unlikely false-positive rate in assays or in detection of unknown agents^18,19. Many professional organizations have recommended the use of reference standards to minimize errors of inappropriate analysis leading to misdiagnosis by reducing bias associated with any method^18–25. These reference standards reflect a wide range of genomic features assessed by the NGS assay, advancing precision and reducing the systematic sequencing errors²⁶.

In case of multigene detection of tumors, a specific NGS test needs to be designed to clearly describe the gene status required for clinical diagnosis and treatment. NGS tests should be fully validated in terms of analysis and technological capabilities, before being used in clinical practice. Several factors affect the quality of NGS results: platform, target region enrichment, library preparation, amplification efficiency, sequencing data volume, bioinformatics analysis pipeline, etc. NGS testing results for specific mutation sites that have low data quality should be validated by other methods before clinical application. Experts performing the testing in laboratories and consultants should have adequate communication channels and informed consent from patients regarding the advantages and disadvantages of conducting a tumor NGS test. Simultaneously, clinicians should understand the medical implications of specific NGS tests.

NGS test content in clinical oncology

In routine clinical practice, along with the differential diagnosis suggested by the physician, a genetic test is recommended so as to obtain a confirmatory diagnosis with the laboratory reporting the wide range of genes analyzed, test methods used, and the performance parameters of every test. Therefore, for higher sensitivity and specificity, the initial testing is done with disease-targeted panels based on the relevant gene regions related to the disease. The referring physicians are recommended to provide detailed phenotypic data, such that the data can aid the laboratory in interpreting the results. In oncology, since a wide range of genomic variants exist within a single tumor type, or similar driver gene mutations are found among different kinds of tumors, the therapeutic strategies could be different for a single tumor, or a similar treatment could be applied to different tumor types^27,28. Thus, molecular classification of tumors based on specific genomic characteristics may move beyond traditional histopathological categorization and become the key step in cancer diagnosis and treatment²⁹. Owing to this genomic phenomenon, elaborate disease panel testing is recommended to track the disease phenotype and correlate it with the genotype. Only those genes with clear scientific evidence of clinical relevance should be included in the disease-targeted gene panel. In case of overlapping phenotype, laboratories should consult with the physicians to restrict the analysis to a subpanel related to sub-phenotype to reduce the number of less relevant variants. Further, during exome and genome sequencing, the laboratories should be aware of the commercially available reagents and refractory areas in the experiment design¹⁹.

In recent years, multi-genotyping of tumors has shown to have a significant impact on drug development strategies with two new designs of targeted-therapy clinical trials: umbrella trials and basket trials. The former involves the same histological cancer with different driver gene mutations that could be potentially treated with different targeted drugs; the latter involves the same genomic mutation found in different histological tumors that could potentially be treated with a single targeted drug^30,31. In both clinical practices and clinical trials, the content of an NGS test for genotyping requires meticulous design and verification of technical reliability. This content, including candidate genes, targeted testing regions, and actionable variants should be well defined within the NGS test.

The establishment and development of the “core gene list” should be patient-oriented, fully integrated with the precision medicine concept from clinical oncologists, and combined with proper application and operability. In lung cancer, the current National Comprehensive Cancer Network (NCCN), Domestic Lung Cancer Clinical Guidelines and National Health and Family Planning Commission Diagnosis and Treatment Norms suggest that some driver gene variants including EGFR mutations, KRAS mutations, ALK rearrangements (fusion), ROS1 rearrangements (fusion), variable shear variations in MET exon 14, MET copy number amplification, RET rearrangements (fusion), HER2 mutations or amplification, and BRAF mutations are the essential parts of the “core gene list”³². The other core genes include HER2, BRCA1, BRCA2, ESR1, and other genes in breast cancer³³; HER2, MSI-related genes like MLH1, MSH2, MSH6, PMS2, PDL1, SMAD4, STK11, APC and other genes for gastric cancer³⁴; TP53, IDH1, IDH2, FGF, KRAS for hepatocellular carcinoma^35,36 and KRAS, NRAS, BRAF and MSI related genes like MLH1, MSH2, MSH6, PMS2, etc. in colorectal and several other cancers³⁷. For hematological malignancies, BCR-ABL, PML-RARA, IDH1, KIT, FLT3, MYC, STAT3, STAT5B, and other potentially actionable or targetable genes should be included in the NGS test.

From the perspective of clinical oncology in the CAGC-POI project, test content (the gene panel and its genomic region) should not only include tumor-specific gene panels, but also a large and comprehensive gene panel. Moreover, because gene mutation profiles of solid tumors and hematologic malignancies are significantly different, comprehensive gene panels for these two different malignancies should be designed separately. “Actionable genes” refer to driver genes and associated upstream and downstream regulators to which the therapeutic drug may be implemented in order to inhibit tumor progression or regress the tumor. In addition, actionable gene variations can also be used to determine molecular subtypes in diagnosis or prognosis. The first category of “actionable gene” is referred to as Level 1 actionable alterations, which includes clinical genomic variants published in various international and domestic guidelines (CSCO, ASCO, NCCN, ESMO, etc.), printed in the drug labels, related to indications approved by the food and drug administration of the USA (FDA)/China Food and Drug Administration CFDA, and having definite molecular diagnostic or prognostic value. The second category, described as Level 2 actionable alterations, includes drug-related targets in ongoing phase 1–3 clinical trials, mutations as inclusion criteria in current or upcoming clinical trials, drug targets of other tumors or indications in international and domestic guidelines, and targets for adjuvant diagnosis or prognosis. In the NGS test, direct analysis of tumor specimens without leukocyte DNA control is accepted for Level 1, while matched leukocyte DNA control is strongly recommended for larger gene sets for Level 2 actionable alterations, or for large groups of targets ranging from hundreds of genes to the whole exome.

Verification of technical parameters is vital to ensure the accuracy of NGS testing results

All NGS measurement parameters should be explicitly stated. The batch and relevant characteristics of analysis specimens to be assayed should be recorded. Testing laboratories have the responsibility of maintaining a structured database to ensure the integrity of the testing platform, branch projects, and sample processing. Each sample should receive a unique identifier during the testing and analysis. During the verification of platform stability, all instruments and reagents should meet quality standards to ensure high accuracy and precision in the investigation of specific driver genes.

The following relevant parameters related to the technical verification process should be recorded: (i) sample-related patient information, unique identifiers, time and date of reception, storage, and processing; (ii) name, manufacturer, and batch number of reagents used in DNA/RNA processing and quality indicators (such as RIN, etc.); (iii) data analysis- and database management-related software (version number, analysis parameters), data-related parameters (data size, QC, etc.), database security, and backup status^{18,23,38–43}.

Sample preparation

A crucial step in the NGS test is the library construction with DNA or cDNA that is reverse-transcribed from RNA. Although fresh tissue specimens are generally preferred for clinical NGS testing, formalin-fixed paraffin-embedded (FFPE) specimens, tumor cytology specimens, and plasma (cell-free DNA/RNA) also can be used in NGS tests^44,45. NGS tests are performed on clinical specimens collected at critical time points during disease management. A multidisciplinary discussion should be undertaken and fully informed consent should be received to allow the use of NGS tests at multiple time points during treatment⁴⁴. The following methods are used for sample collection: (i) Fresh frozen tissues from surgery and biopsy: Snapping frozen tissue in liquid nitrogen is an ideal preservation method for fresh tissues. Otherwise the fresh tissues can be stored in liquid nitrogen or in a –80 °C freezer within 30 minutes after removal from the body to prevent the degradation of nucleic acids such as RNA. Alternatively, fresh tissues can also be stored in preservative reagents and transferred to a –80 °C freezer as soon as possible. The tumor content can be determined by frozen section staining⁴⁶; (ii) FFPE tissues: Handling of tissues should be performed according to standard pathological operating procedures. Tissues should be fixed in 10% neutral formalin solution within 30 minutes after excision, avoiding the usage of fixatives containing acidic or heavy metal ions. Large specimens should be trimmed to proper sizes and fixed for 6 to 48 hours, but not longer than 72 hours. Biopsy specimens should be fixed for 6 to 12 hours, and the tumor content should be determined by H&E staining before NGS testing^47–49; (iii) Cytology samples: the presence of tumor cells in pleural effusion and ascites samples must be confirmed before NGS testing. Samples meeting quality requirements by cytopathological examination can undergo nucleic acid extraction directly, or FFPE preparation for future use^50,51; (iv) Plasma or blood: cell-free/circulating tumor DNA (cf/ctDNA) is often found in plasma, and the proportion of tumor-derived DNA varies dramatically among distinct types and stages of cancers. Evidence indicates that ctDNA assays provide more reliable results at the time of disease progression. However, there is no evidence of clinical utility to suggest that ctDNA assays are useful for diagnosis of early-stage cancer or screening⁵². EDTA anticoagulation vacuum collection tubes can be used in blood sampling for cfDNA preparation. Plasma should be isolated within 2 hours after about 8–10 mL of whole blood is collected and transported under refrigerated condition, after which cfDNA is extracted. cfDNA should be stored either at –80 °C to avoid repeated freeze-thaw cycles or in the commercial cfDNA collection tubes, which can preserve cfDNA for 3 to 7 days at room temperature if peripheral blood samples need to be transported for a long time. The large amount of genomic DNA (gDNA) released from blood cell diminishes the relative frequency of ctDNA dramatically. Therefore, blood samples with obvious hemolytic signs are not suitable for ctDNA NGS testing. cfDNA samples with suspected gDNA contamination should be first determined by the size distribution analysis of nucleic acid fragments before NGS testing⁴⁵.

The evaluation of sample quality has a profound impact on the interpretation of results. Therefore, it is essential to keep a careful record of sample collection circumstances, transportation details, processing before analysis, and pathological assessment. The evaluation for sample integrity should include the tumor content, number of tumor cells, and processing and transportation carried out according to SOPs. Evaluation methods include visual inspection, microscopic observation, and nucleic acid concentration analysis of plasma samples. If tumor-cell–enriched regions are marked for nucleic acid extraction, then microdissection could be carried out whenever required^45,46.

NGS workflow

NGS testing is at present mainly used for driver gene sequencing in clinical cancer practice, which represents an important aspect of precise diagnosis and treatment of tumors. The advantages of NGS technology include its high-throughput workflow, mutation frequency analysis, and relative low cost. The disadvantages include lack of bioinformatics analysis software to meet the clinical application, the interpretation of genotypes depending on the accuracy of bioinformatics results, and the challenge of achieving the sequencing depth due to high overall cost^5,53–56. In terms of the application of NGS technology in cancer diagnosis and disease monitoring, laboratories should pay attention to reagent management, sample QA/QC, testing environment, personnel qualification and proficiency, and NGS standards for technical parameters (including library construction, sequencing process, and QC). SOPs should be applied to every NGS test to ensure robust and accurate NGS testing for specific tumors. All aspects of sequencing technology should have standard laboratory records to establish a structured database for management, which can track platform environmental parameters, reagent usage and performance, sample tracking, and quality control data. Laboratories must ensure the accuracy of testing by daily and periodic QC procedures⁵.

In order to meet the highest quality of clinical needs, the testing laboratory must provide a reasonable backup solution to each step of the workflow that is found to be abnormal or inadequate. Backup solutions could involve adopting another molecular diagnostic platform to detect important sites, validating positive results using other technologies, repeating the high-throughput sequencing process, or requesting new specimens, if this is a possibility.

NGS data generation, management, and bioinformatic analysis

Due to the tremendous volumes of NGS data, powerful computing platforms are needed to support data management, storage, and analysis. SOPs for data QC, bioinformatics analysis, I/O format, storage interfaces, and many other fields should exist. A structured database is needed to manage raw data, quality control data, and results^{53,55,58–63}.

NGS reporting and consulting

Due to the complexity of NGS to generate large volumes of information, the test report should be represented according to the following rules: a concise front page with the essential content, clear results, explicit interpretation for genomic alteration, and sufficient supporting evidence^{19,20,53,65,68,69}. The target range of driver genes should be clarified before NGS testing and included in the information to clinicians in the report. NGS reports should be concise and clear. Results with clinical significance should be prioritized, such as EGFR L858R mutation and mutant allele fraction (MAF) = 15.6%. Basic information of patients such as patient identity, clinical diagnosis and a brief description of testing technology should be included in the front page of the report. The report may contain supplementary material depending on the situation. The “core gene” testing results for each tumor type must be clearly reported. Other genes may be summarized based on these results, and specific reports may be made for particular mutations (e.g., core mutations found in other tumors).

It is recommended that variants are classified into one of the following categories: “pathogenic, likely pathogenic, uncertain significance, likely benign, benign.” Furthermore, it is strongly recommended that variations of unknown significance (VUS) or unclassified variants (UVs) are be recorded and shared among peers.

Genetic variant databases with valid scientific evidence should be used for variant classification, and these databases must meet the requirement of good quality or latest guidance.⁷⁰ This will facilitate future research in pinpointing their clinical value.⁶⁵ Due to the development of many NGS studies, FDA has approved several NGS-based multigene diagnostic services, including Oncomine Dx from Thermo Fisher, Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT) from Memorial Sloan Kettering (MSK), and Foundation One CDx from Foundation Medicine, Inc.(FMI).⁷¹ FMI reports not only the SNP, indel, rearrangement and copy number alterations (CNA) but also microsatellite instability (MSI) and TMB. Further studies and validations are needed to apply these biomarkers to the Chinese population.

It is recommended to integrate NGS data from different types of tumors into the same database, which will be helpful for the referencing of variation information in different tumor types for clinical applications. Therefore, we recommend the format and content of the report to be standardized, while providing enough flexibility to adapt to the development trend in the short term (updates regarding actionable genes and testing content). Appendix 1 is an example of a clinical tumor NGS test report. The report should include patient ID, disease information, sample information, testing methodology information, main results, interpretation of results, and the signature of the operator and the lab expert (molecular oncologist or genetics specialist). The reportable range should be based on NGS testing purposes and qualified detection regions. For example, it is not necessary to report all the genomic alterations by whole-exome sequencing (WES) for lung cancer. However, it is necessary to inform clinicians that there may be potential “non-attended region” variations. The NGS test report should be concise, clear, and understandable, and it should provide the complete necessary information for the clinician to make decisions about the current clinical target-therapy strategy. The interpretation of results must be objective and unbiased, and the genomic variants associated with the tumor should be clearly described. The report should not only describe the curative or predictive correlations from previous studies regarding disease correlation, but also suggest the no-treatment recommendation. Only clinicians or the multidisciplinary team (MDT) can make clinical decisions based on the NGS testing results. Multidisciplinary team discussions are required for genetic counseling, and multiparty communication among the experts in laboratory, cancer genetics, molecular biology, bioinformatics, and pathology is essential for the final clinical decision.

Informed consent

Informed consent form must contain: (1) The basis for testing, including medical guidelines, expert consensus, treatment norms and other content, to inform patients of the significance and purpose of the testing; (2) The conception that the testing process may have limitations, such as the sample volume sufficient for DNA/RNA extraction and failure of test due to bad sample quality. If such a situation occurs to the patient, the patient and family members should be informed as soon as possible, and next steps should be discussed; (3) Gene sequencing can assist in personalized therapy; however, the final decision must be made by the clinician; (4) Testing results only refer to the corresponding samples submitted; (5) The patient’s ID, clinical information, and test results must be kept confidential by the testing institution. Patients must be aware of the above agreement terms and sign informed consent forms before testing^20,72–74.

Differences between research and diagnostic NGS tests

Because of the rapid technology development and improvement of experimental abilities, the boundaries between research NGS and diagnostic NGS have been blurred. However, it should be made explicit whether a specific NGS test is used for research or diagnosis in practical applications. The purpose of diagnostic NGS is to answer the question of whether a patient’s DNA contains a specific genetic variation in the tumor (actionable variation). Normally, only a certified laboratory is qualified to perform diagnostic NGS tests. For hypothesis-driven research purposes, research NGS tests only provide limited or uncertain clinical significance for enrolled patients. The massive quantities of data obtained from WES or WGS not only serve for tumor diagnosis, but also generate new research hypotheses²⁰.

CAGC-POI project NGS test supervision and management

To ensure the same quality and continuity, the CSCO CAGC expert group shall supervise all NGS projects in CAGC-POI clinical practice, clinical trial, and research.

This will entail on-site inspections, document management, process monitoring, and external evaluation of testing samples to ensure that every NGS laboratory executing the CAGC-POI projects is at the highest quality standards.