Evaluation of TP53 mutations in non-cancerous tissue as novel biomarkers of ovarian cancer risk.

非癌组织中TP53突变作为卵巢癌风险新生物标志物的评价 TP53是人类肿瘤中突变最多的基因。令人惊讶的是，最近的研究揭示了TP53 在非癌症组织中也发现了非常低的突变频率。这些突变是否会增加 未来癌症进展的风险目前尚不清楚。然而，这种可能性得到了以下事实的支持： 正常组织中的TP53突变与癌症中的TP53突变相似：它们往往聚集在热点和 大多是有害的。这表明了一个正在进行的积极选择的进化过程 并可能导致某些人的癌症进展。因此，我们假设 体细胞TP53突变负荷增加的个体患癌症的风险可能更高。我们 我将在高级别浆液性卵巢癌(HGSOC)中测试这一假设，HGSOC是一种由TP53驱动的癌症类型 突变，通常在晚期诊断，导致高死亡率。需要更好的HGSOC 对于BRCA1和BRCA2(BRCA1/2)发生生殖系突变的女性来说，风险生物标志物尤其关键 携带者)，分别有约45%和20%的HGSOC终身风险。使用超精确的 一种称为双重测序的测序方法我们最近证明患有HGSOC的女性以及 BRCA1/2携带者血DNA中TP53突变频率较高。类似地，巴氏涂片DNA从 HGSOC患者的TP53癌样突变频率显著高于巴氏涂片 来自未患癌症的女性。这些发现支持了我们的假设，即TP53的体质负荷较高 突变克隆可能会增加HGSOC的风险。我们建议进一步探索这些结果及其潜力。 利用(1)CRISPR-DS，一种基于双重测序的方法作为癌症生物标记物 由我们团队开发的比原始方法更高效、更快、更便宜、需要更少DNA的方法； 和(2)华盛顿大学妇科肿瘤组织库，它有一个广泛的信息库 从疑似妇科手术的妇女身上采集血液和巴氏涂片样本 由于遗传性癌症易感性而导致的肿块或预防性疾病。在目标1中，我们将执行超深(~3,000倍) BRCA1/2携带者和无HGSOC携带者外周血DNA的CRISPR-DS TP53测序 我们将确定TP53突变、HGSOC和生殖系BRCA1/2突变之间的关联。为 在目标2中，我们将测试巴氏涂片DNA中的TP53突变是否也与 HGSOC和/或生殖系突变状态和TP53联合突变信息是否来自 巴氏涂片和血液对癌症的预测价值比任何一种单独检测都要好。这项研究突破了 基于体细胞TP53突变的潜在生物标记物价值，直到最近才被发现 因为它们的频率很低。CRISPR-DS允许它们以高通量方式进行精确测量， 从而使翻译生物标记物的开发能够导致更好的预测和 预防HGSOC以及由TP53突变引起的其他癌症。

Evaluation of TP53 mutations in non-cancerous tissue as novel biomarkers of ovarian cancer risk TP53 is the most mutated gene in human tumors. Surprisingly, recent research has revealed that TP53 mutations are also found at very low frequency in non-cancerous tissues. Whether these mutations increase the risk of future cancer progression is currently unknown. However this possibility is supported by the fact that TP53 mutations in normal tissue are similar to TP53 mutations in cancers: they tend to cluster in hotspots and are mostly deleterious. This indicates an ongoing evolutionary process of positive selection that takes place through life and might result in cancer progression in some individuals. Accordingly, we hypothesize that individuals with an elevated load of somatic TP53 mutations might be at a higher risk of developing cancer. We will test this hypothesis for high grade serous ovarian cancer (HGSOC), a cancer type driven by TP53 mutations and which is typically diagnosed at late stage, causing high mortality. The need for better HGSOC risk biomarkers is especially critical for women with germline mutations in BRCA1 and BRCA2 (BRCA1/2 carriers) who have about 45% and 20% lifelong risk of HGSOC, respectively. Using an ultra-accurate sequencing method called Duplex Sequencing we recently demonstrated that women with HGSOC as well as BRCA1/2 carriers harbor higher frequency of TP53 mutations in blood DNA. Similarly, Pap smear DNA from women with HGSOC contained significantly higher frequency of TP53 cancer-like mutations than Pap smears from women without cancer. These findings support our hypothesis that a constitutionally higher load of TP53 mutant clones might elevate the risk of HGSOC. We propose to further explore these results and their potential utility as cancer biomarkers taking advantage of (1) CRISPR-DS, a Duplex Sequencing based method developed by our group that is more efficient, faster, cheaper, and requires less DNA than the original method; and (2) the University of Washington Gynecologic Oncology Tissue Bank, which has an extensive repository of blood and Pap smear samples collected from women that underwent gynecological surgery for suspected masses or prophylactically due to inherited cancer susceptibility. In Aim 1, we will perform ultra-deep (~3,000x) CRISPR-DS TP53 sequencing of blood DNA from BRCA1/2 carriers and non-carriers with and without HGSOC and we will determine associations between TP53 mutations, HGSOC, and germline BRCA1/2 mutations. For a subset of women, in Aim 2 we will test whether TP53 mutations in Pap smear DNA are also associated with HGSOC and/or germline mutation status and whether the combination of TP53 mutational information from Pap smear and blood has a better cancer predictive value than either test separately. This research breaks ground on the potential biomarker value of somatic TP53 mutations, which were unrecognized until recently due to their very low frequency. CRISPR-DS allows their accurate measurement in a high-throughput manner, thus enabling the development of a translational biomarker that could lead to improved prediction and prevention of HGSOC as well as other cancers driven by TP53 mutations.