Biomarker discovery using scRNA-seq: what's the big deal?【机器翻译】
利用scRNA-seq发现生物标志物:有什么大不了的? 【转： https://www.biomage.net/blog/biomarker-discovery-using-scrnaseq】
Nov 21 Written By Anastasija Slapina
Biomarkers have the potential to save lives.
Biomarkers are simply measurable characteristics of organisms, so this may seem like an overstatement. However, the use of biomarkers is becoming increasingly important both in clinical research and practice. Biomarkers for the early detection of disease and the assessment of prognosis are also key components of precision medicine that allow us to find the best treatment for each individual patient. Especially when it comes to complex diseases like cancer.
生物标记只是生物体可测量的特征，所以这似乎有点言过其实。然而，生物标记的使用在临床研究和实践中变得越来越重要。用于疾病早期检测和预后评估的生物标志物也是精确医疗的关键组成部分，使我们能够为每个患者找到最佳治疗方法。特别是当涉及到像癌症这样的复杂疾病时。
Recently, there has been a growing interest in using single-cell technologies to identify novel biomarkers. Single-cell RNA sequencing (scRNA-seq) is a powerful tool for identifying gene transcripts within individual cells and can be used to detect changes in gene expression that may be indicative of disease or may predict progression or outcome.
最近，人们对使用单细胞技术识别新型生物标志物越来越感兴趣。单细胞RNA测序(scRNA-seq)是一种强大的工具，可用于识别单个细胞内的基因转录本，并可用于检测可能预示疾病或可能预测进展或结局的基因表达变化。
Contents
What are biomarkers?
什么是生物标记物?
A biomarker can be defined as a characteristic of a therapeutic response or a normal or pathological process that can be objectively and reproducibly assessed and measured. The International Programme on Chemical Safety describes biomarkers as “any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease”. Simply - biomarkers tell us something about an organism.
生物标志物可以定义为治疗反应或正常或病理过程的特征，可以客观和重复地评估和测量。国际化学品安全方案将生物标记物描述为"可在体内或其产品中测量并影响或预测结果或疾病发生率的任何物质、结构或过程"。很简单，生物标记告诉我们有机体的一些信息。
There is a wide array of biomarkers. Because of the broad definition, biomarkers include blood pressure and body temperature, as well as more laboratory-measured signs like glucose and viral load. Biomarkers are not a new concept, as medical signs have a long history of use in clinical practice. Currently, biomarkers mostly refer to the most objective, reproducible, and measurable medical signs.
生物标记物种类繁多。由于生物标记的定义很宽泛，它包括血压和体温，以及更多的实验室测量信号，如葡萄糖和病毒载量。生物标记不是一个新概念，因为医学标志在临床实践中有很长的使用历史。目前，生物标志物大多是指最客观、可重复和可测量的医学体征。
It’s important to note that there are different types of biomarkers (Figure 1). Here, we will focus on prognostic biomarkers to delve deeper into this field!
重要的是要注意有不同类型的生物标志物(图1)。在这里，我们将专注于预后生物标志物，以深入研究这一领域!
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Figure 1. Types of biomarkers. The figure outlines the most common types of biomarkers and highlights prognostic biomarkers that are emphasized in this article. Various types of biomarkers serve different purposes. Predictive biomarkers provide information on the probability of a response or the lack of it to a particular therapy. Pharmacodynamic biomarkers indicate the molecular and functional effects produced by a drug on a specific target. Safety biomarkers are measured before or after exposure to a drug or an environmental agent to assess its adverse effects. Risk biomarkers are associated with an increased/decreased risk of developing a disease in the case when an individual doesn’t have the clinical disease yet. Monitoring biomarkers are measured repeatedly over time to mainly assess disease progression. Diagnostic biomarkers detect or confirm the presence of a medical condition of interest.
图1。生物标记的类型。该图概述了最常见的生物标志物类型，并强调了本文所强调的预后生物标志物。不同类型的生物标志物有不同的用途。预测生物标记物提供了对特定治疗产生反应或缺乏反应的可能性的信息。药效学生物标记物是指药物对特定靶点产生的分子和功能效应。在暴露于药物或环境剂之前或之后测量安全生物标记物，以评估其不良影响。当一个人还没有临床疾病时，风险生物标记物与患病风险的增加/降低有关。在一段时间内反复测量监测生物标志物，主要用于评估疾病进展。诊断性生物标志物检测或确认感兴趣的医疗条件的存在。
Prognostic biomarkers
预后的生物标记物
Prognostic biomarkers evaluate the risk of an individual developing a specific disease. They can also be used to state the likely course of the disease or the possibility of disease recurrence. Importantly, an accurate prognosis can lead to appropriate clinical support for patients.
This type of biomarker is often used when talking about cancer. It helps to identify tumor-specific characteristics, such as mRNA levels or circulating tumor cells in the blood. For example, some prognostic biomarkers could be used in staging – i.e. to show how far the disease has progressed. This enables doctors to identify the severity of the disease.
A well-known example of prognostic biomarkers used currently is pathogenic germline mutations in the BRCA1 and BRCA2 tumor suppressor genes which indicate an increased risk for breast, fallopian tube, and ovarian cancers in women, and pancreatic and breast cancers in men [1].
The process of getting the biomarker from the discovery phase to clinical practice is difficult. Before a biomarker can be effectively used in the clinic, it has to go through a rigorous validation process that assesses its statistical and analytical performance. For prognostic biomarkers, changes in the value of the biomarker over time irrespective of treatment need to be repeatedly demonstrated.
Even for a biomarker that has been analyzed or has a strong biological basis, the proof of clinical utility may still require additional evidence. It often takes several trials and studies for a biomarker to be completely validated and approved.
Although many biomarkers are already being used in the clinic, there is still a gap when it comes to identifying specific biomarkers for each individual patient and tumor. For example, serum beta-2-microglobulin is used as a prognostic biomarker in various cancer types, but it lacks patient specificity. Biomarkers with high specificity are going to be the cornerstone of future prognostics and precision medicine!
预后生物标志物评估个体发生特定疾病的风险。它们也可以用来说明疾病的可能过程或疾病复发的可能性。重要的是，准确的预后可以为患者提供适当的临床支持。这种类型的生物标记在谈论癌症时经常被使用。它有助于识别肿瘤特异性特征，如mRNA水平或血液中循环的肿瘤细胞。例如，一些预后生物标志物可用于分期，即显示疾病的进展程度。这使医生能够确定疾病的严重程度。目前使用的预后生物标志物的一个著名例子是BRCA1和BRCA2肿瘤抑制基因的致病性种系突变，这表明女性患乳腺癌、输卵管癌和卵巢癌的风险增加，男性患胰腺癌和乳腺癌的风险增加[1]。从发现阶段到临床实践的过程是困难的。在一种生物标志物能够有效地用于临床之前，它必须经过一个严格的验证过程，以评估其统计和分析性能。对于预后生物标志物，无论治疗与否，生物标志物值随时间的变化都需要反复证实。即使一个生物标记物已经被分析或有很强的生物学基础，其临床效用的证明可能仍然需要额外的证据。通常需要多次试验和研究才能完全验证和批准一种生物标志物。尽管许多生物标记物已经被用于临床，但在为每个个体患者和肿瘤识别特定的生物标记物方面仍有差距。例如，血清-2-微球蛋白被用作各种癌症类型的预后生物标志物，但它缺乏患者特异性。具有高特异性的生物标记物将成为未来预后和精确医学的基石!
Precision medicine
精密(准)医学
This article wouldn’t be complete without mentioning the big picture - precision medicine. Precision medicine is advantageous because of its specificity - it’s changing how diseases are identified, classified, and treated. As a result, patients could receive the right treatment at the right time, improving patient care and survival.
Biomarkers are the foundation of successfully achieving precision medicine across a wide range of diseases and conditions. And precision medicine is the reason why biomarkers hold such importance! Through biomarkers, scientists and doctors can make more precise diagnoses, which then leads to more effective and personalized treatments.
Recent advances in science and technology, such as scRNA-seq, have heightened interest in precision medicine. In diseased tissues, single cells can be analyzed to determine what contributes to individual differences between patients, allowing for personalized treatment and the development of biomarkers.
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这篇文章如果不提到精确医疗这一大图就不完整。精准医疗的优势在于它的特殊性——它正在改变疾病的识别、分类和治疗方式。因此，患者可以在正确的时间接受正确的治疗，改善患者的护理和生存率。生物标记物是成功实现精确医疗的基础，跨越广泛的疾病和条件。精准医疗就是生物标记如此重要的原因!通过生物标记，科学家和医生可以做出更精确的诊断，从而实现更有效和个性化的治疗。最近科学技术的进步，如scRNA-seq，提高了人们对精准医疗的兴趣。在病变组织中，可以通过分析单个细胞来确定是什么导致了患者之间的个体差异，从而实现个性化治疗和生物标志物的开发。了解更多:开放精准医学会议
Where does scRNA-seq come in?
scRNA-seq从何而来?
Traditionally, biomarkers have been identified using various laboratory techniques, such as immunohistochemistry, ELISA, and PCR. For instance, anaplastic lymphoma kinase translocations which are associated with several cancer types can be identified by RT-PCR or ELISA kits. However, these techniques are often limited in their ability to detect low levels of biomarkers or to identify novel biomarkers.
Single-cell transcriptomics provides unprecedented insight into cellular heterogeneity and changes in dynamic cell populations, as well as molecules and pathways linked to various diseases, and that may be “averaged out” using bulk RNA sequencing.
So, scRNA-seq offers a more focused and comprehensive approach for the identification of new biomarkers. For example, scRNA-seq provides the opportunity to identify gene expression changes in specific subsets of cells within a tissue that are associated with disease or disease severity.
With the help of scRNA-seq, scientists are already discovering novel biomarkers that could be used in clinical practice or research in the future.
For example, in a recent study, researchers analyzed lung adenocarcinoma single-cell data in combination with bulk RNA-sequencing data and established a novel prognostic risk model for this cancer type [2]. They screened the marker genes of five important cell populations and found 10 genes related to the prognosis of lung adenocarcinoma - CCL20, CP, HLA-DRB5, RHOV, CYP4B1, BASP1, ACSL4, GNG7, CCDC50, and SPATS2 (Figure 2). This 10-gene signature achieved stable prediction efficiency in datasets from different platforms.
传统上，生物标志物的鉴定使用各种实验室技术，如免疫组化、ELISA和PCR。例如，与几种癌症相关的间变性淋巴瘤激酶易位可以通过RT-PCR或ELISA试剂盒识别。然而，这些技术在检测低水平生物标志物或识别新生物标志物的能力上往往受到限制。单细胞转录组学提供了前所未有的洞察细胞异质性和动态细胞群的变化，以及与各种疾病相关的分子和途径，并可以使用大体积RNA测序“平均”。因此，scRNA-seq为识别新的生物标志物提供了一种更加集中和全面的方法。例如，scRNA-seq提供了识别组织中与疾病或疾病严重程度相关的特定细胞亚群的基因表达变化的机会。在scRNA-seq的帮助下，科学家们已经发现了新的生物标记物，可以在未来的临床实践或研究中使用。例如，在最近的一项研究中，研究人员结合大量rna测序数据分析了肺腺癌单细胞数据，并建立了这种癌症类型[2]的新的预后风险模型。他们筛选了5个重要细胞群的标记基因，发现了10个与肺腺癌预后相关的基因——CCL20、CP、HLA-DRB5、RHOV、CYP4B1、BASP1、ACSL4、GNG7、CCDC50、SPATS2(图2)。这10个基因签名在不同平台的数据集中实现了稳定的预测效率。
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Figure 2. The risk score, survival time, survival status, and gene expression in samples based on the 10-gene signature. The risk score reflects the level of risk of disease in the presence of the signature. Analysis of the prognostic classification of the risk score concludes that patients with a higher risk score had a poorer prognosis. Image adapted from Xu et al. (2022).
图2。基于10个基因特征的样本风险评分、生存时间、生存状态和基因表达。风险评分反映了存在签名时的疾病风险水平。对危险评分的预后分类分析，危险评分越高的患者预后越差。图片改编自Xu等人(2022)。
In another study, autophagy-related 16 like 1 gene (ATG16L1) was associated with poor prognosis in patients with osteosarcoma [3]. So, ATG16L1 is a potential prognostic biomarker in osteosarcoma patients. This gene seems to affect osteosarcoma by mediating CD8+ T cells, especially in patients with metastases.
Since scRNA-seq is a relatively new method, for many diseases, researchers are still in the biomarker discovery phase. In the previously mentioned works, researchers explored cancer single-cell data to find potential prognostic biomarkers. This means that the reliability of these biomarkers still needs to be investigated.
However, there are currently several ongoing clinical trials that aim to discover and validate molecular biomarkers that show reproducible and reliable results using scRNA-seq. For example, in a currently recruiting clinical trial researchers are trying to identify prognostic and diagnostic biomarkers according to the characteristics and evolution of systemic sclerosis and inflammatory myopathies (ClinicalTrials.gov Identifier: NCT04917705).
In the future, these prognostic biomarkers identified by scRNA-seq can allow for the reliable identification of individuals with a more aggressive tumor progression and, thus, a higher chance of disease recurrence and death. As a result, medical practitioners could develop management strategies that ensure these patients receive appropriate and personalized care.
Learn more about other uses of scRNA-seq: Top 5 most revolutionary uses of single-cell RNA sequencing
在另一项研究中，自噬相关的16样1基因(ATG16L1)与[3]骨肉瘤患者的不良预后相关。因此，ATG16L1是骨肉瘤患者潜在的预后生物标志物。该基因似乎通过介导CD8+ T细胞影响骨肉瘤，特别是在转移患者中。由于scRNA-seq是一种相对较新的方法，对于许多疾病，研究人员仍处于生物标志物的发现阶段。在前面提到的工作中，研究人员探索了癌症单细胞数据，以寻找潜在的预后生物标志物。这意味着这些生物标记物的可靠性仍然需要研究。然而，目前有几个正在进行的临床试验旨在发现和验证分子生物标记物，使用scRNA-seq显示可复制和可靠的结果。例如，在一项目前正在招募的临床试验中，研究人员正试图根据系统性硬化症和炎症性肌病的特征和进化来确定预后和诊断性生物标志物(ClinicalTrials.gov Identifier: NCT04917705)。在未来，这些由scRNA-seq识别的预后生物标志物可以可靠地识别肿瘤进展更严重的个体，因此，疾病复发和死亡的几率更高。因此，医疗从业人员可以制定管理策略，以确保这些患者得到适当和个性化的护理。了解更多关于scRNA-seq的其他用途:单细胞RNA测序的五大最具革命性的用途
Biomarker data analysis
Single-cell transcriptomics data can be complex, noisy, and large-scale. This presents challenges in scRNA-seq analysis and thus biomarker discovery. If you are looking for an easier way to explore your data, try out Cellenics®!
Researchers already use the Cellenics® data analysis platform to discover new biomarkers by exploring clinical trial data. It’s an open-source and cloud-based scRNA-seq analysis software that allows you to analyze your dataset without prior programming knowledge. It’s fast, user-friendly, and free for academic researchers with datasets of up to 300 thousand cells!
单细胞转录组学数据可能是复杂的、有噪声的和大规模的。这给scRNA-seq分析和生物标志物发现带来了挑战。如果您正在寻找一种更简单的方式来探索您的数据，请尝试Cellenics®!研究人员已经使用Cellenics®数据分析平台通过探索临床试验数据来发现新的生物标志物。它是一款开源的、基于云计算的scRNA-seq分析软件，允许您在不具备编程知识的情况下分析数据集。它是快速的，用户友好的，和免费的学术研究人员的数据集高达30万细胞!
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References
[1] https://oncologypro.esmo.org/education-library/factsheets-on-biomarkers/brca1-and-brca2-in-ovarian-cancer
[2] Xu, Y., Wang, Y., Liang, L. and Song, N., 2022. Single-cell RNA sequencing analysis to explore immune cell heterogeneity and novel biomarkers for the prognosis of lung adenocarcinoma. Frontiers in Genetics, 13. doi: 10.3389/fgene.2022.975542.
[3] Qin Z, Luo K, Liu Y, Liao S, He J, He M, Xie T, Jiang X, Li B, Liu H, Huang Q, Tang H, Feng W, Zhan X. ATG16L1 is a Potential Prognostic Biomarker and Immune Signature for Osteosarcoma: A Study Based on Bulk RNA and Single-Cell RNA-Sequencing. Int J Gen Med. 2022;15:1033-1045. https://doi.org/10.2147/IJGM.S341879
