Overview- 'The imaging data and how it will be related to apoptosis alteration could have been more strongly stated, and 'Investigators demonstrate their ability to carry out the extensive imaging experiments proposed, but they do not really explain why they are necessary or how they will help in the planned human trials.'We agree that in the previous application we did not provide adequate biological rationale for the proposed imaging modalities (including apoptosis, proliferation, EGFR expression, and VEGFR expression). Here, we have revised the Background and Significance section to explicitly state the biological rationale for each of the proposed imaging metrics with respect to the utilized treatments. This rationale is further emphasized by the inclusion of additional preliminary in vitro and in vivo data that has been generated since the previous submission. Although beyond the scope of the proposed pre-clinical investigations, we envision that highly effective non-invasive imaging readout(s) of treatment response will provide valuable information towards optimizing individualized patient therapy. More directly, non-invasive imaging metrics could be employed clinically as markers of treatment response in place of invasive biopsy procedures. Thus, in the revised application, we have addressed the clinical utility and intended use of the imaging metrics developed here and prioritize their clinical implementation. 'The potential impact of sequential CT studies and radiation dose was not discussed and should either be dropped or further considered.'The potential impact of sequential CT, as well as the effective radiation dose, is discussed in the revised application (Background and Significance and Research Design and Methods), and we now emphasize that only two CT scans will be collected per animal during the study. These scans will be used for anatomical co-registration with PET images ([18F]-FLT or [18F]-FDG) as are routinely done in PET/CT. 'Criteria for determining the relationships of imaging variables to response are not well described, and there appears to be limited use of statistical input.'In the revised experimental plan, we have carefully described procedures that will be used to validate the proposed imaging metrics, particularly at the tissue level using several well-accepted immunohistochemical (IHC) methods. Dr. Kay Washington, an expert in Gl pathology, has been added as key personnel and will be responsible for the IHC analysis of tissues. Furthermore, our statistical analysis plan has been revised considerably and will be strengthened by the new Statistics Core that will be directed by Dr. Yu Shyr. 'Reduced enthusiasm [was expressed] for the lack of focus in Research Project 1.'We have made a major concerted effort to focus the aims of Project 1. We hypothesize that the most relevant biological readouts to be studied in Project 1 include apoptosis, proliferation, EGFR expression and VEGFR expression. Therefore, we have considerably focused the project by reducing the total number of imaging metrics studied, eliminating tangentially relevant metrics such as PBR imaging with NIR-conPK11195 and DCE MRI. Furthermore, we have focused our therapeutic strategies. Previously, the therapeutic regimen described in Aim 1 relied on the hypothesis that sequential administration of two mechanistically distinct EGFR axis inhibitors would result in improved therapeutic efficacy. Additionally, Aim 2 focused on a novel combination of EGFR and HDAC inhibition. While we remain optimistic with respect to these innovative strategies, we recognize that the focus of this application is upon the development of novel imaging methods and not the establishment of innovative therapeutic regimens. Therefore, our experimental plan now utilizes a much simpler therapeutic strategy with established readouts of clinical response.
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