Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 10⁴ wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

中文翻译：

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声阵列生物芯片结合等位基因特异性 PCR 用于组织和液体活检中的多种癌症突变分析

定期筛查癌性点突变对于癌症管理和治疗选择非常重要。尽管可以使用新一代测序和液滴数字 PCR 等优秀技术，但这些技术仍然缺乏速度、简单性和成本效益。这里提出了一种新方法，其中等位基因特异性 PCR (AS-PCR) 与新型高基频石英晶体微天平 (HFF-QCM) 阵列生物传感器相结合，分别用于癌症点突变的扩增和检测。对于概念验证，该方法应用于筛选BRAF V600E 和KRAS掺入样品和临床样品中的 G12D 突变。关于BRAF目标，证明了0.01%的分析灵敏度，即在超过10 ^{4 个}野生型分子中检测到基因组DNA的1个突变拷贝；此外，当采用灵敏度为 0.05% 的优化分析时，在 KRAS 检测过程中获得了定量结果。使用从黑色素瘤、结肠直肠癌和肺癌患者获得的组织和血浆样本对这些测定进行了验证。结果与 Sanger 测序和液滴数字 PCR 完全一致，证明了BRAF和KRAS 的有效检测等位基因频率低于 1% 的样本中的突变。该方法的高灵敏度和技术就绪水平，以及多样本分析（24 阵列生物芯片）的能力、成本效益和与常规工作流程的兼容性，有望实现这种 AS-PCR/声学方法在临床肿瘤学中作为组织和液体活检的工具。