Current practices and guidelines for clinical next-generation sequencing oncology testing

:::info Cancer Biol Med (IF: 4.25; Q2). 2016 Mar;13(1):3-11. doi: 10.28092/j.issn.2095-3941.2016.0004. :::

Abstract

Next-generation sequencing (NGS) has been rapidly integrated into molecular pathology, dramatically increasing the breadth genomic of information available to oncologists and their patients. This review will explore the ways in which this new technology is currently applied to bolster care for patients with solid tumors and hematological malignancies, focusing on practices and guidelines for assessing the technical validity and clinical utility of DNA variants identified during clinical NGS oncology testing.
下一代测序（NGS）已迅速融入分子病理学，极大地增加了肿瘤学家及其患者可获得的基因组信息的广度。本综述将探讨这项新技术目前应用于支持实体瘤和血液系统恶性肿瘤患者护理的方式，重点关注于评估临床NGS肿瘤学检测中DNA变体的技术有效性和临床实用性的实践和指南。
Keywords: Cancer genomics, next-generation sequencing, molecular diagnostics

Introduction

Massively parallel sequencing of nucleic acids enables DNA and RNA analysis on a grand scale. A natural implementation of this “next-generation sequencing (NGS)” technology is to assess the unique and complex set of genomic alterations that occur in malignant neoplasms, with the goal of improving patient care through personalized diagnosis, prognosis, and therapy.
大规模的核酸平行测序使DNA和RNA分析得以大规模进行。这种“下一代测序（NGS）”技术的自然实现是评估恶性肿瘤中发生的一组独特而复杂的基因组改变，目标是通过个性化诊断、预后和治疗改善患者护理。
The most prevalent current implementation of NGS for oncology is mutation detection via targeted panels1-5. These assays use molecular methods such as multiplex polymerase chain reactions (PCR) to isolate clinically relevant segments of the genome, such as mutation hotspots or coding exons of entire genes. These panels range from a few hundred target loci to many thousands. In these assays, raw sequence reads are first aligned to the reference human genome. Variant calling is then performed to identify small mismatches in these alignments which may represent mutations present in the specimen. Variant analysis and interpretation must then be performed to assess the technical validity and clinical utility of each variant (Figure 1).
目前，NGS在肿瘤学中最普遍的应用是通过靶向面板进行突变检测。这些检测使用多重聚合酶链式反应（PCR）等分子方法分离基因组的临床相关片段，如突变热点或整个基因的编码外显子。这些面板的范围从几百个目标位点到数千个。在这些分析中，原始序列首先与参考人类基因组对齐。然后进行变异调用，以识别这些比对中可能代表样本中存在的突变的小错配。然后必须进行变异分析和解释，以评估每个变异的技术有效性和临床效用（图1）。

Before any variant analysis is performed, the data must be checked for overall assay performance and quality. As many surgical pathology specimens have limited tissue amounts and quality, and have been treated with formalin fixation, assay failures occur with some regularity. As there are many different ways to measure assay performance, each assay requires a unique set of parameters established during validation6,7.
在进行任何变异分析之前，必须检查数据的总体分析性能和质量。由于许多外科病理标本的组织数量和质量有限，并且已经用福尔马林固定治疗，因此检测失败的发生具有一定的规律性。由于有许多不同的方法来测量分析性能，每个分析都需要在验证期间建立一组独特的参数。
Technical validity and clinical utility are the two major issues that must be resolved for every variant identified via NGS somatic variant detection. If a detected variant fails to meet or exceed predetermined validity and utility criteria, it should not be clinically reported as medically relevant. While simple in principal, there exists deep complexity in these areas which merit close consideration.
技术有效性和临床实用性是通过NGS体细胞变异检测确定的每个变异必须解决的两个主要问题。如果检测到的变异未能达到或超过预定的有效性和实用性标准，则不应将其作为医学相关的临床报告。虽然原则上很简单，但这些领域存在着深刻的复杂性，值得仔细考虑。

Terminology

NGS introduces a new suite of vocabulary into the clinical lab, with some specific to oncology testing. The following glossary is provided as reference and to facilitate further discussion in this review.
NGS在临床实验室中引入了一套新的词汇，其中一些专门用于肿瘤检测。以下术语表仅供参考，以便于本次审查中的进一步讨论。

Genomic analyst (GA)

GA is typically a doctorate or masters level specialist who performs some or all of the initial review of assay quality, technical validity assessment of detected variants, clinical utility assessment of variants, and generating draft reports. Final reports must be signed out by either a board certified MD clinical pathologist or PhD with clinical laboratory board certification.
GA通常是一名博士或硕士级别的专家，负责对分析质量进行部分或全部初步审查，对检测到的变异进行技术有效性评估，对变异进行临床效用评估，并生成报告草稿。最终报告必须由委员会认证的MD临床病理学家或具有临床实验室委员会认证的博士签署。

Base call quality score (Q score)

Q score is a PHRED-scaled probability ranging from 0-20 inversely proportional to the probability that a single sequenced base is correct. For example, a T base call with Q of 20 is considered likely correct with a confidence P-value of 0.01. Any base call with Q<20 should be considered low quality, and any variant identified where a substantial proportion of reads supporting the variant are of low quality should be considered potentially false positive.
Q分数是一个分段标度概率，范围为0-20，与单个序列碱基正确的概率成反比。例如，Q为20的T-base调用被认为可能是正确的，置信P值为0.01。Q<20的任何基本调用都应被视为低质量调用，如果支持该变体的大部分reads都是低质量的，则任何识别出的变体都应被视为潜在的假阳性。

Read depth

Read depth (or coverage, conventionally a number followed by “×”) is the number of independent reads with overlapping alignment at a locus of interest. This is often expressed as an average or percentage exceeding a cutoff over a set of intervals (such as exons, genes, or panels). For example, a clinical report might say that a panel average coverage is 1,105× with 98% of targeted bases covered >100×.
测序深度（或覆盖率，通常是一个数字后跟“×”）是在感兴趣的基因位点上重叠对齐的独立reads。这通常表示为在一组时间间隔（如外显子、基因或面板）内超过临界值的平均值或百分比。例如，一份临床报告可能会说，一个小组的平均覆盖率为1105×，其中98%目标碱基覆盖率>100×。

Mutations

Mutations are events that result in changes of genomic DNA. Variants are deviations from the human genome reference sequence observed in a specimen. The goal of NGS cancer panel testing is to identify variants which are extremely likely to be caused by somatic mutation. The difference is subtle; the two terms are often used interchangeably.
突变是导致基因组DNA变化的事件。变异是在样本中观察到与人类基因组参考序列的偏差。NGS癌症小组测试的目标是识别极有可能由体细胞突变引起的变异。差别是微妙的；这两个术语经常互换使用。

Variant read number (variant reads)

Variant reads is the number of independent sequence reads supporting the presence of a variant. Due to the high error rate of NGS at the per-base call level, calls supported by fewer than 5 variant reads are typically considered to be likely false positive calls.
突变reads是支持变体存在的独立序列reads数。由于NGS在每个碱基call水平的错误率很高，由少于5个突变reads支持的calls通常被认为可能是假阳性。

Variant allele frequency (VAF)

VAF is the percentage of sequence reads observed matching a specific DNA variant divided by the overall coverage at that locus. Because NGS provides a near random sample, VAF is thus a surrogate measure of the proportion of DNA molecules in the original specimen carrying the variant. For constitutional genetic testing, VAF is a measure of diploid zygosity: heterozygous loci should be near 50% VAF, homozygous loci should be near 100%, and reference loci should be near 0%. Deviations from these three expected values should be considered suspicious as potential errors due to incorrect base calls or alignment. For somatic testing, contamination from normal cells and tumor heterogeneity combine to cause unpredictable VAFs. In some clinical scenarios, such as testing a patient for therapeutic resistance mutations, the desired sensitivity dictates that variants with low VAF be included in downstream analysis. This all means that in order to maintain an acceptable level of sensitivity, variant filtering for somatic variant must be highly permissive from a technical validity perspective.
VAF是观察到的与特定DNA变体匹配的序列读取百分比除以该位点的总覆盖率。由于NGS提供了一个近乎随机的样本，因此VAF是携带该变体的原始样本中DNA分子比例的替代指标。对于体质基因检测，VAF是衡量二倍体合子性的指标：杂合子位点应接近50%VAF，纯合子位点应接近100%，参考位点应接近0%。与这三个预期值的偏差应被视为可疑的，因为不正确的基本调用或对齐可能会导致错误。在体细胞检测中，来自正常细胞的污染和肿瘤异质性共同导致不可预测的VAF。在某些临床情况下，如检测患者的治疗性耐药突变，预期的敏感性要求将VAF低的变异纳入下游分析。这都意味着，为了保持可接受的灵敏度水平，从技术有效性的角度来看，对体细胞变异的变异筛选必须是高度容许的。

Variant quality scores (QUAL)

QUAL are generated during the variant calling step and a requisite component of the Variant Call File (VCF). Generally speaking, QUAL scores are transformed log-scaled (PHRED) values where, for example, a score of 90 supports the variant call with a P-value of 1×10-9. It is critical to keep in mind that there are significant multiple testing considerations due to the large number of variants that are detected by NGS. The scale for QUAL values vary widely and depend on assay platform, capture method, and variant calling software. As such, it is strongly recommended at this time that each laboratory performing NGS independently assess the performance characteristics of QUAL scores for each assay, preferably by orthogonal testing.
QUAL是在变量调用步骤和变量调用文件（VCF）的必要组件期间生成的。一般来说，QUAL分数是经过转换的对数标度（PHRED）值，例如，90分支持P值为1×10-9的变量调用。关键是要记住，由于NGS检测到大量变体，因此存在大量的多重测试考虑因素。QUAL值的范围变化很大，取决于分析平台、捕获方法和变量调用软件。因此，强烈建议此时每个进行NGS的实验室独立评估每个分析的QUAL分数的性能特征，最好通过正交试验。

Strand bias (SB)

SB is a measure of how far the observed variant reads deviate from an expectation of equal likelihood of sequencing the plus and minus strands. A high SB score indicates that the variant call may be caused by an artifact of the alignment process rather than a true mutation8. Certain targeting methodologies, including those using Illumina TruSight capture techniques selectively amplify one target strand9; for these assays, SB is not a meaningful measure of quality.
SB是一种测量所观察到的变异读到多少偏离预期的同等可能性测序正负链。较高的SB得分表明变异调用可能是由对齐过程的人为因素引起的，而不是真正的突变。某些靶向方法，包括使用Illumina TruSight捕获技术的方法，有选择地放大一个目标链9;对于这些检测，SB不是一个有意义的质量测量。

Technical validity

To date, there are no NGS oncology in vitro diagnostics (IVD) cleared by the United States Food and Drug Administration (FDA). As such, all current clinically validated NGS oncology assays are classified as “laboratory developed” tests (LDT) which can vary highly in content and quality from lab to lab10. In the US, LDTs must be performed in laboratories with Clinical Laboratory Improvement Amendments (CLIA) licensure and are subject to state and federal regulations. The New York State Department of Health has issued guidelines for NGS oncology testing, setting a very high bar for clinical validation and ongoing assay performance (Table 1)11.
Table1 Summary of valuable references and guidelines relevant to clinical NGS oncology testing

For labs without New York approval, there are no matching federal guidelines at this time. As such, it is strongly recommended to prefer laboratories with optional College of American Pathology (CAP) accreditation. The major benefit of CAP accreditation comes from proficiency testing participation, wherein laboratories performing similar assays are given identical reference materials for comparison of results. This proficiency testing gives labs a mechanism and incentives to continually improve their test methodologies by direct comparison with others (Table 1)12-16.
Orthogonal confirmatory testing can validate that detected variants is a direct means of assuring technical validity. This requires a laboratory to have access to duplicate sequencing of entire panels using alternate methodologies (e.g. sequencing using both Life Technologies and Illumina platforms for every specimen), or to have a single-locus test of equivalent sensitivity for every targeted interval. As neither approach is simple or inexpensive to implement, many laboratories eschew such confirmatory testing during routine analysis. While PCR-based dideoxy sequencing (“Sanger” sequencing) is a generally accepted method of confirmatory testing for constitutional clinical NGS17-20, its sensitivity is limited to approximately 10%-20% VAF. As cancer NGS testing routinely identifies clinically relevant mutations from 5%-10% VAF, it Sanger is not an ideal method for confirmation for all variants.
正交验证试验可以验证检测到的变异是确保技术有效性的直接手段。这要求实验室能够使用替代方法（例如，使用Life Technologies和Illumina平台对每个样本进行测序）对整个面板进行重复测序，或者对每个目标间隔进行等效灵敏度的单位点测试。由于这两种方法都不简单或成本低廉，许多实验室在常规分析过程中避免了此类验证性测试。虽然基于PCR的双脱氧测序（“Sanger”测序）是公认的体质性临床NGS验证性检测方法，但其敏感性仅限于约10%-20%的VAF。由于癌症NGS检测通常识别5%-10%VAF的临床相关突变，因此它不是确认所有变异的理想方法。
Manual review of aligned reads in a genome browser (“variant inspection”) can greatly reduce the risk of false positive or incorrect results (Figure 2). While variant calling software has become increasingly sophisticated, it still cannot yet perform as well as a GA when it comes to pattern recognition. For cancer NGS, it is best to think of the role of variant calling software as to alert the GA to loci of interest. From there, it is the job of the GA to assess first whether any variant is present and if the variant called is the correct change. In some cases, variant inspection will reject dubious variant calls which have received a high QUAL score; this is the necessary cost of setting high sensitivity. In other cases, the variant observed will differ from that called. Complex insertion/deletion events are especially susceptible to this type of error. Given how common this mutation type is in certain tumors, laboratories must be prepared for this issue to be routine rather than exceptional.
在基因组浏览器中手动检查对齐读取（“变体检查”）可以大大降低假阳性或错误结果的风险（图2）。虽然变体调用软件已经变得越来越复杂，但在模式识别方面，它的性能仍然不如GA。对于癌症NGS，最好考虑变体调用软件的作用，即提醒GA注意感兴趣的位点。在此基础上，遗传算法的工作是首先评估是否存在任何变体，以及调用的变体是否是正确的更改。在某些情况下，变体检查将拒绝收到高质量分数的可疑变体呼叫；这是设置高灵敏度的必要成本。在其他情况下，观察到的变体将不同于所称的变体。复杂的插入/删除事件尤其容易受到此类错误的影响。考虑到这种突变类型在某些肿瘤中是多么常见，实验室必须为这一问题做好常规而非例外的准备。

There are many metrics that can be used to assess if a variant call is likely a true positive result. VAF, QUAL, SB, and other metrics may be available depending on the variant callers used (Figure 1). As each LDT NGS assay performs differently, it is necessary to establish filtering criteria anew for all new tests. If hard cutoffs are to be used to filter out low quality variants, a rigorous validation should be performed, including accuracy and limit of detection experiments to determine and test the cutoffs. Alternatively, very inclusive filters can be set to minimize false negative risk. This will increase the burden of confirmatory analysis and/or testing required for each case. Both options can be rate-limiting for small laboratories.
During the validation phase of assay development, it is valuable to test assay performance using reference materials. DNA or false FFPE blocks from positive control cell lines are commercially available for many of the most recurrent mutations. Some reference materials now available contain a variety of mutations with different VAF, genes, and mutation types. These are designed to be run during validation and/or during routine testing to ensure ongoing assay performance. Acrometrix (a division of Thermo Fisher Sciences Inc. Waltham, MA, United States), and Horizon Diagnostics (a division of Horizon Discovery Group plc. Waterbeach, United Kingdom) are two commercial vendors specializing in this area.

Clinical validity

It is tempting to think of tumors as “gene positive” or “gene negative”. This is a convenient way to discuss certain classical oncogenic changes such as fusion genes, but it glosses over a significant layer of complexity we are only now beginning to understand. For example, some mutations in EGFR render a tumor more susceptible to targeted tyrosine kinase inhibitor (TKI) therapy; other mutations confer resistance to the same21-24. Certain KRAS mutations are known to be targetable driver mutations while others are commonly observed but seem to have little to no impact on drug response (Figure 1)1,25,26. These now classic examples are the vanguard of a new way of thinking about cancer genes and mutations.
For each detected variant, there are multiple different types of considerations a GA should weigh.
人们很容易将肿瘤视为“基因阳性”或“基因阴性”。这是讨论某些经典致癌变化（如融合基因）的便捷方式，但它掩盖了我们现在才开始理解的一个重要的复杂层面。例如，EGFR的一些突变使肿瘤更容易接受靶向酪氨酸激酶抑制剂（TKI）治疗；其他突变赋予对相同的抗性。已知某些KRAS突变是靶向驱动突变，而其他KRAS突变通常被观察到，但似乎对药物反应几乎没有影响（图1）。这些现在的经典例子是思考癌症基因和突变的新方法的先锋。

对于每个检测到的变体，遗传算法应该权衡多种不同类型的考虑因素。

Somatic vs. germline

If normal tissue is available and tested, variant subtraction can be used to directly assess which DNA variants arose in somatically in the tumor. As this is not always feasible (consider hematological malignancies, where blood specimens contain neoplastic cells) or practical, indirect measures should be taken to avoid reporting benign germline variants as relevant for oncology. The front-line tool for indirectly filtering out germline variants is population allele frequency. Any variant that is present on >1% of normal human chromosomes is almost certainly not clinically relevant for cancer. VAF databases such as the Exome Aggregation Consortium (ExAC)27 or dbSNP (which uses 1,000 genome project allele frequencies)28 should be used to perform this filtering. As they match expectations for heterozygous and homozyous frequencies respectively, variants with VAF at nearly 50% or 100% should be considered potentially germline during analysis. Hard filtering for VAF however is not recommended as true mutations can certainly be observed at low VAF.
如果正常组织可用并经过检测，变异减影法可用于直接评估肿瘤中哪些DNA变异是在体细胞中产生的。由于这并不总是可行（考虑血液恶性肿瘤，血液样本中含有肿瘤细胞）或实用，因此应采取间接措施，避免报告与肿瘤相关的良性种系变异。间接筛选种系变异的前线工具是群体等位基因频率。任何存在于超过1%正常人类染色体上的变异几乎肯定与癌症无关。VAF数据库，如外显子组聚合联合会（ExAC）或dbSNP（使用1000个基因组计划等位基因频率），应用于执行该过滤。由于它们分别符合杂合子和纯合子频率的预期，在分析过程中，VAF接近50%或100%的变异应被视为潜在的种系。然而，不建议对VAF进行硬筛选，因为在低VAF时肯定可以观察到真正的突变。

Tumor type

Many mutations impact different tumor types in different ways, or have only been studied rigorously in a specific set of tumor types but are recurrently observed in other types. This means that variant annotation and interpretation procedures must take tumor type into account prior to rendering a clinical report. Resources such as COSMIC, The Cancer Genome Atlas (TCGA), and MyCancerGenome.org (MCG) have publically available databases with tumor-type specific mutation information and should be referred to when performing variant analysis. Literature review may be necessary for are mutations.
许多突变以不同的方式影响不同的肿瘤类型，或者仅在特定的肿瘤类型中严格地研究，但在其他类型中经常观察到。这意味着在提升临床报告之前必须考虑肿瘤类型的变体注释和解释程序。诸如宇宙，癌症基因组Atlas（TCGA）和MyCancergenome.org（MCG）等资源具有肿瘤型特定突变信息的公开可用数据库，并且在进行变体分析时应被提及。文献综述可能是突变所必需的。

VAF and tumor cellularity

If the VAF and percent tumor cell prediction from pathology are highly discordant, it could indicate an invalid or unreportable variant. If the VAF is far higher than expected, it could indicate that the variant is either germline or in a region of loss of heterozygosity (LoH). If the variant is in a gene where LoH is not anticipated (e.g. an oncogene where activating mutations are the mechanism), germline may be the more likely explanation. A VAF far lower than expected could indicate either false positivity or low level tumor heterogeneity. If the technical validity of a lower than expected VAF variant is very strong, tumor heterogeneity is more likely. If this is the case, the relevance of the mutation may depend on contextual considerations such as mutation type, tumor type, and patient treatment history. For example, the observation of a low relative VAF of a known recurrent acquired resistance mutation is of much stronger clinical validity in a treated patient than the same observation in a treatment naive patient.
如果VAF和病理学预测的肿瘤细胞百分比高度不一致，则可能表明存在无效或不可移植的变异。如果VAF远高于预期，则可能表明该变体是种系或杂合性缺失（LoH）区域。如果该变异存在于一个不存在LoH的基因中（例如一个致癌基因，激活突变是其机制），那么可能是种系细胞较多。VAF远低于预期可能表明假阳性或低水平的肿瘤异质性。如果低于预期的VAF变体的技术有效性非常强，则肿瘤异质性更可能存在。如果是这种情况，突变的相关性可能取决于背景因素，如突变类型、肿瘤类型和患者治疗史。例如，对已知复发性获得性耐药突变的低相对VAF的观察在接受治疗的患者中比在未接受治疗的患者中的相同观察具有更强的临床有效性。
It is also important to remember that percent tumor cell prediction, especially surgical pathology/histology, are essentially qualitative assessments never intended to have highly accurate or precise measurements. This is not an indictment of pathologists, rather a statement of fact that the methods used are not high enough resolution to be used in this way. Genomic analysts, who may or may not have any training in anatomic pathology, should be aware that these assessments may be off by as much as 10%-20% when performed by the best practitioners. Flow cytometry assessment of hematological malignancies should be a much more precise and accurate quantitation, though the correlation between flow cytometry results and NGS VAFs is as yet poorly understood.
同样重要的是要记住，肿瘤细胞百分比预测，尤其是外科病理学/组织学，本质上是定性评估，从来没有打算进行高度准确或精确的测量。这并不是对病理学家的指控，而是一种事实陈述，即所用方法的分辨率不够高，无法以这种方式使用。基因组分析员可能接受过解剖病理学方面的任何培训，也可能没有接受过任何培训，他们应该知道，如果由最好的从业者进行评估，这些评估可能会偏离10%-20%。流式细胞术对血液系统恶性肿瘤的评估应该是更精确和准确的定量，尽管流式细胞术结果与NGS VAFs之间的相关性尚不清楚。

Highly recurrent mutations

The observation of certain mutations, even at borderline technical quality, represents a “smoking gun” and must be reported. Examples such as BRAF p.V600E and KRAS codon 12/13 mutations should come to mind immediately for experienced cancer genomic analysts. These variants must be reported.
对某些突变的观察，即使是在技术质量的边缘，也代表了“确凿的证据”，必须予以报告。对于有经验的癌症基因组分析人员来说，BRAF p.V600E和KRAS 12/13密码子突变等例子应该立即出现在脑海中。必须报告这些变异。

Clinically significant mutation types

In-frame insertions/deletions (indels) and loss of function (LoF) mutations are two mutation types which can present as apparently novel variants at the DNA level despite having well-known implications. Examples include in-frame indels in EGFR exon 19 or LoF mutations in TP53. As practically all genomic analysis software solutions perform comparisons at the DNA (or predicted mRNA) level, this pattern can cause patient variants to be incorrectly classified as of uncertain clinical significance. Until such a times as more sophisticated software tools are available, it is the role of the genomic analyst to determine whether apparently novel variants are in fact of known clinical significance.
框架内插入/删除（indels）和功能丧失（LoF）突变是两种突变类型，尽管具有众所周知的含义，但它们在DNA水平上仍可以表现为明显的新变体。例子包括EGFR外显子19的框内indels或TP53的LoF突变。由于几乎所有基因组分析软件解决方案都在DNA（或预测的mRNA）水平上进行比较，这种模式可能会导致患者变异被错误地归类为具有不确定的临床意义。在出现更复杂的软件工具之前，基因组分析员的职责是确定明显的新变种是否具有已知的临床意义。
This area can become quite complex. For example, acute myeloid leukemia (AML) patients positive for biallelic LoF mutations in CEBPA have a favorable prognosis only if one mutation is in the C terminal region and the other is in the N terminal region29. This means that a specific mutation seen alone should be considered of uncertain significance, whereas that same mutation in conjunction with a second mutation would be clinically relevant to prognosis. This pattern can be challenging to convey in standard databases, requiring either highly specialized software or manual review by experts.
这一领域可能变得相当复杂。例如，CEBPA双等位基因LoF突变阳性的急性髓系白血病（AML）患者只有在一个突变位于C末端区域，另一个位于N末端区域时，才会有良好的预后。这意味着单独观察到的特定突变应被认为具有不确定的意义，而同一突变与第二个突变的结合将与临床预后相关。这种模式很难在标准数据库中表达，需要高度专业化的软件或专家手动审查。

Variants of uncertain clinical significance (VUS)

The number of VUS identified in a given specimen closely correlates with the scale of targets analyzed. As more genes or hotspots are added to tests, more VUS will be observed. Whether and how to report VUS is highly laboratory and test dependent: some labs will always report all VUS, some will never report any, and some will report only when specific conditions are met. The ordering oncologist should be aware of VUS reporting policies and utilize the test that best addresses the clinical needs of their patient. Contacting a lab directly is the best way to ascertain VUS reporting policies; if no such policy exists or cannot be readily ascertained, it may be wise to consider a provider with better clarity. However, due to a lack of evidence supporting the clinical utility of VUS, reporting of VUS should not be considered a necessary component of tumor sequencing analysis.
在给定的样本中识别VUS的数量与分析的目标的规模密切相关。随着更多的基因或热点被添加到测试中，更多的VUS将被观察到。是否报告VUS以及如何报告VUS高度依赖于实验室和测试:一些实验室总是报告所有的VUS，一些永远不会报告任何VUS，还有一些只在满足特定条件时才报告。肿瘤科医生应该了解VUS报告政策，并利用最能满足患者临床需求的测试。直接联系实验室是确定VUS报告政策的最好方法;如果不存在这样的政策或不能很容易地确定，则明智的做法是考虑更清楚的提供者。然而，由于缺乏支持VUS临床应用的证据，VUS的报道不应被认为是肿瘤测序分析的必要组成部分。

Integration of cytogenetics information

There are clinically significant cytogenetic abnormalities or nearly all major malignancies routinely detected by karyotype or FISH. Some of these have a significant impact on clinical interpretation of NGS results. For example, there are multiple mechanisms of TP53 loss, including point mutation (detectable by NGS) and gene deletion.
有临床意义的细胞遗传学异常或几乎所有常规核型或FISH检测到的主要恶性肿瘤。其中一些对NGS结果的临床解释有重大影响。例如，TP53缺失有多种机制，包括点突变（NGS可检测）和基因缺失。

Clinical trials

The unfortunate reality for many patients is that standard of care treatments available are unlikely to result in a positive outcome. A major goal of precision medicine initiatives is to improve and expand on these treatments through the use of targeted therapies, either small molecules or biologics designed to attack cells expressing specific mutations. While there are now several such drugs available, the modest gains achieved so far are hopefully the vanguard of a much larger and more effective suite of targeted therapies.
对许多患者来说，不幸的现实是，现有的标准护理治疗不太可能产生积极的结果。精准医学计划的一个主要目标是通过使用靶向疗法来改进和扩大这些治疗，无论是小分子疗法还是旨在攻击表达特定突变的细胞的生物制剂。虽然现在有几种这样的药物可供使用，但迄今为止取得的少量进展有望成为一套更大、更有效的靶向疗法的先锋。
To assist in managing the complexities of drug development, discovery, and evaluation, the National Cancer Institute (NCI) has created the NCI Match program where patients are connected with clinical trials for which their mutation status makes them eligible. This nationwide collaboration among academic labs, pharmaceutical companies, oncologists, and patients hold great promise. From the molecular pathologist perspective, these trials offer a window into which targets may be clinically utile in the near future, pending their results. In the meantime, labs are developing or even offering testing of targets that remain in the trial phase both to prepare for therapies which may come available in the near future and to enable their patients to enroll in a clinical trial.
为了帮助管理药物开发、发现和评估的复杂性，国家癌症研究所（NCI）创建了NCI匹配计划，在该计划中，患者与临床试验相关，其突变状态使其符合资格。学术实验室、制药公司、肿瘤学家和患者之间的这种全国性合作前景广阔。从分子病理学家的角度来看，这些试验提供了一个窗口，在不久的将来，靶点可能会在临床上使用，等待他们的结果。与此同时，实验室正在开发甚至提供仍处于试验阶段的靶点测试，以便为可能在不久的将来提供的治疗做准备，并使患者能够参加临床试验。

Hereditary cancer syndrome variants遗传性癌症综合征变异

There is significant overlap between the genes known to have clinically relevant somatic alterations and hereditary cancer predisposition30,31. As such, panel testing may reveal germline variants of clinical relevance to the patient and the patient’s family. At this time, such variants are considered incidental, as patients are not typically consented or counseled that such information may be identified during testing. As screening for hereditary cancer syndromes is only recommended in high risk patients (e.g. those with family history or atypical presentation), this is a moral grey area with minimal guidance from professional societies or governmental agencies at this time.
已知具有临床相关躯体改变和遗传性癌症易感性的基因之间存在显著重叠。因此，小组测试可能会揭示与患者及其家人临床相关的种系变异。目前，这种变异被认为是偶然的，因为患者通常不同意或不建议在测试期间识别这种信息。由于遗传性癌症综合征筛查仅推荐给高危患者（如有家族史或非典型表现的患者），这是一个道德灰色地带，目前专业协会或政府机构的指导很少。

Conclusions

Clinical NGS testing in cancer is a new field experiencing rapid change. As a result, the current landscape is extremely varied, with each laboratory deciding how and when to report detected variants. For now, labs must perform extensive assay performance testing and complex validations to ensure a high level of reliability, accuracy, and sensitivity.
癌症临床NGS检测是一个快速变化的新领域。因此，目前的情况千差万别，每个实验室都决定如何以及何时报告检测到的变异。目前，实验室必须进行广泛的分析性能测试和复杂的验证，以确保高水平的可靠性、准确性和灵敏度。
It is also important to recognize the current limitations of NGS analysis. Due to the limited number of prospective studies and sample sizes, mutation screening has very limited negative predictive value. This means that at present NGS testing cannot discern between benign and malignant neoplasms from solid or liquid biopsy samples. Rather, as we have seen, it can help guide therapy and management for individuals known to have malignancies.
认识到NGS分析目前的局限性也很重要。由于前瞻性研究和样本量有限，突变筛查的阴性预测价值非常有限。这意味着目前NGS检测无法从固体或液体活检样本中区分良性和恶性肿瘤。相反，正如我们所看到的，它可以帮助指导已知患有恶性肿瘤的患者的治疗和管理。
Genome-wide sequencing, including genome and exome, has enabled major advances in our understanding of the molecular basis of cancer. Some findings, such as the roles of calreticulin (CALR) mutations in myeloproliferative neoplasms32 and IDH1 mutations in gliomas33 have led to rapidly adopted, high-impact clinical tests. Thus the current common practice is to use the results of genome-wide sequencing as a resource to inform clinical test development.
全基因组测序，包括基因组和外显子组，使我们对癌症分子基础的理解取得了重大进展。一些发现，如钙网蛋白（CALR）突变在骨髓增生性肿瘤中的作用，以及IDH1突变在胶质瘤中的作用，导致了迅速采用的高影响临床试验。因此，目前的普遍做法是将全基因组测序结果作为一种资源，用于指导临床试验的开发。
While the benefit of these research efforts have been tremendous, the utility of performing the same tests on clinical specimens is less certain. At present, the total number of genes relevant to targeted therapies, diagnostic and prognostic implications, and clinical trials is likely in the hundreds. For any given cancer type, this number may be far lower: in some cases only half a dozen or so. As such, the majority of variants or mutations detected by genome-wide sequencing of neoplastic specimens cannot be interpreted in a clinical setting and are of limited clinical utility. However, several commercial and academic laboratories offer clinical exome or genome sequencing, and have reported some early success34. So far though, the vast majority of relevant mutations detected by genome-wide sequencing are within genes on standard panels. For rare cancer types or types with unusually broad mutational spectra, genome-wide sequencing may already have clinical use enough above panels to merit semi-routine implementation.
虽然这些研究工作带来了巨大的好处，但在临床标本上进行相同测试的效用还不太确定。目前，与靶向治疗、诊断和预后影响以及临床试验相关的基因总数可能有数百个。对于任何特定的癌症类型，这个数字可能要低得多：在某些情况下，只有六种左右。因此，通过对肿瘤标本进行全基因组测序检测到的大多数变体或突变不能在临床环境中解释，且临床实用性有限。然而，一些商业和学术实验室提供临床外显子组或基因组测序，并报告了一些早期成功34。然而，到目前为止，通过全基因组测序检测到的绝大多数相关突变都在标准面板上的基因范围内。对于罕见的癌症类型或具有异常广泛突变谱的类型，全基因组测序可能已经具有足够的临床应用价值，值得半常规实施。
Genome-wide sequencing may confer additional benefits, such as structural rearrangement detection (copy number changes, loss of heretozygosity, ploidy, etc.) and tumor purity and heterogeneity estimation. At this time, the bioinformatics required to perform these additional analyses are not mature enough for clinical diagnostics. Eventually, the benefits of genome-wide sequencing will increase (along with the decrease in the cost of sequencing) to meet a crossing point where panels are no longer sufficient. Thus it is likely a matter of “when?” - not “if?” - genome-wide sequencing replaces panel testing.
全基因组测序可能带来额外的好处，例如结构重排检测（拷贝数变化、杂合性丢失、倍性等）以及肿瘤纯度和异质性估计。目前，进行这些额外分析所需的生物信息学还不够成熟，无法用于临床诊断。最终，全基因组测序的好处将增加（同时测序成本降低），以满足一个交叉点，即面板不再足够。因此，这可能是一个“何时”的问题不是“如果？”-全基因组测序取代了面板测试。
Over time, labs will coalesce around similar methods and approaches. The driving forces behind this will be increasingly sophisticated professional guidelines, more powerful reference materials, and availability of in vitro diagnosis (IVD) tests. Professional societies such as the College of American Pathologists (CAP) and the US Centers for Disease Control and Prevention (CDC) have laid the early groundwork for regulations and guidelines that will likely grow more specific and rigorous in years to come. If the pace at which commercial vendors have set continues, the rate-limiting step of mutation-positive control specimen acquisition will be reduced. And it is likely that the US FDA will continue to approve more NGS IVD tests including cancer panels.
随着时间的推移，实验室将围绕类似的方法和途径进行整合。这背后的驱动力将是越来越复杂的专业指南、更强大的参考材料和体外诊断（IVD）测试的可用性。美国病理学家学院（CAP）和美国疾病控制和预防中心（CDC）等专业协会已经为法规和指南奠定了早期基础，这些法规和指南在未来几年可能会变得更加具体和严格。如果商业供应商设定的速度继续下去，突变阳性对照样本采集的限速步骤将减少。美国食品和药物管理局很可能会继续批准更多的NGS IVD测试，包括癌症小组。
All of these trends will combine to make clinical cancer NGS testing less costly and complex to offer, thus making them more accessible to laboratories and patients. Due to mainly economic concerns, DNA testing of solid tumors and hematological malignancies is typically limited to high level tertiary care centers and/or patients with refractory disease at this time. As the cost of testing comes down and as more targeted therapies are available, its net benefit to population health will rise dramatically.
所有这些趋势都将使临床癌症NGS检测的成本和复杂性降低，从而使实验室和患者更容易获得这些检测。由于主要是出于经济考虑，目前对实体瘤和血液系统恶性肿瘤的DNA检测通常仅限于高级三级医疗中心和/或难治性疾病患者。随着检测成本的降低和更多靶向治疗的出现，其对人群健康的净效益将显著增加。