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.
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.
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