Molecular Determinants Affecting FLT-PET in Colorectal Cancer

描述(由申请人提供)：本申请涉及广泛的挑战领域(03)生物标记物的发现和验证，以及特定的挑战主题，03-CA-110：已知生物标记物的验证。这项综合实验室和临床的建议试图验证影响[18F]-Flt PET成像的分子决定因素作为结直肠癌(CRC)靶向治疗反应的生物标记物。分子靶向疗法的引入凸显了开发和验证高度特异和强大的生物标记物以分析这些干预措施的临床和生物学活性的迫切需要。许多分子靶向治疗被设计来减少肿瘤细胞的增殖。传统的评估肿瘤细胞增殖的方法需要侵入性地获取有限数量的组织，由于异质性而伴随着风险和抽样误差。此外，需要连续活检来纵向评估治疗反应，在许多情况下，这在临床上是不可行的。由于非侵入性成像有可能绕过这些限制，人们对将能够评估细胞增殖的成像方法推向临床非常感兴趣。广泛使用的PET示踪剂[18F]-FDG用于测量葡萄糖代谢，是癌症检测和分期的重要工具。然而，葡萄糖代谢与增殖是间接相关的，这表明需要新的成像方法来更直接地测量增殖。利用PET示踪剂3‘-脱氧-3’[18F]-氟代胸苷，[18F]-Flt，一种快速出现的测量细胞增殖的分子成像方法。理论上，[18F]-Flt通过报告胸苷挽救活性作为增殖的替代标记物，胸苷挽救是一种复杂的细胞周期驱动机制，将脱氧核糖核苷从细胞外环境中隔离出来，为分裂细胞提供DNA前体。在内化后，[18F]-Flt在胞浆酶胸苷激酶1(TK1)的催化下被单磷酸化，从而导致细胞内的捕获和积累。在正常组织中，TK1的活性在转录、翻译和翻译后水平上受到调控，并且它的活性与细胞周期的DNA合成阶段(通常是G1晚期到S)密切相关。相比之下，癌细胞经常在控制细胞周期调节的分子机制中存在突变，从而影响TK1活性的调节和由此产生的[18F]-Flt PET成像，其方式尚不清楚。例如，大约80%的癌组织存在P53突变，P53是一种肿瘤抑制基因，具有负向调节TK1表达和增殖的能力。最近，我们在人类CRC细胞系和临床前CRC小鼠模型中研究了功能失调的P53对TK1和由此产生的[18F]-Flt PET的细胞周期调节的影响，特别是在EGF受体(EGFR)阻断的情况下。基于这些研究，很明显，在接受[18F]-Flt PET成像作为该病增殖的生物标志物之前，迫切需要更好地了解细胞周期、TK1活性和[18F]-Flt摄取之间的关系。这项建议有两个特定的目标：目的1.在临床前人类结直肠癌小鼠模型中，确定影响[18F]-Flt PET成像的分子决定因素。我们将利用概括人类结直肠癌临床特征的小鼠模型，包括p53和KRAS的基因突变，在EGFR抑制和联合EGFR抑制和SRC抑制的治疗背景下验证[18F]-Flt PET成像。[18F]-Flt PET的验证将在基因组、蛋白质组和细胞水平上进行，使用从治疗和未治疗队列中收集的成像匹配的肿瘤组织。微阵列分析将被用来识别其表达谱随着[18F]-Flt PET的变化而发生变化的协调调控基因簇。通过聚类分析定性鉴定的基因将使用qRT-PCR进行量化。与蛋白质印迹分析一起，这些数据将被用来定量评估遗传和蛋白质组分子事件与肿瘤组织内评估的相应[18F]-Flt PET成像读数之间的关系。目的2.探讨[18F]-Flt PET在晚期结直肠癌新辅助试验中抑制EGF受体和联合应用EGFR/SRC的临床和生物学效应。作为Vanderbilt GI孢子计划的扩展，我们将把相关的治疗前和治疗后[18F]-Flt PET成像作为目前在我们机构登记的两个II期新辅助试验的一部分。一项试验将评估局部进展期直肠癌的新辅助阻断EGFR，另一项试验将评估EGFR和联合阻断EGFR/SRC联合阻断可切除肝转移的结直肠癌患者。与AIM 1类似，[18F]-Flt PET的验证将在基因组、蛋白质组和细胞范围内进行，使用在治疗前和手术切除后收集的影像匹配的肿瘤组织。 公共卫生相关性：这项综合实验室和临床提案试图验证影响[18F]-Flt PET成像的分子决定因素作为结直肠癌(CRC)靶向治疗反应的生物标记物。我们预计，所提出的综合研究将整合基因组、蛋白质组和细胞水平的验证，将有助于更好地理解细胞周期、TK1活性和[18F]-Flt摄取之间的复杂关系，并可能加速临床接受[18F]-Flt PET成像作为这种疾病增殖的生物标志物。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03) Biomarker Discovery and Validation and specific Challenge Topic, 03-CA-110: Validation of Known Biomarkers. This integrated laboratory and clinical proposal seeks to validate molecular determinants affecting [18F]-FLT PET imaging as a biomarker of response to targeted therapeutics in colorectal cancer (CRC). The introduction of molecularly targeted therapies to treat cancer has underscored a critical need to develop and validate highly specific and robust biomarkers to assay the clinical and biological activity of these interventions. Many molecularly targeted therapies are designed to reduce tumor cell proliferation. Conventional methods to assess tumor cell proliferation require invasive procurement of limited amounts of tissue with attendant risks and sampling errors due to heterogeneity. Furthermore, serial biopsy is required to assess treatment response longitudinally and is clinically impractical in many instances. Since noninvasive imaging has the potential to circumvent these limitations, there is tremendous interest in advancing imaging methods capable of assessing cellular proliferation to the clinic. The widely used PET tracer [18F]-FDG, which is used to measure glucose metabolism, is an important tool for cancer detection and staging. However, glucose metabolism is indirectly related proliferation, suggesting a need for novel imaging methods that measure proliferation more directly. A rapidly emerging molecular imaging approach to measure cellular proliferation utilizes the PET tracer 3'- deoxy-3'[18F]-fluorothymidine, [18F]-FLT. Theoretically, [18F]-FLT serves as a surrogate marker of proliferation by reporting the activity of thymidine salvage, a complex cell cycle-driven mechanism that sequesters deoxyribonucleosides from the extracellular environment to provide dividing cells with DNA precursors. Upon internalization, [18F]-FLT is monophosphorylated in a reaction catalyzed by the cytosolic enzyme thymidine kinase 1 (TK1) which results in intracellular trapping and accumulation. In normal tissues, TK1 activity is regulated at transcriptional, translational, and post-translational levels and its activity is closely correlated with the DNA synthesis phase of cell cycle (typically late G1 through S). In contrast, cancer cells frequently harbor mutations in the molecular machinery that govern cell cycle regulation, thereby affecting regulation of TK1 activity and resultant [18F]-FLT PET imaging in ways not well understood. For example, approximately 80% of CRCs harbor mutations in p53, a tumor suppressor gene with the capacity to negatively regulate TK1 expression and proliferation. Recently, we have studied the effects of dysfunctional p53 on cell cycle regulation of TK1 and resultant [18F]-FLT PET in both human CRC cell lines and preclinical CRC mouse models, particularly within the context of EGF receptor (EGFR) blockade. Based upon these studies, it is clear that an improved understanding of the relationship between cell cycle, TK1 activity, and [18F]-FLT uptake in CRC cells is urgently needed prior to acceptance of [18F]-FLT PET imaging as a biomarker of proliferation in this disease. This proposal has two Specific Aims: Aim 1. To identify molecular determinants that affect [18F]-FLT PET imaging in preclinical mouse models of human CRC. We will utilize mouse models that recapitulate clinical features of human CRC, including genetic mutations in p53 and KRAS, to validate [18F]-FLT PET imaging within the therapeutic contexts of EGFR inhibition and combined EGFR inhibition and SRC inhibition. Validation of [18F]-FLT PET will be performed at genomic, proteomic, and cellular scales utilizing imaging-matched tumor tissues collected from treated and untreated cohorts. Micro-array analysis will be used to identify clusters of coordinately regulated genes whose expression profile changes concomitantly with [18F]-FLT PET. Genes identified qualitatively via cluster analysis will be quantified using qRT-PCR. Together with western-blot analysis, these data will be used to quantitatively evaluate the relationship between genetic and proteomic molecular events and corresponding [18F]-FLT PET imaging readouts assessed within tumor tissue. Aim 2. To explore the utility of [18F]-FLT PET to assess clinical and biological effects of inhibiting EGF receptor and combined EGFR/SRC in neoadjuvant trials of patients with advanced CRC. As an extension of the Vanderbilt GI SPORE program, we will incorporate correlative pre- and post-treatment [18F]- FLT PET imaging as part of two Phase II neoadjuvant trials presently enrolling at our institution. One trial will evaluate neoadjuvant blockade of EGFR in locally advanced rectal cancer and the other trial will evaluate EGFR and combined EGFR/SRC blockade CRC patients with resectable liver metastases. Analogous to Aim 1, validation of [18F]-FLT PET will be performed at genomic, proteomic, and cellular scales utilizing imaging- matched tumor tissues collected pre-treatment and following surgical resection. PUBLIC HEALTH RELEVANCE: This integrated laboratory and clinical proposal seeks to validate molecular determinants affecting [18F]-FLT PET imaging as a biomarker of response to targeted therapeutics in colorectal cancer (CRC). We envision that the comprehensive studies proposed, which integrate validation at genomic, proteomic, and cellular scales, will enable improved understanding of the complex relationship between cell cycle, TK1 activity, and [18F]-FLT uptake in CRC cells and may accelerate clinical acceptance of [18F]-FLT PET imaging as a biomarker of proliferation in this disease.