Abstracts: AACR Special Conference on Advances in Liquid Biopsies; January 13-16, 2020; Miami, FL The last few years have witnessed exceptional advances in liquid biopsies based on the isolation and analysis of diverse tumor-derived material circulating in the blood, such as cell-free DNA (cfDNA) and extracellular vesicles (EVs). However, the isolation of different tumor-derived circulating components from a single aliquot of patients’ plasma and the consequent accurate detection of cancer biomarkers is still challenging. By taking advantage of a recently published nickel-based EVs isolation (NBI) protocol (1) that allows for recovery of cfDNA after EVs isolation, we generated a high-sensitivity molecular assay to accurately detect ERBB2 amplification and consequent HER2 overexpression on a limited volume of plasma collected from 20 breast cancer patients (stage I, II, and III) at diagnosis. The amplification of ERBB2 gene and the overexpression of the relevant encoded protein HER2 are currently used in the clinic for breast cancer classification at diagnosis and subsequent treatment decision with the anti-HER2 monoclonal antibody trastuzumab. By performing droplet digital PCR (ddPCR), we were able to detect ERBB2 amplifications in plasma cfDNA from 20 patients selected based on in situ evaluation of tissue biopsies (IHC and FISH). Moreover, we set up an antibody-based affinity reaction to detect HER2 protein on the surface of the isolated EVs that confirmed HER2 overexpression on a subset of patients. Most importantly, we also succeeded in absolute quantification of HER2 transcripts enclosed within EVs by performing ddPCR in samples of patients showing a range of circulating tumor-derived material. The yields and detection performance of this novel methodologic assay were tested on a cohort of healthy individuals (n=20) and on a cohort of triple-negative (and therefore HER2-negative) breast cancer patients (TNBC; n=20; stage: I, II, III). Overall, here we report a pilot study on a novel multimodal noninvasive method for breast cancer-specific biomarker detection from a minimal amount of plasma (1,5mL) integrating cfDNA-derived information with EVs-derived RNA and proteins analysis. This proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. Reference: 1. Notarangelo M et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine 2019;43:114–26. Citation Format: Vera Mugoni, Caterina Nardella, Orsetta Quaini, Yari Ciani, Michela Notarangelo, Antonella Ferro, Orazio Caffo, Vito D'Agostino, Francesca Demichelis. Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B60.

中文翻译：

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摘要B60：分离cfDNA和循环的细胞外囊泡可在单个等份的乳腺癌患者血浆中检测生物标志物

摘要：AACR液体活检进展特别会议；2020年1月13日至16日；基于分离和分析血液中循环的多种肿瘤来源的物质，例如无细胞DNA（cfDNA）和细胞外囊泡（EVs），过去几年来液体活检取得了非凡的进步。然而，从患者血浆的等分试样中分离出不同的肿瘤来源的循环成分以及随之而来的准确检测癌症生物标志物仍然是挑战。通过利用最近发布的镍基电动汽车隔离（NBI）协议（1），该协议允许在电动汽车隔离后恢复cfDNA，我们产生了一种高灵敏度的分子测定法，以在诊断时准确地从20例乳腺癌患者（I，II和III期）收集的有限体积血浆中准确检测ERBB2扩增和随后的HER2过表达。目前，ERBB2基因的扩增和相关编码蛋白HER2的过表达目前在临床上用于乳腺癌的诊断和抗HER2单克隆抗体曲妥珠单抗的治疗决策的分类。通过执行液滴数字PCR（ddPCR），我们能够根据组织活检（IHC和FISH）的原位评估，从20例患者中检测血浆cfDNA中的ERBB2扩增。此外，我们建立了基于抗体的亲和反应，以检测分离出的电动汽车表面上的HER2蛋白，从而证实了一部分患者中HER2过表达。最重要的是，我们还通过对表现出一系列循环肿瘤来源物质的患者样本进行ddPCR，成功地对EV内的HER2转录本进行了绝对定量。在一组健康个体（n = 20）和一组三阴性（因此为HER2阴性）乳腺癌患者（TNBC； n = 20；阶段：n）中测试了这种新型方法的产率和检测性能。一，二，三）。总的来说，在这里我们报告了一项新的多模式非侵入性方法的初步研究，该方法可从最小量的血浆（1.5mL）中整合cfDNA衍生信息与EVs衍生RNA和蛋白质分析，用于乳腺癌特异性生物标志物检测。该概念证明可以最终转化为用于疾病诊断以及治疗选择和疾病进展监测的相关临床应用。参考：1. Notarangelo M等。通过基于镍的血液中多分散细胞外囊泡的镍基分离来超灵敏地检测癌症生物标志物。EBioMedicine 2019; 43：114–26。引用格式：薇拉·穆戈尼（Vera Mugoni），卡特琳娜·纳德拉（Caterina Nardella），奥尔塞塔·奎尼（Orsetta Quaini），亚里·奇亚尼（Maria Notarangelo），安东尼奥·费罗（Antonella Ferro），奥拉齐奥·卡菲（Orazio Caffo），维托·达戈斯蒂诺（Vito D'Agostino），弗朗西斯卡·德米凯里斯（Francesca Demichelis）。cfDNA的分离和循环的细胞外囊泡可在一小份乳腺癌患者血浆中提取生物标志物[摘要]。在：AACR液体活检进展特别会议录中；2020年1月13日至16日；佛罗里达州迈阿密。费城（PA）：AACR；Clin Cancer Res 2020; 26（11_Suppl）：