Tracking the evolution of breast cancer through single cell analyses of premalignant breast tissues from women at high risk for cancer development

预防性乳房切除术是目前唯一的乳腺癌预防策略的妇女遗传BRCA 1 或BRCA 2突变。虽然这种策略通常有效，但它会显著影响生活质量， 在其它组织中产生的癌症的发展（例如，胰腺癌和前列腺癌）。因此， 迫切需要拦截战略。父母补助金的总体目标是， 乳腺癌高危人群乳腺组织的改变，并为 发现新的预防措施。我们与Sam Aparicio博士的合作研究使用单细胞复制 数量分析已经鉴定了从BRCA 1/2扩增的表面上正常的乳腺上皮细胞群 携带拷贝数改变（CNA）的突变携带者。有趣的是，这些CNA是那些 最常与早期（DCIS）和确诊（IBC）形式的乳腺癌相关， BRCA 1/2杂合性缺失（洛），提示这些变化可能使乳腺上皮细胞 肿瘤引发的基因改变。本补充提案旨在检验总体假设 在BRCA 1或BRCA 2的洛缺失之前发生的遗传改变允许细胞在最终丧失BRCA 1或BRCA 2的情况下存活。 BRCA 1或BRCA 2的两个拷贝，从而增加其细胞适应性和恶性癌症的易感性 随后由同源重组中的缺陷引起的额外遗传改变的积累。我们 我计划通过1）通过确定单个核苷酸来补充我们的CNA分析来解决这个假设 使用Tapestri平台（MissionBio）的癌前乳腺组织的变异和突变景观， 最先进的靶向单细胞DNA和蛋白质测序技术，以及2）利用3D乳腺类器官 模型，以确定CNA/SNV在BRCA 1/2缺失后生存中的功能作用，以及是否有额外的 下游的改变导致对肿瘤发展的敏感性。更好地了解早期 CNA/SNV影响肿瘤发生可以导致识别这些细胞的脆弱性， 在癌症进展之前消除它们，以及开发检测异常细胞扩增的策略 作为监测癌前细胞扩张的手段。重要的是，实现本提案的目标将 使奥列芬特博士能够进一步加强分析单细胞多组DNA所需的计算技能， seq和蛋白质表达数据集独立，以及利用3D类器官模型来识别和 更有效地制定可行的治疗策略。这也将为奥列芬特博士提供机会， 参与各种职业发展经验，包括参加会议和扩大 他的专业网络。总的来说，拟议项目的资金将确保奥列芬特博士扩大他的 科学和专业培训，同时为可行的独立学术研究奠定基础 程序.

Prophylactic mastectomy is currently the only breast cancer prevention strategy for women with inherited BRCA1 or BRCA2 mutations. While often effective, this strategy significantly impacts quality of life and does not prevent the development of cancers arising in other tissues (e.g., pancreatic and prostate cancers). Thus, better interception strategies are urgently needed. The overall goal of the parent grant is to characterize the earliest alterations in breast tissues in individuals at high risk of developing breast cancer and to lay the groundwork for the discovery of new prevention measures. Our collaborative studies with Dr. Sam Aparicio using single-cell copy number analyses have identified expanded population of ostensibly normal breast epithelial cells from BRCA1/2 mutation carriers that carry copy number alterations (CNAs). Interestingly, these CNAs are among those that are most commonly associated with early (DCIS) and established (IBC) forms of breast cancer and are present prior to BRCA1/2 loss of heterozygosity (LOH), suggesting that these changes may predispose mammary epithelial cells to tumor-initiating genetic alterations. This supplement proposal aims to test the overarching hypothesis that the genetic alterations that occur prior to LOH of BRCA1 or BRCA2 allow cells to survive eventual loss of both copies of BRCA1 or BRCA2, thereby increasing their cellular fitness and predisposition to malignant cancer following accumulation of additional genetic alterations caused by defects in homologous recombination. We plan to address this hypothesis by 1) complementing our CNA analysis by determining the single nucleotide variant and mutational landscape of precancerous breast tissues using the Tapestri Platform (MissionBio), a state-of-the-art, targeted single cell DNA and protein sequencing technology and 2) utilizing 3D breast organoid models to determine the functional role of CNAs/SNVs in survival after loss of BRCA1/2 and whether additional alterations downstream result in sensitization to tumor development. A better understanding of how early CNAs/SNVs influence tumorigenesis can lead to the identification of vulnerabilities of these cells in order to eliminate them before cancer progresses, as well as to develop strategies to detect expansion of aberrant cells as a means of monitoring pre-malignant cell expansion. Importantly, accomplishing the goals of this proposal will enable Dr. Oliphant to further strengthen the computational skills needed to analyze single-cell multiomic DNA- seq and protein expression datasets independently, as well as leverage 3D organoid models to identify and develop actionable therapeutic strategies more effectively. It will also provide Dr. Oliphant with opportunities for engagement in various career development experiences including participating at conferences and expanding his professional network. Overall, the funding of the proposed project will ensure that Dr. Oliphant expands his scientific and professional training, while establishing the foundation for a viable independent academic research program.