Developing Bioinformatic and Microfluidic Single Cell Methods for Studying Intratumoral Heterogeneity in Acute Myeloid Leukemia

项目摘要/摘要 据推测，化疗耐药性反映了对肿瘤突变克隆的选择。 由于其独特的遗传学，对化疗具有内在抵抗力的细胞。然而，最近有报道称， 显示急性髓系白血病的基因型别与化疗耐药性之间仅有微弱的相关性。 作为另一种选择，我们认为肿瘤内的异质性(ITH，即克隆性多样性)可能是预测 化疗耐药。虽然人们可能会假设ITH水平的增加与不那么容易驯服有关 疾病，几乎没有数据可以明确地将ITH与结果联系起来，更不用说 存在遗传多样性和基因表达或其他表型变化。 虽然克隆进化在白血病发展和治疗中的作用一直是一个关注的焦点 在许多研究途径中，推断单个样品的克隆成分的能力一直是 受到大量肿瘤样本数据使用的限制。许多对克隆的遗传评估都是在 癌症标本将要求克隆的最终描述由单细胞数据提供信息，而不仅仅是 依靠克隆结构的计算去卷积。不幸的是，尽管测序成本很高 减少，从单个单元格生成有统计意义的数据的挑战使大多数技术无法 为此目的，成本效益高或完全不能提供信息。除了成本之外，巨大的技术和 生成和分析数据时存在的计算挑战限制了单细胞分析的研究 实验室通常不参与临床研究。 完善技术能力，以适当地获取批量和单个单元级别的准确数据 处理和解释这些数据，并将这种方法应用于更大规模的患者队列，这是至关重要的下一步 从ITH分析中得出相关的生物学结论。我们的目标是通过将大宗- 利用我们的新型微流控芯片进行单细胞靶向基因分析的水平ITH去卷积，并扩展 这项技术包括下游转录评估。识别遗传多样性、追踪 通过治疗，并将这种多样性与相应的基因表达变化联系起来，都将提供一种 我们在临床上对ITH在癌症治疗中的作用的认识有了实质性的提高。有可能 更直接地询问这两个模型系统中的遗传变异性和可能的转录影响 就像在原始人类标本中一样，我们可以更清楚地了解肿瘤内的异质性是什么作用 在人类的恶行中扮演了重要角色。旨在为所有克隆人创造公平进化竞争环境的替代疗法 并将其频率降低到可控制的水平，可以从根本上将急性疾病转变为慢性疾病 一。对于特别有毒或耐受性差的疗法，这种可能性将是一种有价值的新的临床选择。 旨在废除所有经常导致更具侵略性的复发疾病的克隆。

PROJECT SUMMARY/ABSTRACT It has been hypothesized that chemotherapy resistance reflects selection for a mutant clone of tumor cells that is intrinsically resistant to chemotherapy due to its unique genetics. However, recent reports demonstrate only a weak correlation between acute myeloid leukemia genotype and chemotherapy resistance. As an alternative, we propose that intratumoral heterogeneity (ITH, i.e. clonal diversity) may be a predictor of chemotherapy resistance. While one might hypothesize that increased levels of ITH relates to less tractable disease, little data is available that definitively links ITH with outcome, let alone a relationship between the presence of genetic diversity and gene expression or other phenotypic changes. While the role of clonal evolution during leukemia development and therapy has been a focus for a number of avenues of research, the ability to deduce the clonal composition of individual samples has been limited by the use of data from bulk tumor samples. Many of the genetic assessments of clonality performed on cancer specimens will require the final description of clonality to be informed by single cell data rather than solely relying on computational deconvolution of the clonal structure. Unfortunately, despite sequencing cost reductions, the challenge of generating statistically meaningful data from single cells makes most techniques not cost effective or flat out uninformative for this purpose. Beyond the costs, the enormous technical and computational challenges that exist for generating and analyzing the data limit single cell analysis to research laboratories often not involved with clinical research. Refining the technical ability to derive accurate data at the bulk and single cell levels, appropriately process and interpret these data, and apply this approach to larger cohorts of patients are crucial next steps for making relevant biological conclusions from ITH analyses. We aim to address this challenge by combining bulk- level ITH deconvolution with single cell targeted genetic analysis using our novel microfluidic chip, and extending this technique to include downstream transcriptomic assessments. The ability to identify genetic diversity, track it through therapy, and connect this diversity with corresponding gene expression changes would all provide a substantial improvement in our clinical understanding of the role of ITH in cancer therapy. With the possibility to more directly query the genetic variability and possible transcriptomic implications of this in both model systems as well as in primary human specimens, we can more clearly understand what role intratumoral heterogeneity plays in human malignancy. Alternative therapies designed to level the evolutionary playing field for all clones and reduce their frequency to a manageable level, could essentially transform an acute disease to a chronic one. This possibility would be a valuable new clinical option for especially toxic, or poorly tolerated therapies designed to abolish all clones that often result in more aggressive relapsed disease.