Background Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

中文翻译：

---

Surfaceome分析可以从胰腺癌患者的液体活检样本中分离出特定于癌症的外泌体。

背景技术由于循环肿瘤DNA相对缺乏，尤其是在患者积极接受治疗时，循环肿瘤DNA的检测可能受到限制。外泌体提供了一种媒介物，通过该媒介物可以从循环的非肿瘤组织衍生的核酸的浓缩物中富集癌症特异性物质。我们对胰腺导管腺癌（PDAC）外泌体'surfaceome'进行了全面的分析，以鉴定表面蛋白，这些蛋白将使液体活检适合于癌症衍生的外泌体富集，用于下游分子分析。患者和方法通过液相色谱-质谱法在13个人类PDAC和2非肿瘤细胞系中分析了表面外泌体蛋白。从103名PDAC患者中总共采集了173份预期采集的血液样本进行了外来体分离。在74例患者（总共136个外泌体样本）上使用了液滴数字PCR来确定患者在治疗期间的基线KRAS突变检出率。然后，使用针对所选蛋白质的抗体混合物对另外29位患者（37个样品）进行PDAC特异性外泌体捕获，然后进行液滴数字PCR分析。使用分子条形码，靶向测序面板分析了对治疗耐药的PDAC患者的外泌体DNA，以确定用于全面分子分析的富集核酸材料的实用性。结果外泌体“表面组”的蛋白质组学分析显示了多种PDAC特异性生物标志物候选物：CLDN4，EPCAM，CD151，LGALS3BP，HIST2H2BE和HIST2H2BF。在接受积极治疗的患者中，总外泌体中的KRAS突变检出率为44.1％，相比之下，使用选定的生物标记物捕获外泌体后为73.0％。外泌体货物的富集适合于分子谱分析，阐明了携带BRCA2突变的患者对PARP抑制剂治疗有抗性的推定机制。结论外泌体为液体活检提供了独特的机会，可以丰富循环中的肿瘤特异性物质。我们介绍了PDAC外泌体的全面表面组表征，它允许捕获和分子分析肿瘤来源的DNA。阐明了携带BRCA2突变的患者对PARP抑制剂治疗有抗性的推定机制。结论外泌体为液体活检提供了独特的机会，可以丰富循环中的肿瘤特异性物质。我们介绍了PDAC外泌体的全面表面组表征，它允许捕获和分子分析肿瘤来源的DNA。阐明了携带BRCA2突变的患者对PARP抑制剂治疗有抗性的推定机制。结论外泌体为液体活检提供了独特的机会，可以丰富循环中的肿瘤特异性物质。我们介绍了PDAC外泌体的全面表面组表征，它允许捕获和分子分析肿瘤来源的DNA。