High Definition Clonal Analyses of Archival Pancreatic Adenocarcinoma Samples

描述（由申请人提供）：基因组不稳定性似乎与达尔文选择合作，通过基因组畸变以加速速率发生的过程促进癌症形成，并且提供选择性生长优势的那些改变导致克隆进化和扩增。因此，癌症中的基因组畸变模式可能差异很大，甚至在同一器官部位产生的肿瘤中也是如此。目前癌症基因组研究的一个基本假设是，根据每个样本中存在的选定畸变及其靶向途径，肿瘤细胞对可用的临床干预措施具有不同的抗性或敏感性。因此，患者样品中选定畸变的鉴定将有助于开发新的治疗靶点，其可以被推进用于改善的更个性化的癌症治疗方法。基因组技术的最新进展提供了对感兴趣的样品的高度详细的分析。例如，寡核苷酸CGH阵列可以在基因内作图分辨率下区分整个基因组中的单拷贝变化，探针错误率<5%。研究复杂组织的一个挑战是细胞混合物的存在和人类肿瘤的多克隆性质。此外，除了掩盖关键的基因组畸变之外，活组织检查中肿瘤细胞群体的克隆混合物的存在使得难以辨别哪些基因组畸变同时发生并全面定义患者样品的基因组背景。福尔马林固定石蜡包埋（FFPE）组织是临床注释样本的巨大资源，具有患者随访数据，包括诊断和治疗结果。因此，这些样本代表了高度期望的和信息丰富的材料，用于应用高清晰度基因组学，可以改善患者管理，并提供个性化治疗选择的分子基础。然而，迄今为止，使用这些样品进行高分辨率基因组分析的主要限制是从感兴趣的样品中提取的DNA的质量高度可变。流式细胞术已被用于使用客观的可定量标记物从各种组织中的原发性活检中鉴定和分离肿瘤克隆。一旦鉴定出单个群体，可以将其流动纯化至大于95%的纯度，用于随后的分子分析。我们最近开发的方法，适用于单参数流式细胞术的新鲜冷冻样品的高清晰度克隆aCGH分析胰腺癌。本申请的总体目标是通过开发和验证与高清晰度阵列CGH分析和下一代临床样本测序兼容的多参数流式细胞术测定来扩展这些方法。这些将包括来自胰腺癌的固定石蜡包埋（FFPE）样本的二倍体和非整倍体细胞群。

DESCRIPTION (provided by applicant): Genomic instability appears to cooperate with Darwinian selection to promote cancer formation through a process in which genomic aberrations occur at accelerated rates, and those alterations that provide a selective growth advantage lead to clonal evolution and expansion. Consequently the patterns of genomic aberrations in cancers can vary extensively, even in tumors arising in the same organ site. A fundamental hypothesis of current cancer genome efforts is that tumor cells become differentially resistant or sensitive to available clinical interventions according to selected aberrations present in each sample and the pathways that they target. Thus the identification of selected aberrations in patient samples will help develop novel therapeutic targets that can be advanced for improved more personalized approach to the treatment of cancer. Recent advances in genomic technologies provide highly detailed analyses of samples of interest. For example oligonucleotide CGH arrays can distinguish single copy changes across an entire genome at intragenic mapping resolution with probe error rates of <5%. A challenge in studying complex tissues is the presence of admixtures of cells and the polyclonal nature of human neoplasias. Furthermore, in addition to masking critical genomic aberrations, the presence of clonal mixtures of neoplastic cell populations in biopsies makes it prohibitively difficult to discern which genomic aberrations occur concurrently and to comprehensively define the genomic contexts of patient samples. Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of clinically annotated samples with patient follow-up data including diagnostic and therapeutic outcomes. As such, these samples represent highly desirable and informative materials for the application of high definition genomics that could improve patient management and provide the molecular basis for the selection of personalized therapeutics. However a major limitation to the use of these samples for high resolution genomic analyses to date is the highly variable quality of the DNA extracted from samples of interest. Flow cytometry has been used to identify and isolate neoplastic clones from primary biopsies in a variety of tissues using objective quantifiable markers. Once identified individual populations can be flow purified to greater than 95% purity for subsequent molecular analyses. We have recently developed methods that adapt single parameter flow cytometry of fresh frozen samples to high definition clonal aCGH analyses of pancreatic cancer. The overall objective of this application is to extend these methods by developing and validating multiparameter flow cytometry assays that are compatible with high definition array CGH analyses and next generation sequencing of clinical samples. These will include diploid and aneuploid cell populations from formulin fixed paraffin embedded (FFPE) samples of pancreatic adenocarcinomas.