Volume: 15, Issue: 2

Preface

Disease Biomarkers for Precision Medicine: Challenges and Future Opportunities

Edwin Wang, William C.S. Cho, S.C. Cesar Wong, Siqi Liu

Page 57-58

Review

Circulating Tumor DNA as Biomarkers for Cancer Detection

Xiao Han, Junyun Wang, Yingli Sun

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.
随着精准医学的发展，血液循环肿瘤DNA（circulating tumor DNAs, ctDNAs）的检测逐渐走入公众视线。相比于传统的物理、化学检测手段，ctDNA检测具有非侵入、无创性等优点，能够更方便、更有效地应用于临床癌症诊断、预后评估和用药指导。那么： 1. ctDNA的检测指标有哪些？ 2. 目前ctDNA相关的疾病研究有何进展？ 3. ctDNA研究方法都有哪些？ 4. ctDNA作为潜在生物标志物有怎样的应用前景和可能面临的挑战？ 在这篇文章中，我们对以上问题进行了逐一解答和探讨，综述了近几年来ctDNA检测相关研究（包括乳腺癌、结直肠癌、非小细胞性肺癌等）并介绍了一些代表性的研究方法（数字PCR、iDES-enhanced CAPP-Seq、MCTA-seq等）。本文为如何看待ctDNA检测及其未来的发展方向提供了一个新的视角。

Page 59-72

Review

Protein Array-based Approaches for Biomarker Discovery in Cancer

Yi Huang, Heng Zhu

Biomarkers are deemed to be potential tools in early diagnosis, therapeutic monitoring, and prognosis evaluation for cancer, with simplicity as well as economic advantages compared with computed tomography and biopsy. However, most of the current cancer biomarkers present insufficient sensitivity as well as specificity. Therefore, there is urgent requirement for the discovery of biomarkers for cancer. As one of the most exciting emerging technologies, protein array provides a versatile and robust platform in cancer proteomics research because it shows tremendous advantages of miniaturized features, high throughput, and sensitive detections in last decades. Here, we will present a relatively complete picture on the characteristics and advance of different types of protein arrays in application for biomarker discovery in cancer, and give the future perspectives in this area of research.
相比CT与穿刺活检，生物标志物检测在癌症早期诊断、治疗监测与预后评估方面，无疑具有操作简便、经济实惠的明显优势。然而，目前使用的大部分肿瘤标志物均存在着敏感性和/或特异性不足的缺陷。因此，寻找新的肿瘤标志物成为摆在广大科研人员面前的一项迫切任务。作为最令人激动的技术之一，蛋白质芯片凭借其近十年来表现出的微型化、高通量、高敏感性的特点，为肿瘤蛋白质组学的研究提供了一个多用途的强大研究平台。在这篇综述里，我们将向广大读者整体呈现不同类型蛋白芯片技术在寻找新肿瘤标志物应用中所具有的特点与优势，并介绍这个研究领域未来的发展前景。

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Review

The Promise of Pharmacogenomics in Reducing Toxicity During Acute Lymphoblastic Leukemia Maintenance Treatment

Shoshana Rudin, Marcus Marable, R. Stephanie Huang

Pediatric acute lymphoblastic leukemia (ALL) affects a substantial number of children every year and requires a long and rigorous course of chemotherapy treatments in three stages, with the longest phase, the maintenance phase, lasting 2–3 years. While the primary drugs used in the maintenance phase, 6-mercaptopurine (6-MP) and methotrexate (MTX), are necessary for decreasing risk of relapse, they also have potentially serious toxicities, including myelosuppression, which may be life-threatening, and gastrointestinal toxicity. For both drugs, pharmacogenomic factors have been identified that could explain a large amount of the variance in toxicity between patients, and may serve as effective predictors of toxicity during the maintenance phase of ALL treatment. 6-MP toxicity is associated with polymorphisms in the genes encoding thiopurine methyltransferase (TPMT), nudix hydrolase 15 (NUDT15), and potentially inosine triphosphatase (ITPA), which vary between ethnic groups. Moreover, MTX toxicity is associated with polymorphisms in genes encoding solute carrier organic anion transporter family member 1B1 (SLCO1B1) and dihydrofolate reductase (DHFR). Additional polymorphisms potentially associated with toxicities for MTX have also been identified, including those in the genes encoding solute carrier family 19 member 1 (SLC19A1) and thymidylate synthetase (TYMS), but their contributions have not yet been well quantified. It is clear that pharmacogenomics should be incorporated as a dosage-calibrating tool in pediatric ALL treatment in order to predict and minimize the occurrence of serious toxicities for these patients.
每年有数量可观的儿童被确诊为小儿急性淋巴细胞白血病（ALL），该病需要接受分三阶段的长期严格的化疗，其中时间最长的维持期一般持续2-3年。维持期使用的主要药物6-巯基嘌呤（6-MP）和甲氨蝶呤（MTX）对降低该病复发风险是必须的药物治疗，但这两种药物具有潜在的严重毒性，包括可能危及生命的骨髓抑制及胃肠道毒性。目前已经明确药物基因组因素能够解释不同患者对这两种药物毒性反应存在的大量变异，并且可以作为ALL的维持治疗期间有效的毒性预测因子。6-MP的毒性与编码硫嘌呤甲基转移酶（TPMT）、Nudix水解酶15（NUDT15）和潜在的肌苷三磷酸酶（ITPA）的基因在不同种族之间的多态性有关。此外，MTX毒性与编码溶质载体有机阴离子转运蛋白家族成员1B1（SLCO1B1）和二氢叶酸还原酶（DHFR）的基因多态性有关。其他可能与MTX毒性有关的多态性也被确认，包括编码溶质载体家族19成员1（SLC19A1）和胸苷酸合成酶（TYMS）的基因多态性，但其贡献尚未得到很好的量化。显然，药物基因组学应作为儿科ALL治疗中的剂量校准工具，以预测和最小化严重毒性在患者中的发生。

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Review

Vitamin Pharmacogenomics: New Insight into Individual Differences in Diseases and Drug Responses

Hai-Yan He, Mou-Ze Liu, Yue-Li Zhang, Wei Zhang

Vitamins are vital to sustain normal physiological function, metabolism, and growth for all living organisms. Being an integral component of coenzyme, vitamins can affect the catalytic activities of many enzymes and the expression of drug transporters. Genetic variations in metabolism and/or transporter genes of drugs can influence the exposure of the human body to drugs and/or their active metabolites, thus contributing to the variations in drug responses and toxicities. Nonetheless, pharmacogenomics studies on nutrients have been rarely summarized. In this article, we reviewed recent progress on vitamin pharmacogenomics, for a better understanding on the influence of vitamin-related gene polymorphisms on inter-individual differences in diseases and drug efficacy and safety.
维生素是所有生物维持正常的生理功能、代谢和生长至关重要的成分之一。 维生素通常以辅酶形式存在的，影响许多代谢酶催化活性以及药物转运体的表达。 药物基因组研究表明，药物代谢酶和转运体基因的遗传多态性能影响身体内药物和/或活性代谢产物的浓度水平，从而导致药物反应及其毒性的个体差异。维生素药物基因组学，顾名思义，即探讨影响维生素ADME（吸收、分布、代谢、排泄）及其效应受体基因多态性对维生素水平及其效应的影响。越来越多的研究表明维生素ADME多态性与疾病和药物治疗预后相关。 维生素D的生成和代谢途径（图一）中， CYP27B1（编码1-羟化酶） 和CYP24A1（编码24-羟化酶）发挥重要作用，研究发现，CYP27B1 rs28934604和CYP24A1 rs6068812突变基因型，酶的催化活性分别仅为野生型的55%和31%[1]。该突变可能与结直肠癌、乳腺癌和前列腺癌等的风险相关[2]。此外，维生素D受体（VDR）SNP rs7968585中，患者每携带一个最小等位，其复合终点的风险（长期随访的髋部骨折，心肌梗死，癌症和死亡率）增加40%[3]。脂质转运蛋白APOA5 （Apolipoprotein A5） rs662799 (-1131T>C) 遗传多态性影响维生素E水平。突变杂合子的维生素E水平比其他人群高13%，且其中-1131C等位携带者与代谢综合征的风险相关（OR=1.77）[4]。APOE（Apolipoprotein E）E3/4+E4/4基因型个体比E2/3 or E3/3基因型显著要高[5]。维生素K环氧化物还原酶（vitamin K epoxide reductase）基因VKORC1 -1639G>A变异显著影响过度抗凝患者中维生素K的作用，G等位携带者国际标准化比值(INR)下降更快[6]，此外VKORC1 -1639G>A通过影响维K循环，与华法林抗凝治疗的疗效相关[6, 7]。 水溶性维生素中，FUT2 （Fucosyl transferase）基因rs602662(772G>A)，维生素B12转运蛋白TCN2 rs1801198 (776C>G) 变异影响维生素B12水平[8, 9]；维C转运体基因SVCT2/SLC23A2 rs1776964 和rs6139591影响维C水平，与女性患者急性冠脉综合症相关[10]。亚甲基四氢叶酸还原酶基因MTHFR rs1801133变异中，TT基因型患者心血管疾病的风险显著要高[11]。详见（表二）。 近年来维生素药物基因组学的研究表明维生素PK、PD遗传多态性通过调控维生素水平，参与疾病和药物反应个体差异的调控。维生素药物基因组学研究将为未来精准医疗的实施提供参考和指导。

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Review

Omics Approaches to Identify Potential Biomarkers of Inflammatory Diseases in the Focal Adhesion Complex

Johanne Brooks, Alastair Watson, Tamas Korcsmaros

Inflammatory diseases such as inflammatory bowel disease (IBD) require recurrent invasive tests, including blood tests, radiology, and endoscopic evaluation both to diagnose and assess disease activity, and to determine optimal therapeutic strategies. Simple ‘bedside’ biomarkers could be used in all phases of patient management to avoid unnecessary investigation and guide further management. The focal adhesion complex (FAC) has been implicated in the pathogenesis of multiple inflammatory diseases, including IBD, rheumatoid arthritis, and multiple sclerosis. Utilizing omics technologies has proven to be an efficient approach to identify biomarkers from within the FAC in the field of cancer medicine. Predictive biomarkers are paving the way for the success of precision medicine for cancer patients, but inflammatory diseases have lagged behind in this respect. This review explores the current status of biomarker prediction for inflammatory diseases from within the FAC using omics technologies and highlights the benefits of future potential biomarker identification approaches.
炎症性疾病（Inflammatory disease）是十分常见而又重要的基本病理过程，体表的外伤感染和各器官的大部分常见病和多发病都属于炎症性疾病，比如炎性肠病（inflammatory bowel disease，IBD）。临床上通常经过反复血液检测、X-射线、内窥镜来综合诊断IBD的病情以及确定治疗策略，这些侵入性检测对患者的身心会造成一定程度的伤害。因此，炎症性疾病的‘bedside’生物标志识别就显得尤为重要。黏着斑复合体（Focal Adhesion Complex；FAC）作为一种通过整合蛋白或者其它受体将细胞内信号向细胞外进行转导的蛋白体，能通过如IL-1信号通路等来调控炎性基因表达，进而影响炎症过程。癌症研究已经利用多组学技术来识别标志物，但是炎症性疾病在这一方面还是相对落后的。这篇综述从基因组学、转录组学、蛋白质组学等方面来阐述使用单一组学手段来研究FAC作为炎性疾病预测生物标志物的缺点和不足，同时强调了采用多组学技术以系统生物学的方式来识别潜在生物标志物的优势和必要性。未来可以利用多组学技术在FAC中识别出可以作为多种炎症性疾病如IBD等临床预测疾病发生发展的新生物标志物。

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Review

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders

Lijun Zhang, Xiaofang Jia, Jun-O Jin, Hongzhou Lu, Zhimi Tan

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases.
人免疫缺陷病毒-1（HIV-1）主要依靠宿主因素来完成其生命周期。因此，鉴定受HIV调控的宿主细胞蛋白质对了解HIV感染具有非常重要的意义。蛋白质组学因其高通量与高灵敏度的特点为HIV发病机制及相关疾病的研究提供了新思路。本文全面综述了目前蛋白质组学技术的进展，包括iTRAQ标记，SILAC标记，亚细胞蛋白质组学以及蛋白质翻译后修饰技术。此外，我们对蛋白质组学技术在HIV相关疾病、HIV感染机制方面的应用进行了系统的综述。蛋白质组学发现的蛋白质可能为HIV感染与相关疾病的诊断与治疗提供新的标志分子。

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Original Research

Network Analysis Reveals A Signaling Regulatory Loop in the PIK3CA-mutated Breast Cancer Predicting Survival Outcome

Shauna R. McGee, Chabane Tibiche, Mark Trifiro, Edwin Wang

Mutated genes are rarely common even in the same pathological type between cancer patients and as such, it has been very challenging to interpret genome sequencing data and difficult to predict clinical outcomes. PIK3CA is one of a few genes whose mutations are relatively popular in tumors. For example, more than 46.6% of luminal-A breast cancer samples have PIK3CA mutated, whereas only 35.5% of all breast cancer samples contain PIK3CA mutations. To understand the function of PIK3CA mutations in luminal A breast cancer, we applied our recently-proposed Cancer Hallmark Network Framework to investigate the network motifs in the PIK3CA-mutated luminal A tumors. We found that more than 70% of the PIK3CA-mutated luminal A tumors contain a positive regulatory loop where a master regulator (PDGF-D), a second regulator (FLT1) and an output node (SHC1) work together. Importantly, we found the luminal A breast cancer patients harboring the PIK3CA mutation and this positive regulatory loop in their tumors have significantly longer survival than those harboring PIK3CA mutation only in their tumors. These findings suggest that the underlying molecular mechanism of PIK3CA mutations in luminal A patients can participate in a positive regulatory loop, and furthermore the positive regulatory loop (PDGF-D/FLT1/SHC1) has a predictive power for the survival of the PIK3CA-mutated luminal A patients.
同种类型的癌症患者间的突变基因很少有一致性，因此，利用基因组测序数据预测临床特征(表型)是一个非常有挑战的工作。PIK3CA是肿瘤中相对突变率高的少数基因之一。例如，超过46.6％的Luminal A 型 乳腺癌具有PIK3CA突变，而35.5％的所有乳腺癌样本中有PIK3CA突变。为了揭示Luminal A 型 乳腺癌中PIK3CA突变的功能，我们应用了我们最近提出的癌症标志网络框架来研究携带PIK3CA突变的Luminal A 型乳腺癌样本中的网络模块。我们发现超过70％的携带PIK3CA突变的Luminal A 型乳腺癌样本包含一个正调节循环（一个3基因网络模块），包括主调因子（PDGF-D），第二调因子（FLT1）和输出节点（SHC1）共同作用。重要的是，我们发现携带PIK3CA突变以及这种阳性调节环的Luminal A 型乳腺癌患者比在肿瘤中只具有PIK3CA突变的那些肿瘤患者存活时间更长。这些研究结果表明，Luminal A 型乳腺癌患者的PIK3CA突变可以能参与这个阳性调节循环，因此这个阳性调节环（即PDGF-D / FLT1 / SHC1）能够预测PIK3CA突变的Luminal A 型乳腺癌患者的存活率。

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Method

eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood

Jinfeng Zou, Edwin Wang

With the technology development on detecting circulating tumor cells (CTCs) and cell-free DNAs (cfDNAs) in blood, serum, and plasma, non-invasive diagnosis of cancer becomes promising. A few studies reported good correlations between signals from tumor tissues and CTCs or cfDNAs, making it possible to detect cancers using CTCs and cfDNAs. However, the detection cannot tell which cancer types the person has. To meet these challenges, we developed an algorithm, eTumorType, to identify cancer types based on copy number variations (CNVs) of the cancer founding clone. eTumorType integrates cancer hallmark concepts and a few computational techniques such as stochastic gradient boosting, voting, centroid, and leading patterns. eTumorType has been trained and validated on a large dataset including 18 common cancer types and 5327 tumor samples. eTumorType produced high accuracies (0.86–0.96) and high recall rates (0.79–0.92) for predicting colon, brain, prostate, and kidney cancers. In addition, relatively high accuracies (0.78–0.92) and recall rates (0.58–0.95) have also been achieved for predicting ovarian, breast luminal, lung, endometrial, stomach, head and neck, leukemia, and skin cancers. These results suggest that eTumorType could be used for non-invasive diagnosis to determine cancer types based on CNVs of CTCs and cfDNAs.
目前，在血液、血清和血浆中检测循环肿瘤细胞（circulating tumor cells, CTCs）和细胞外游离DNA（cell-free DNAs, cfDNAs）的技术日益发展，为无创肿瘤诊断提供光明前景。一些研究报道，肿瘤组织与循环肿瘤细胞和细胞外游离DNA所含信号具有相关性。然而，仅依赖于肿瘤循环细胞或细胞外游离DNA，很难识别原发肿瘤类型。为此，我们提出eTumorType，基于肿瘤始发克隆（founding clone）的拷贝数变化（copy number variations, CNVs）识别肿瘤类型。eTumorType整合了肿瘤特征（cancer hallmark）和计算技术，如随机梯度提升（stochastic gradient boosting）、投票、重心法（centroid）和领选模式（leading patterns）。eTumorType基于18种常见肿瘤，共包含5327个样本的大型数据集，进行训练和验证。对于直肠、脑、前列腺和肾肿瘤，eTumorType预测肿瘤类型显示了高准确率（accuracy, 0.86-0.96）和高覆盖率（recall rate, 0.79-0.92）。另外，对于卵巢、乳腺（Luminal亚型）、肺、子宫内膜、胃、头颈、白血病和皮肤肿瘤，eTumorType也显示了较高的肿瘤预测准确率（0.78-0.92）和和覆盖率（0.58-0.95）。结果表明，eTumorType可以被用于基于循环肿瘤细胞和细胞外游离DNA的拷贝数变化进行肿瘤类型预测。

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Application Note

Lirex: A Package for Identification of Long Inverted Repeats in Genomes

Yong Wang, Jiao-Mei Huang

Long inverted repeats (LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is highly complicated when mismatches and indels between the repeats are permitted. Long inverted repeat explorer (Lirex) was developed and introduced in this report. Written in Java, Lirex provides a user-friendly interface and allows users to specify LIR searching criteria, such as length of the region, as well as pattern and size of the repeats. Recombinogenic LIRs can be selected on the basis of mismatch rate and internal spacer size from identified LIRs. Lirex, as a cross-platform tool to identify LIRs in a genome, may assist in designing following experiments to explore the function of LIRs. Our tool can identify more LIRs than other LIR searching tools. Lirex is publicly available at http://124.16.219.129/Lirex.
长反向重复(LIRs)是参与RNA干扰，DNA重组以及基因重复的重要进化和功能结构。当重复序列间引入错配和插入缺失，LIRs的鉴定会变的很复杂。由此我们开发了长反向重复检索工具—Lirex。Lirex是由java编写的，拥有友好用户界面，并允许用户自行设定搜索标准的长反向重复检索包。用户可以根据不同的需求设定搜索区域的长度，模式和重复序列的长度。 在检测到的LIRs中，Lirex可以根据这些LIRs的错配率和重复序列间隔长度来判断其是否属于重组的LIRs。因此Lirex可用来检测基因组中存在的LIRs,用于后续的LIRs功能实验。经与其他LIRs检索工具的比较，Lirex可以准确鉴定出更多的LIRs。Lirex公开网址：http://124.16.219.129/Lirex。

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