液滴数字PCR在肿瘤个体化治疗方面的应用

个体化治疗可以提高肿瘤病人的生存质量和生存时间,而肿瘤患者基因状态指导个体化用药,商业化的液滴数字PCR（droplet digital PCR,ddPCR）已经上市,并且可以在一个样本中对核酸精确定量。它是通过将模板稀释到20000个水包油微滴实现的,每个液滴可被微滴阅读器判定为阳性或阴性,因此某个等位基因的浓度可以被精确计算,它能够用于检测稀有突变体以及定量。与传统的实时PCR（qPCR）相比,ddPCR具有许多优点,尤其是灵敏且能够获得精确定量,例如,可以检测石蜡包埋样品中基因组扩增;可以以非侵入性的方式通过体液,如血液和痰来检测肿瘤基因突变;也可通过ddPCR检测拷贝数变异,从而研究其与癌症的相关性。     

二代测序技术在乳腺癌精准治疗中的应用

二代测序技术推动了乳腺癌精准医疗的发展。在乳腺癌基因组学方面,二代测序技术可以识别乳腺癌驱动基因,发现新的易感基因及突变类型,有助于乳腺癌发病风险评估和管理。在乳腺癌个体化治疗方面,二代测序技术可以检测和分析乳腺癌相关治疗靶点及信号通路,指导靶向药物和治疗方案的选择,实现精准用药。二代测序结合液体活组织检查技术,在乳腺癌动态监测方面也有着重要的临床意义。笔者总结了二代测序技术在乳腺癌精准治疗方面的研究进展,有助于更全面深入地探究乳腺癌的本质,最终实现精准医疗。     

循环肿瘤DNA检测在乳腺癌诊断中的临床应用

随着二代测序技术的发展,循环肿瘤DNA(circulating tumor DNA,ctDNA)检测在乳腺癌中的应用得到越来越多的关注。目前国内外大量研究显示ctDNA检测技术在监测肿瘤负荷、疗效预测、早期诊断、预后分析等方面具有广阔的应用价值。在乳腺癌诊疗走向个体化精准的时代,ctDNA检测能够为患者提供更为精准的诊断,指导临床治疗。     

晚期乳腺癌基因检测热点问题中国专家共识(2021版)

晚期乳腺癌预后较差, 疾病特征复杂, 后线解救治疗较为困难, 但是通过选择有效的治疗方案, 部分患者可以实现长期带瘤生存, 获得较好的生存质量。近年来, 随着分子生物学和基因检测技术的飞速发展, 晚期乳腺癌相关研究不断深入, 治疗手段不断丰富, 基因靶向治疗使晚期乳腺癌患者生存期显著延长。基因检测是分子分型诊断、遗传风险预测、疗效监测、耐药提示以及治疗方案选择的重要手段, 对晚期乳腺癌患者的分子病理诊断、靶向药物选择以及治疗模式优化具有重要意义。共识专家委员会基于循证医学证据, 归纳晚期乳腺癌基因检测的热点问题, 深入探讨晚期乳腺癌基因检测的适用人群和临床意义、不同分子分型患者的检测基因和分子标志物、循环肿瘤DNA、全外显子基因检测的应用, 总结规范二代测序技术在临床中使用的注意事项, 指导临床医师合理应用基因检测, 为晚期乳腺癌患者提供更全面的基因检测信息, 从而制定更精准的治疗策略。     

晚期乳腺癌基因检测热点问题中国专家共识(2021版)

晚期乳腺癌预后较差,疾病特征复杂,后线解救治疗较为困难,但是通过选择有效的治疗方案,部分患者可以实现长期带瘤生存,获得较好的生存质量。近年来,随着分子生物学和基因检测技术的飞速发展,晚期乳腺癌相关研究不断深入,治疗手段不断丰富,基因靶向治疗使晚期乳腺癌患者生存期显著延长。基因检测是分子分型诊断、遗传风险预测、疗效监测、耐药提示以及治疗方案选择的重要手段,对晚期乳腺癌患者的分子病理诊断、靶向药物选择以及治疗模式优化具有重要意义。共识专家委员会基于循证医学证据,归纳晚期乳腺癌基因检测的热点问题,深入探讨晚期乳腺癌基因检测的适用人群和临床意义、不同分子分型患者的检测基因和分子标志物、循环肿瘤DNA、全外显子基因检测的应用,总结规范二代测序技术在临床中使用的注意事项,指导临床医师合理应用基因检测,为晚期乳腺癌患者提供更全面的基因检测信息,从而制定更精准的治疗策略。     

精准医学时代乳腺癌外科治疗的热点问题

肿瘤的精准医学是通过综合应用组学分析、分子检测、分子影像、分子病理、以及大数据分析等技术手段,实现手术、放疗、化疗及生物治疗的微创、精准、合理利用。微创精准外科理念,不仅是将原来的大切口变成小切口,而是要求结合肿瘤基因的分型、大数据分析等技术手段,开展精确的诊断分期、精细的手术方案设计、微创而精湛的手术技术研究和精细的术后管理,这也是区别于传统仅围绕手术技术创新的理念创新。本文就乳腺癌外科治疗的最新进展来探讨微创、精准医学理念指导下乳腺癌的外科治疗策略的变化。     

28基因检测在早期乳腺癌中应用的现状与展望

【摘要】辅助治疗是早期乳腺癌的重要治疗手段。如何精准评估乳腺癌患者的复发转移风险、筛选辅助治疗的获益人群，是亟待解决的临床问题。精准医学理念的发展和分子生物学技术的进步推动了多基因表达谱检测在早期乳腺癌患者中的应用。28基因（RecurIndex）是目前唯一一个结合临床病理因素和基因表达情况的亚洲乳腺癌患者复发风险预测评估模型。随着相关临床研究的开展，28基因检测在中国乳腺癌患者中的应用具有独特的优势。该文就28基因检测的相关研究进行综述，探讨其在指导辅助治疗中的应用价值和发展前景。     

基于靶标指导乳腺癌精准治疗标志物临床应用专家共识（2022版）

 乳腺癌是一种分子水平异质性很高的恶性肿瘤,病理分型结合分子标志物成为常规诊断方式。分子分型可助力乳腺癌的分类分层精准治疗,但在临床实践中乳腺癌标志物的检测方法、评价体系与临床解读等尚存在一定局限性。为了进一步促进基于靶标指导的乳腺癌精准治疗,中国抗癌协会肿瘤标志专业委员会乳腺癌标志物协作组组织临床、病理、分子检测等领域专家,综合国内外乳腺癌临床应用共识和指南、重要文献及临床实践编写本共识,对基于靶标指导乳腺癌精准治疗给出专家组意见,以期提高临床上以标志物为靶标的乳腺癌精准诊断和治疗水平,为乳腺癌患者的系统治疗提供新视野。      

精准医学时代乳腺癌外科治疗的热点问题

肿瘤的精准医学是通过综合应用组学分析、分子检测、分子影像、分子病理、以及大数据分析等技术手段，实现手术、放疗、化疗及生物治疗的微创、精准、合理利用。微创精准外科理念，不仅是将原来的大切口变成小切口，而是要求结合肿瘤基因的分型、大数据分析等技术手段，开展精确的诊断分期、精细的手术方案设计、微创而精湛的手术技术研究和精细的术后管理，这也是区别于传统仅围绕手术技术创新的理念创新。本文就乳腺癌外科治疗的最新进展来探讨微创、精准医学理念指导下乳腺癌的外科治疗策略的变化。     

乳腺癌精准医疗大数据的可视化分析

目的分析乳腺癌精准医疗的既往概况以及动态演化历程,探索未来乳腺癌的精准医疗研究的前沿热点和发展趋势。方法①以中国万方数据库和美国的Web of Science核心数据库关于乳腺癌精准医疗相关文献为信息来源,可视化分析相关文献的发表年份、国家、机构、作者、期刊、基金以及关键词分布,同时比较不同分子分型以及不同靶点乳腺癌研究的文献分布差异;②检索中国临床试验注册中心、北美临床试验注册中心、中国国家药品监督管理局以及美国食品药品监督管理局乳腺癌精准医疗的临床注册以及药物信息。结果①在中国共有450篇与乳腺癌精准医疗有关的研究文章,而在国际上有2303篇与乳腺癌精准治疗有关的研究文章,在中国以及全球均于2016年出现有关乳腺癌精准治疗研究文章的爆发式增长;Luminal A型、Luminal B型、HER2过表达型、基底细胞型(Basal-like型)以及三阴性乳腺癌中以三阴性乳腺癌精准医疗的研究文章数量最多,而乳腺癌精准治疗的主要靶点为HER2,EGFR,CDK4/6,PIK3CA和PARP,其中文献量最大的为HER2;②在全球范围内,美国的哈佛大学是发表乳腺癌精准治疗最多的机构,其次为法国国家健康与医学研究院,而后是加州大学以及德克萨斯大学,而中国发文机构的排名还不太高,仍有很大前进的空间;③美国是乳腺癌精准治疗研究的主要国家,发表超过900篇研究;④中国发文量最多的研究作者为解放军总医院第五医学中心(原解放军第307医院)乳腺肿瘤科的江泽飞教授,而在全球范围内发文量最多的是ANDRE F(巴黎第十一大学)和KATOH M(日本国立癌症研究中心);⑤《中华乳腺病杂志(电子版)》是中国发表乳腺癌精准治疗研究文章最多的期刊,全球发表乳腺癌精准治疗文章的主要期刊为《Personalized Medicine》;⑥全球范围中发表乳腺癌精准治疗研究文章的最主要资助机构为美国卫生与公共服务部以及美国国立卫生研究院,其次为美国国家癌症研究所和中国国家自然科学基金委员会;⑦中国临床注册中心收录的最新乳腺癌研究有177项,而北美临床注册中心收录的最新乳腺癌研究达3663项,主要集中在美国、欧洲和中国;⑧目前已上市的乳腺癌精准治疗药物的治疗靶点主要为HER2,也存在靶点为PARP-1,2,3、PIK3CA、PD-L1、mTOR、CDK4/6、EGFR、HER4的药物,研发机构主要为诺华、辉瑞等跨国药企。结论建立全国甚至全球性精准乳腺癌信息服务集成整合平台将有助于推动乳腺癌精准医疗的发展,同时有助于提升中国企业乳腺癌精准治疗药物的研发能力。     

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa? =?0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3–17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6–3.6, p?<?0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.     

IDH1 mutation detection by droplet digital PCR in glioma

Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.     

Highly sensitive detection of <Emphasis Type="Italic">ESR1</Emphasis> mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing

Purpose

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB–NGS) targeting the hotspot segment (c.1600–1713).

Methods

The sensitivity of MB–NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB–NGS. The results of MB–NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR).

Results

MB–NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB–NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB–NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB–NGS were found to have no mutations by ddPCR.

Conclusion

In conclusion, MB–NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

     

TMSB4Y is a candidate tumor suppressor on the Y chromosome and is deleted in male breast cancer

Male breast cancer comprises less than 1% of breast cancer diagnoses. Although estrogen exposure has been causally linked to the development of female breast cancers, the etiology of male breast cancer is unclear. Here, we show via fluorescence in situ hybridization (FISH) and droplet digital PCR (ddPCR) that the Y chromosome was clonally lost at a frequency of ~16% (5/31) in two independent cohorts of male breast cancer patients. We also show somatic loss of the Y chromosome gene TMSB4Y in a male breast tumor, confirming prior reports of loss at this locus in male breast cancers. To further understand the function of TMSB4Y, we created inducible cell lines of TMSB4Y in the female human breast epithelial cell line MCF-10A. Expression of TMSB4Y resulted in aberrant cellular morphology and reduced cell proliferation, with a corresponding reduction in the fraction of metaphase cells. We further show that TMSB4Y interacts directly with β-actin, the main component of the actin cytoskeleton and a cell cycle modulator. Taken together, our results suggest that clonal loss of the Y chromosome may contribute to male breast carcinogenesis, and that the TMSB4Y gene has tumor suppressor properties.     

IGF2基因在乳腺癌中的研究进展

乳腺癌目前是中国女性发病率第一位的恶性肿瘤， 严重威胁着患者的健康。与乳腺癌相关基因和治疗靶点的研究一直是业界热潮。胰岛素样生长因子2(insulin-like growth factor 2，IGF2)与多种恶性肿瘤的发生与发展存在相关性。随着精准医疗的发展，乳腺癌的治疗越来越趋于个体化，IGF2基因与乳腺癌的侵袭性、发生风险、进展速度、预后治疗是否存在着联系与相关性，笔者总结了IGF2基因在乳腺癌研究的重要成果，包括IGF2基因结构与功能、IGF2基因克隆与表达及IGF2基因与乳腺癌的关系等。     

BEAMing ddPCR与Super ARMS检测EGFR酪氨酸激酶抑制剂治疗后非小细胞肺癌患者循环肿瘤DNA EGFR基因突变的比较研究

背景与目的:BEAMing微滴数字PCR(droplet digital PCR,ddPCR)被认为是灵敏度极高的基因突变检测方法,探讨该方法和扩增阻碍突变系统(Super amplification refractory mutation system,Super ARMS)检测表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)治疗非小细胞肺癌(non-small cell lung cancer,NSCLC)患者后循环肿瘤DNA(circulating tumor DNA,ctDNA)EGFR基因突变的灵敏度差异,进而为指导临床ctDNA检测方法的选择提供更多参考。方法:收集复旦大学附属肿瘤医院2017-2018年接受过EGFR TKI治疗的NSCLC患者血浆33例及国家病理质控评价中心(PathologyQualityControlCenter,PQCC)模拟血浆10例,采用凯杰血浆游离核酸纯化试剂盒进行循环游离DNA(circulating free DNA,cfDNA)提取,分别用ddPCR和Super ARMS方法进行EGFR基因19 del、T790M和L858R突变检测,两种结果进行对比和统计学分析。结果:33例临床样本应用两种方法检测19 del、T790M和L858R突变,阳性率分别为27.3%vs 27.3%(P=1.00)、42.4%vs 27.3%(P=0.23)和27.3%vs 27.3%(P=1.00)。10例PQCC样本结果与标准结果相比,ddPCR检测T790M符合率显著高于Super ARMS(P=0.01)。ddPCR和Super ARMS检测T790M的样本突变丰度范围分别为0.04%~7.66%和0.05%~7.66%。11例T790MddPCR(+)/Super ARMS(-)的平均突变丰度为0.19%(0.04%~0.76%),与10例Super ARMS(+)平均突变丰度[1.73%(0.05%~7.66%)]差异有统计学意义(P=0.03)。EGFR TKI耐药突变T790M突变丰度在0.01%~0.20%、0.20%~1.00%和>1.00%区间的分布分别为42.9%、14.2%和42.9%。结论:BEAMing ddPCR法较Super ARMS法具有更高的灵敏度,可检出更多T790M耐药患者,可能为更多患者选择最有效的靶向治疗方案提供参考。     

Absolute quantification of cell-free microRNAs in cancer patients

The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.