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）生物标志物发现和验证以及特定的挑战主题，03-CA-110：已知生物标志物的验证。这项综合实验室和临床提案旨在验证影响[18 F]-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志物。引入分子靶向疗法来治疗癌症已经强调了开发和验证高度特异性和稳健的生物标志物以测定这些干预措施的临床和生物活性的迫切需要。许多分子靶向疗法被设计成减少肿瘤细胞增殖。评估肿瘤细胞增殖的常规方法需要侵入性地获取有限量的组织，伴随着风险和由于异质性导致的取样误差。此外，需要连续活检来纵向评估治疗反应，并且在许多情况下在临床上不切实际。由于非侵入性成像具有规避这些限制的潜力，因此对能够评估临床细胞增殖的先进成像方法有极大的兴趣。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，葡萄糖代谢是间接相关的增殖，这表明需要新的成像方法，更直接地测量增殖。一种快速出现的测量细胞增殖的分子成像方法利用PET示踪剂3 '-脱氧-3'[18F]-氟胸苷，[18F]-FLT。理论上，[18 F]-FLT通过报告胸苷补救的活性作为增殖的替代标志物，胸苷补救是一种复杂的细胞周期驱动机制，其从细胞外环境中螯合脱氧核糖核苷，为分裂细胞提供DNA前体。内化后，[18 F]-FLT在胞质酶胸苷激酶1（TK 1）催化的反应中单磷酸化，导致细胞内捕获和蓄积。在正常组织中，TK 1活性在转录、翻译和翻译后水平上受到调节，其活性与细胞周期的DNA合成期（通常为G1晚期至S期）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携带突变，从而以尚不清楚的方式影响TK 1活性的调节和由此产生的[18 F]-FLT PET成像。例如，大约80%的CRC在p53中具有突变，p53是一种能够负调节TK 1表达和增殖的肿瘤抑制基因。最近，我们在人CRC细胞系和临床前CRC小鼠模型中研究了功能失调的p53对TK 1的细胞周期调节和所得[18 F]-FLT PET的影响，特别是在EGF受体（EGFR）阻断的背景下。基于这些研究，很明显，在接受[18 F]-FLT PET成像作为这种疾病增殖的生物标志物之前，迫切需要进一步了解CRC细胞中细胞周期，TK 1活性和[18 F]-FLT摄取之间的关系。该提案有两个具体目标：目标1。确定影响人CRC临床前小鼠模型中[18 F]-FLT PET成像的分子决定因素。我们将利用重现人CRC临床特征（包括p53和KRAS基因突变）的小鼠模型，在EGFR抑制和EGFR抑制与SRC抑制联合治疗背景下验证[18 F]-FLT PET成像。[18 F]-FLT PET的验证将在基因组、蛋白质组和细胞规模上进行，使用从治疗和未治疗队列中采集的成像匹配肿瘤组织。微阵列分析将用于鉴定表达谱随[18 F]-FLT PET变化的协同调节基因簇。通过聚类分析定性鉴定的基因将使用qRT-PCR定量。与蛋白质印迹分析一起，这些数据将用于定量评价遗传和蛋白质组分子事件与肿瘤组织内评估的相应[18 F]-FLT PET成像读数之间的关系。目标二。探索[18 F]-FLT PET在晚期CRC患者新辅助治疗试验中评估抑制EGF受体和EGFR/SRC联合治疗的临床和生物学效应的实用性。作为范德比尔特GI SPORE项目的延伸，我们将纳入相关的治疗前和治疗后[18 F]- FLT PET成像，作为我们机构目前正在招募的两项II期新辅助治疗试验的一部分。一项试验将评估局部晚期直肠癌中EGFR的新辅助阻断，另一项试验将评估EGFR和EGFR/SRC联合阻断的可切除肝转移的CRC患者。与目标1类似，将利用治疗前和手术切除后收集的成像匹配肿瘤组织，在基因组、蛋白质组和细胞规模上进行[18 F]-FLT PET验证。 公共卫生相关性：这项综合实验室和临床提案旨在验证影响[18 F]-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志物。我们设想，所提出的综合性研究，整合了在基因组、蛋白质组和细胞尺度上的验证，将能够更好地理解CRC细胞中细胞周期、TK 1活性和[18 F]-FLT摄取之间的复杂关系，并可能加速临床接受[18 F]-FLT PET成像作为这种疾病中增殖的生物标志物。

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.