Protein and proteolytic activity biomarkers of early stage pancreatic cancer

摘要 识别非常早期的疾病，包括原位癌，是目前最有前途的方法， 降低胰腺导管腺癌（PDAC）死亡率。早期乳腺癌血清生物标志物的开发 诊断对于位于胰腺的肿瘤特别重要，胰腺是一个相对难以接近的器官。 考试在PDAC中寻找临床上有用的生物标志物一直具有挑战性。明确定义的 从正常导管上皮到侵袭性PDAC的遗传途径表明， 侵袭性和不可治愈的癌症的发展。因此，有可能检测所得蛋白质， PTM在肿瘤发展到不可治愈的阶段之前发生变化。不幸的是，所有的生物标志物 迄今为止所研究的方法缺乏灵敏度和特异性以作为临床上有用的筛选试验。这 因此，提案解决了一个主要的未满足的临床需求：目前，尚未制定任何策略， 有效检测早期PDAC。我们最近开发了两种互补的方法， 潜在的生物标志物。第一种方法是使用免疫组织化学方法鉴定和定量血清中PDAC衍生的蛋白质生物标志物。 由培养的PDAC细胞产生和/或分泌的同位素质量标记的蛋白质组，作为内标。 第二种方法是对选定的蛋白酶的活性进行定量，这是一类在蛋白酶中起重要作用的酶。 在癌症生物学中的作用。我们建议扩展我们现有的血清蛋白和酶生物标志物， 通过提高分析灵敏度和分析仔细收集的，统计相关的， 性别和年龄匹配的样本集（PDAC;癌前病变;慢性胰腺炎等）。我们努力的核心 将是（1）基于质谱的蛋白质定量和基于荧光的蛋白酶活性测定，（2） 癌细胞、分泌组和微小细胞的蛋白质组和磷酸化蛋白质组的深入定量分析 量的PDAC肿瘤组织，和（3）肿瘤细胞类型特异性细胞和分泌蛋白参考 标准品，称为Super-SILAC和SEC-super-SILAC，能够准确相对定量选择的 生物体液和肿瘤组织中的蛋白质。我们建议在以下三个条件下检验这些假设 高度整合的研究目的：（1）我们将表征细胞，分泌和肿瘤组织蛋白质组 特异于PDAC以及血清蛋白酶组，并对候选生物标志物进行优先排序，以进一步 基于四个标准的开发。(2)然后，我们将研究蛋白质和蛋白酶生物标志物候选人， 人类血清我们假设这些生物标志物候选物的一个子集在细胞中差异表达或活跃。 早期PDAC与良性胰腺疾病的血清。(3)有希望的生物标志物将受到 涉及通过使用更大的样本群组来建立混合（即，蛋白质和蛋白酶活性）多变量 PDAC预后模型。该模型的性能特征将被确定和验证， 临床应用。开发有效的非侵入性生物标志物，用于筛查有PDAC风险的患者， 目前还不存在，将对这种疾病的治疗和临床过程产生重大影响。

ABSTRACT Identification of very early stage disease, including cancer in situ, is currently the most promising approach to reduce pancreatic ductal adenocarcinoma (PDAC) mortality. Development of serum biomarkers for early diagnosis is particularly significant for tumors located in the pancreas, an organ that is relatively inaccessible to examination. The search for clinically useful biomarkers in PDAC has been challenging. The well-defined genetic pathway from normal ductal epithelium to invasive PDAC suggests that genetic alterations predate development of invasive and incurable cancer. As such, there is the potential to detect the resulting protein and PTM changes before the tumor has progressed to an incurable stage. Unfortunately, all biomarkers investigated to date lack the sensitivity and specificity to serve as a clinically useful screening test. This proposal therefore addresses a major unmet clinical need: currently, no strategy has been developed to effectively detect early stage PDAC. We have recently developed two complementary approaches to identify potential biomarkers. The first seeks to identify and quantify PDAC-derived protein biomarkers in serum using an isotopic mass-labeled proteome, produced and/or secreted by cultured PDAC cells, as an internal standard. The second approach quantifies activities of selected proteases, a class of enzymes that plays an important role in cancer biology. We propose to expand upon the serum protein and enzymatic biomarkers that we have discovered, by improving the analytical sensitivity and by analyzing carefully collected, statistically relevant and gender- and age-matched sample sets (PDAC; pre-cancerous; chronic pancreatitis; etc.). Central to our efforts will be (1) mass spectrometry-based protein quantitation and fluorescence-based protease activity assays, (2) deep and quantitative proteome and phosphoproteome analysis of cancer cells, secretomes and minute amounts of PDAC tumor tissues, and (3) tumor cell type-specific cellular and secreted protein reference standards, termed Super-SILAC and SEC-super-SILAC, that enable accurate relative quantitation of selected proteins in biological fluids and tumor tissues. We propose to test these hypotheses under the following three highly integrated study aims: (1) We will characterize panels of cellular, secreted, and tumor tissue proteins specific for PDAC as well as panels of serum proteases and prioritize candidate biomarkers for further development based on four criteria met. (2) We will then study protein and protease biomarker candidates in human serum. We hypothesize a subset of these biomarker candidates are differentially expressed or active in serum from early PDAC versus benign pancreatic disease. (3) Biomarkers that show promise will be subjected to verification by using larger sample cohorts to build mixed (i.e., proteins and protease activities) multivariate PDAC prognostic models. Performance characteristics of this model will be determined and validated for clinical use. Development of effective, noninvasive biomarkers for screening patients at risk for PDAC, where none currently exist, will have a significant impact on the treatment and clinical course of this disease.