Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell-free (cf) DNA offers a noninvasive approach to detect mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cfDNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end-to-end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom amplicon sequencing. Our approach relies on open-software tools to select primers suitable for multiplex PCR using minimal cfDNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cfDNA. We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cfDNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient-specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cfDNA correlate well with disease progression. Finally, we demonstrate that sequencing of cfDNA can reveal mechanisms of resistance to anti-Epidermal Growth Factor Receptor(EGFR) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cfDNA.

中文翻译：

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通过定制扩增子测序检测大肠癌无细胞DNA中的突变模式。

在癌症治疗期间监测实体瘤的突变模式是肿瘤学的主要挑战。无细胞（cf）DNA中的突变分析提供了一种非侵入性方法来检测可能预后疾病存活或可预测原发或继发耐药性的突变。cfDNA作为诊断工具的应用面临的主要挑战是癌症的多样化突变格局。在这里，我们开发了灵活的端到端实验和生物信息学工作流程，以使用定制扩增子测序分析cfDNA中的突变。我们的方法依靠开放软件工具，以最少的cfDNA作为输入来选择适合于多重PCR的引物。此外，我们开发了一个强大的线性模型，可以从cfDNA的测序数据中识别特定的遗传变异。我们使用我们的工作流程设计了一个定制扩增子板，适用于检测与大肠癌相关的热点突变，并分析了来自34名晚期大肠癌患者的试验队列的连续cfDNA样品中的突变。使用我们的方法，我们可以检测大多数患者的复发性和患者特异性突变模式。此外，我们表明，cfDNA中突变等位基因频率的动态变化与疾病进展密切相关。最后，我们证明了cfDNA的测序可以揭示抗表皮生长因子受体（EGFR）抗体治疗的抗性机制。因此，我们的方法提供了一种简单且高度可定制的方法来探索cfDNA中的遗传变异。