Photolithographic Tumor DNA Isolation

摘要：光刻肿瘤DNA的提取 个性化肿瘤学的基础是癌症中的突变决定了最佳治疗。 使用新的下一代测序仪和生物信息学进行突变检测越来越有可能，但 目前，DNA提取的第一步是即席、手动和非标准化的。人类肿瘤是复杂的 正常细胞和肿瘤细胞的混合，以及突变检测将变得更可靠和更具重复性 提取了近乎纯净的肿瘤DNA。在这里，我们建议开发一个自动化系统，它可以提取 从常规HE染色显微镜载玻片中提取90%纯净的肿瘤DNA 高分辨率幻灯片扫描仪、图像分析算法和现代3D打印机。机器学习图像 算法可以区分肿瘤和正常细胞，这些信息将被传输到3D打印机， 它将不透明物质直接放置在载玻片上的肿瘤细胞核上。然后，幻灯片暴露在短波中 紫外线破坏未受保护的正常细胞中的DNA，而肿瘤DNA则有选择地受到 光刻掩模。可以从整个玻片中提取DNA，只有受保护的肿瘤细胞中的DNA 可以测序(整个外显子组或靶向测序)或测量CpG甲基化。现场 3D打印机的分辨率约为40微米，因此地形非常不规则复杂，体积很小 像单个肿瘤腺体这样的特征可以通过光刻来保护。整个系统(扫描、分析、 打印、照射、提取)可以在大约24小时内从H&E载玻片中提取90%的纯肿瘤DNA。 转型的潜力是，该系统将目前临时的、高度劳动密集型的 迈向自动化、文档齐全且可重现的提取的技术步骤，可以添加信息和 学习是因为准确提取的细胞数量、它们的表型和空间位置是已知的。因为 它使用图像算法来选择肿瘤细胞，任何人都可以进行“相同”的DNA分离 世界上任何地方。此外，图像算法可以根据反馈更好地提取肿瘤DNA 从DNA测序中。将该系统集成到测序管道中将改善 通过提取几乎纯净(90%)的肿瘤DNA，突变呼叫的可靠性、文献性和重复性， 这将促进可复制癌症的研究和精确肿瘤学的临床翻译。

Abstract: Photolithographic Tumor DNA Isolation Personalized oncology is based on the idea that the mutations in a cancer determine optimal therapy. Mutation detection is increasingly possible with newer next generation sequencers and bioinformatics, but currently the first step of DNA isolation is ad hoc, manual, and non-standardized. Human tumors are complex mixtures of normal and tumor cells, and mutation detection would become more reliable and reproducible if nearly pure tumor DNA was extracted. Here we propose to develop an automated system that can extract >90% pure tumor DNA from conventional H&E stained microscope slides by integrating photolithography with high resolution slide scanners, image analysis algorithms, and modern 3D printers. Machine learning image algorithms can distinguish tumor from normal cells, and this information will be transferred to the 3D printer, which places opaque material directly over tumor nuclei on the slide. The slide is then exposed to short wave UV light to destroy the DNA in unprotected normal cells whereas tumor DNA is selectively protected by the photolithographic mask. DNA can be extracted from the entire slide, and only DNA in the protected tumor cells can be sequenced (whole exome or targeted sequencing) or measured for CpG methylation. The spot resolution of the 3D printer is about 40 microns, and therefore very irregular complex topography and small features like a single tumor gland can be protected by photolithography. The entire system (scan, analyze, print, irradiate, extract) could yield >90% pure tumor DNA from an H&E slide in about 24 hours. The transformational potential is that the system converts a currently ad hoc, highly labor-intensive technical step into an automated, well-documented and reproducible extraction that can add information and learning because the exact extracted cell numbers, their phenotypes and spatial locations are known. Because it uses an image algorithm to select the tumor cells, the “same” DNA isolation can be performed by anyone anywhere in the world. Moreover, image algorithms can “learn” to better extract tumor DNA based on feedback from the DNA sequencing. The integration of this system into a sequencing pipeline would improve the reliability, documentation, and reproducibility of mutation calling by extracting nearly pure (>90%) tumor DNA, which will advance both reproducible cancer research and the clinical translation of precision oncology.