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Although this is officially an annual report, the release of the funds was delayed until July 2014 because of the PI s transfer from Beth Israel Deaconess Medical Center to the Mayo Clinic. Activities since then have succeeded in obtaining ICACUC and ACURO approvals, which was the first task in the statement of work, months 0-3. We have also initiated work on the second task, months 3-6 in which the design of the adjustable stiffness fixators will be finalized and the manufacturing initiated. One such fixator has been tested on a cadaveric sheep tibia. In the unlocked, loose position, the axial stiffness of the tibia and fixator was 102 N/mm. In the locked, stiff position it was 218 N/mm. This result is better than expected and close to the target of a 2.5-fold increment suggested by our previous studies using rats. This aspect of the project is the present focus of attention, and additional cadaver legs will be tested in due course., The original document contains color images.

Polytrauma is most often caused from explosive devices and accounts for about 65 percent of injuries to our military personnel. The patients who have polytrauma are at increased risk of developing either bleeding and/or a clot in their veins which cause a life-threatening event known as venous thromboembolism (VTE). We began enrollment of patients into the study on 2 February 2011. As of 1 October 2014, we have successfully enrolled and collected blood samples on 1139 patients and 89 healthy volunteers. We have thus far analyzed plasma samples of over 443 patients and 89 volunteers. In our preliminary analysis of thrombin generation and procoagulant microvesicle analysis, we have observed that thrombin generation is accelerated early after traumatic injury and there are greater numbers of procoagulant microvesicles noted after traumatic injury relative to healthy volunteers., The original document contains color images.

The goal of this project is to develop biopsy based assays to assess the probability that patients with a negative biopsy or with a prostate cancer (CaP) Gleason score 6 (GS6) biopsy actually have significant CaP of Gleason score 7 or higher which was missed during the biopsy evaluations due to insufficient sampling. In 2014, we considerably expanded the size of the discovery samples which will improve the robustness of biomarkers and developed methodologies for improving the detection of epigenetic modification in rare cells. Also, we tested several methods for multiplex analyses of transcriptome and DNA methylation changes. Further, we have secured additional funding which will allow us to expand transcriptome and epigenome profile of indolent and significant prostate tumors. Finally, we have applied for major grant initiatives which if funded, will help expanding the scope of this study through the use of nano-scale devices and quantum dot based assays. We are expecting the publications of our research results in the coming year., The original document contains color images.

The breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 (BRCA1/2) are key components of the Fanconi anemia (FA)/homologous recombination (HR) pathway of DNA repair. Previous work had shown that cancer cells with deleterious FA/HR pathway mutations are hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Importantly, however, only about half of the cancer patients with germline FA/HR pathway mutations respond to PARP inhibitors, raising the question of why a substantial fraction of HR-deficient cancers are resistant to these agents in the clinic. Based on previous work in the Swisher and Kaufmann laboratories, we proposed to test the hypothesis that two different conditions must be met for ovarian cancer to be hypersensitive to platinum and PARP inhibitors: The FA/HR pathway must remain disabled and NHEJ must remain intact and functional. Although we proposed two aims, the aim in previously banked specimens was removed before the present grant was awarded, leaving us with the following aim: Correlate biomarkers of HR deficiency and NHEJ pathway integrity in pre-treatment biopsies with response to a PARPi in a prospective single-agent PARPi phase 2 clinical trial in sporadic ovarian carcinoma. Over the past 11 months we have i) completed IRB and HRPO review of our project, ii) developed and performed rigorous validation of our IHC assays, and, as of a few weeks ago, iii) begun accessioning samples from the phase 2 rucaparib trial (Ariel 2. ClinicalTrials.gov identifier NCT01891344)., The original document contains color images.

The specific aim of this proposal is to develop improved small-molecule botulinum neurotoxin serotype A endopeptidase (BoNTAe) inhibitors with Ki values of 100 nM. We have developed BoNTAe inhibitors MHC and HAB (see US Patent 8,404,728 B2) that showed significant 6-hour-post-exposure protection of mice against 5 LD50 BoNTA. HAB also showed significant 4-hour-post-exposure protection of zebrafish against 5 LD50 BoNTA. Our kinetics and affinity analyses using the surface plasmon resonance technique showed that the Ki value of our BoNTAe inhibitor AHP (see US Patent 8,404,728 B2) is 71 + or - 26 nM (2 independent experiments with chi square values of 0.393 and 0.396). In addition, we have developed a generic approach to cysteine-targeting irreversible inhibitors of pathogenic enzymes (Adv. Insect Physiol. 46, 435-494, 2014) that enables conversion of our reversible BoNTAe inhibitors to irreversible inhibitors that target Cys164 in the BoNTAe active site to effectively counteract BoNTA that has an unusually long in vivo half life of 31 days.

The goal of this grant proposal was to: 1) construct a prostate tissue-specific expression quantitative trait loci (eQTL) dataset; and 2) utilize this dataset to identify candidate genes for existing prostate cancer (PC) risk-single nucleotide polymorphisms (SNPs) that could then be followed up in future studies. To accomplish this goal we performed a genomewide SNP analysis (Illumina Human Omni 2.5M SNP array) and a genome-wide mRNA expression analysis (RNA sequencing) on a common set of 500 samples of normal prostate tissue sampled from men with PC. Of 500 processed samples, 471 samples passed stringent quality control (QC) and were available for further analysis. Our primary analysis focused on identifying eQTLs for 146 PC risk-SNPs, including all SNPs in linkage disequilibrium with each risk-SNP (r2 0.5), resulting in 100 unique risk-intervals. Furthermore, we focused on cis-acting associations only where the transcript was located within 2Mb (+/-1Mb) of the risk-SNP interval. Of all SNPs located in the 100 risk-intervals (N=6324 SNPs), 1,718 demonstrated a significant eQTL signal after adjustment for sample histology (% lymphocytes and % epithelial cells) and meeting a Bonferroni-adjusted p-value threshold of 1.96 e-7 (ranged from 1.96 e-7 to 1.52 e-91). Of the 100 PC riskintervals, 31 (31%) demonstrated a significant eQTL signal and these were associated with 54 genes., The original document contains color images.

Cognitive dysfunction is increasingly recognized in multiple sclerosis (MS), and the role of cortical gray matter lesions as possible causative factors for cognitive dysfunction, and possibly as a main disease initiating factor has recently been proposed, partially by our team's observations in MS neuropathology studies. Our main hypothesis is that cortical lesions determine the formation of white matter lesions along tracts directly originating from them, and that the extent of cortical dysfunction will be directly proportional to the location and overall load of cortical lesions. In order to detect cortical lesions more efficiently, our team has developed a customized pulse sequence we termed GM-DIR, with increased sensitivity for cortical lesion detection. Utilizing standard DIR, our custom-made GM-DIR and 60-direction DTI studies, we have been able to determine in an early pilot study that connections between lesions do exist. During the study period, we have processed data on 25 early MS patients (less than 5 years of disease activity) and compared that with 24 controls, and concluded that white matter lesions do in deed appear to be connected with cortical lesions. Our first abstract related to these novel observations has been submitted to the upcoming ACTRIMS/ECTRIMS meeting. Furthermore, we started collecting and analyzing neuropsychiatric outcome measures using the MACFIMS battery. Our first results are presented herein this report. Correlative analysis of MRI and MACFIMS data has been initiated and is ongoing in our laboratory. Enrollment has been steady; we don't anticipate any problems meeting our target during the study duration. We believe that our observations will meaningfully contribute not only to our understanding of cognitive dysfunction in early MS, but also to key disease initiating events, which may pave the way to the development of more specific disease modifying therapies.

The U.S. military is providing amputees with state-of-the-art prosthetic devices that allow them to more easily adapt to the different surfaces and environments they are faced with in the community. In addition to walking and changing direction, amputees must be able to manage uneven terrain, crowded environments, stairs, ramps, and hills. The largest problem for a lower extremity amputee is falls. Even with the newer technologies, falls and injuries due to falling are still an issue. Falling history and balance confidence are associated with reduced mobility capability and social activity. The goal of this research effort is to rehabilitate individuals with lower extremity amputations to reduce falls using a novel training method. The training program utilizes a microprocessor-controlled treadmill designed to deliver task-specific training perturbations. The training consists of six, 30 minute sessions delivered over a 2-week period. Trunk motion and velocity were assessed using a perturbation test in an immersive virtual environment, since trunk kinematics has been shown to determine fall likelihood. We have enrolled 18 research subjects at the Naval Medical Center San Diego. Mean trunk flexion angle and velocity significantly improved after participating in the training program. The improved performance was maintained up to 6 months. Subjects reported decreased uncontrolled and semi-controlled falls in their free-living environment outside of the research laboratory. These results indicate that task-specific training is an effective rehabilitation method to reduce falls in persons with lower limb amputations., The original document contains color images.

Polytrauma is most often casused from explosive devices and accounts for about 65 percent of injuries to our military personnel. The patients who have polytrauma are at increased risk of developing either bleeding and/or a clot in their veins which cause a life-threatening event known as venous thromboembolism (VTE). We began enrollment of patients into the study on 2 February 2011. As of 1 October 2013, we have thus far analyzed plasma samples of over 426 patients and volunteers. In our preliminary analysis of thrombin generation and procagulant microvesicles noted after traumatic injury relative to healthy volunteers., The original document contains color images.

The goal of this project is to develop biopsy based assays to assess the probability that patients with a negative biopsy or with a prostate cancer (CaP) Gleason score 6 (GS6) biopsy actually have significant CaP of Gleason score 7 or higher which was missed during the biopsy evaluations due to insufficient sampling. According to the DOD regulations, all research activities began after the approval of the research protocol by the HRPO at the end of March 2012. Since then, we have completed the transcriptomic profiling of the samples described in the original protocol. Furthermore, through additional funding and collaborations, we have significantly expanded the size and scope of the samples for genomic profiling. Transcriptomic profile (RNA-seq) of the expanded set of samples has been generated and mapped. We encountered some challenges in generating RRBS libraries from LCM samples which we overcame and we expect to have the RRBS data of the expanded set available shortly. Validation experiments were started by two new members of our team and are proceeding rapidly. Also, with the assistance of the biostatisticians in our group, case selection for all steps of the project is completed. Other research engagements funded separately, such as investigations of DNA-BPDE adducts and specific non-coding RNA in the benign tissues have promise in improving the accuracy of assays developed in this proposal. We are encouraged by our progress and are excited about the prospects of this research in improving the care of patients at risk of prostate cancer.

The goals of this grant proposal are to: 1) construct a prostate tissue-specific expression quantitative trait loci (eQTL) dataset; and 2) utilize this dataset to identify candidate genes for existing prostate cancer (PC) risk-single nucleotide polymorphisms (SNPs) that can then be followed up in future studies. To accomplish this goal we will perform a genome-wide SNP analysis (Illumina Human Omni 2.5M SNP array) and a genome-wide mRNA expression analysis (RNA sequencing) on a common set of 500 samples of normal prostate tissue sampled from men with PC. To date, we have pre-screened normal prostate tissue with the use of H&E stained sections from 4000 men having a radical prostatectomy in order to identify those cases meeting our strict selection criteria for further processing (tissue localized to the posterior region of the prostate, no tumor, no high grade PIN, no BPH, 1% lymphocytes, and the final percent of epithelial cells present 40%). Furthermore, the RNA / DNA purification and DNA genotyping / RNA expression analyses proposed have also been completed. We are now in the final phase of the project. We have completed the quality-control analysis of the genotyping and RNA sequencing results and are now completing the statistical analysis required for the construction of the eQTL dataset., The original document contains color images.

Individuals with germline mutations in BRCA1 have an elevated but incomplete risk of developing ovarian cancer suggesting the presence of genetic modifiers of ovarian cancer in this population. A genome wide association study (GWAS) for ovarian cancer in BRCA1 mutation carriers has identified several novel modifiers of ovarian cancer risk for BRCA1 mutation carriers that can be used for individualized ovarian cancer risk assessment., The original document contains color images.

Breast cancer develops from breast mucosa and breast mucosa has intact immune system to maintain epithelial integrity. In this study our goal was to study the immune subsets associated with breast mucosa and develop the strategies to populate mucosa with immune effectors in order to prevent breast cancer. Data obtained from our studies suggest that T cells constitute the majority of immune cells in breast mucosa and this includes conventional CD4 T cells, CD8+ T cells and significant fraction of unconventional double positive (DP) CD4+CD8+ T cells. We also observed that intramammary immunization induces antigen-specific immune responses in breast mucosa. Currently, studies are being done to characterize these double positive T cells to determine whether these are regulatory or cytotoxic in nature and their role in prevention of breast cancer. In addition to this, we are also investigating the ability of intramammary immunization in prevention of breast cancer and the feasibility of translating this approach into preventive breast cancer vaccine setting., The original document contains color images.

The U.S. military is providing amputees with state-of-the-art prosthetic devices. In addition to walking and changing direction, amputees must be able to manage uneven terrain, crowded environments, stairs, ramps, and hills. The largest problem for a lower extremity amputee is falls. Falling history and balance confidence are associated with reduced mobility capability and social activity. The goal of this research effort is to rehabilitate individuals with lower extremity amputations to reduce falls using a novel training method. The training program utilizes a microprocessor-controlled treadmill designed to deliver task-specific training perturbations. The training consists of six, 30 minute sessions delivered over a 2-week period. Trunk motion and velocity was assessed using a perturbation test in an immersive virtual environment, since trunk kinematics has been shown to determine fall likelihood. We have enrolled 14 research subjects at the Naval Medical Center San Diego. Mean trunk flexion angle and velocity significantly improved after participating in the training program. The improved performance was maintained up to 6 months. Subjects reported decreased uncontrolled and semi-controlled falls in their free-living environment outside of the research laboratory. These results indicate that task-specific training is an effective rehabilitation method to reduce falls in persons with lower limb amputations., The original document contains color images.

Polytrauma is most often caused from explosive devices and accounts for about 65 percent of injuries to our military personnel. The patients who have polytrauma are at increased risk of developing either bleeding and/or a clot in their veins which cause a life-threatening event known as venous thromboembolism (VTE). We began enrollment of patients into the study on 2 February 2011. As of 1 October 2012, we have successfully enrolled and collected blood samples on 684 patients and 64 healthy volunteers. We have thus far analyzed plasma samples of over 230 patients and 64 volunteers. In our preliminary analysis of thrombin generation and procoagulant microvesicle analysis, we have observed that thrombin generation is accelerated early after traumatic injury and there are greater numbers of procoagulant microvesicles noted after traumatic injury relative to healthy volunteers.

Electronic Medical Records (EMRs) have greatly expanded the potential for the evidence-based improvement of clinical practice by providing a data source for computable medical information. The Text REtrieval Conference 2012 Medical Records Track (TREC-med) explored how information retrieval may support clinical research by providing an efficient means to identify cohorts for clinical studies. A shared task called participants to find cohorts of relevant patients for 50 different topic queries. The users in TREC-med information retrieval systems would be medical experts who are searching for cohorts. In our previous work, we have collaborated with such experts on specific queries; the assortment of 50 queries makes this competition a standardized benchmark task. Thus, techniques that have shown case-by-case improvement can be tested against a much larger number of queries. We have taken this opportunity to investigate three core questions around which many of our algorithms are designed: 1. What is the relative value of structured data (e.g., fields in EMRs, or document metadata) compared to clinical text? 2. Are extensive information extraction (IE) efforts any benefit when we consider the applied question of information retrieval (IR)? 3. Can distributional semantics help supply missing information in a query?, Presented at the Twenty-First Text REtrieval Conference (TREC 2012) held in Gaithersburg, Maryland, November 6-9, 2012. The conference was co-sponsored by the National Institute of Standards and Technology (NIST) the Defense Advanced Research Projects Agency (DARPA) and the Advanced Research and Development Activity (ARDA). U.S. Government or Federal Rights License

This is a preclinical evaluation of non-invasive medical monitoring devices to predict blood loss and hemorrhage in humans. The goal is to develop technologies that can enhance decision support and resuscitation algorithms to treat combat casualties. In the last 12 months we have initiated the protocol and begun to compare simulated hemorrhage with lower body negative pressure (LBNP) with real blood loss of 1 L. Two subjects have been studied to date and we are finding a remarkably tight correlation between LBNP levels of -15, -30, and -45mmHg with blood loss of 333 ml, 666 ml, and 1 L total. Notably, the changes in central venous pressure evoked by LBNP are similar to those evoked by real blood loss. Additionally, we are performing coagulation studies and also making detailed measurements of arterial pressure and heart rate. This primary data will be subject to further analysis including machine learning approaches designed to enhance the decision support algorithms developed by the U.S. Army Institute of Surgical Research. It should also be noted that the original project schedule has been delayed as a result of significant delays in the IRB process for review and approval of the experimental protocol., The original document contains color images.

The goal of this idea award was to characterize one novel long non-coding transcript that we had identified in a screen using whole-genome tiling arrays. This transcript which is called Long Stress-Induced Non-Coding Transcript 5 (LSINCT5) was one of 12 long non-coding transcripts that we had identified. This transcript was chosen over the other 11, as it had increased expression in most of the breast cancer cell lines and primary tumors that we analyzed. There were three Specific Aims to our original proposal aimed at characterizing this transcript and determining what role it played in the development of breast cancer. While we have been successful in completing most of our original Specific Aims we have struggled in the past year because the level of expression of this transcript is so low. This has made the characterization of this transcript difficult, but we have now been able to reproducibly measure this transcript. In the final year of this Idea Award we propose to finish up the remaining work from our original Specific Aims, but also have several additional things we d like to do in order that we can successfully compete for extramural funding from the National Institutes of Health.

The goal of this project is to develop biopsy based assays to assess the probability that patients with a negative biopsy or with a prostate cancer (CaP) Gleason score 6 (GS6) biopsy actually have significant CaP of Gleason score 7 or higher which was missed during the biopsy evaluations due to insufficient sampling. Based on the concepts of the CaP field effect , this goal will be achieved by gene expression and epigenetic analysis of preneoplastic lesions and non-cancerous areas of prostate tissues from patients with CaP and benign prostate tissues from patients free of CaP. According to the DOD regulations, all research activities began after the approval of the research protocol by the HRPO in March 20 2012. Since then, we have identified the appropriate cases for the study and completed collection by laser captured microdissection (LCM) of all but two of the samples needed for genomic profiling. The collection of samples by LCM was an important part of the experiments in the first year of this project. Procedures for the collection of benign prostate tissues from patients free of CaP have been established. Internally funded collaborations have been established which will enhance the chances of success in this project by expanding the scope of genomic profiling. Two 2012 publications describe our findings related to the CaP field effect. Encouraged by these findings, we are actively pursuing the objectives of this project.

To test the idea that ovarian cancer arises from oviductal epithelium, spreads to the ovary, and presents as ovarian cancer, we generated a mouse model in which we can target genetic deletions to the oviductal epithelium. We are currently performing selective genetic deletion of ovarian cancer genes, such as TP53, Rb, and BRCA1 in oviductal epithelium in this mouse model., The original document contains color images.

Individuals with germline mutations in BRCA1 have an elevated but incomplete risk of developing ovarian cancer suggesting the presence of genetic modifiers of ovarian cancer in this population. A genome wide association study (GWAS) for ovarian cancer in BRCA1 mutation carriers was initiated in an effort to identify common genetic variants that modify ovarian cancer risk. Discovery and validation studies have identified several novel modifiers of ovarian cancer risk for BRCA1 mutation carriers that can be used for individualized ovarian cancer risk assessment., The original document contains color images.

Predisposition to rheumatoid arthritis (RA) is associated with the presence of genetic factors, HLA class II molecules, DR4 and DQ8, being the strongest. Patients with RA show an imbalance of gut microbiota suggesting its role in regulation of disease. We have used HLA-DR4/ DQ8 mice to test our hypothesis that treatment with commensal bacteria like Prevotella histicola can modulate Collagen-induced arthritis (CIA). In vitro study showed that treatment of mice with P. histicola in CII-immunized mice led to suppression of antigen-specific immune response and reduction in production of inflammatory cytokines. We carried out rtPCR for various cytokines and regulatory proteins in guts of mice treated with p histicola and controls. Our data showed that treatment with p histicola led to an increase in Tregulatory protein FoxP3 as well cytokine IL-10, a cytokine produced by Treg cells and suppression of pro-inflammatory IL-23. To determine if susceptibility is associated with proinflammatory cytokines in na ve mice, we analyzed cytokines in jejenum of arthritis-susceptible *0401 and resistant *0402 mice. Our data showed that arthritis-susceptible mice had higher level of proinflammatory cytokines compared to resistant mice. These data suggest that P histicola induced immune responses in the gut can induce tolerance in periphery leading to systemic immune suppression and protection from arthritis and may be a potential target for therapy., The original document contains color images.

The goals of this grant proposal are to: 1) construct a prostate tissue-specific expression quantitative trait loci (eQTL) dataset; and 2) utilize this dataset to identify candidate genes for existing prostate cancer (PC) risk-single nucleotide polymorphisms (SNPs) that can then be followed up in future studies. To accomplish this goal, we will perform a genome-wide SNP analysis (Illumina Human Omni 2.5M SNP array) and a genome-wide mRNA expression analysis (Illumina humanht-12 BeadChip) on a common set of 500 samples of normal prostate tissue sampled from men with PC. To date, we have pre-screened normal prostate tissue with the use of H&E stained sections from 4000 men having a radical prostatectomy in order to identify those cases meeting our strict selection criteria for further processing (tissue localized to the posterior region of the prostate, no tumor, no high grade PIN, no BPH, 1% lymphocytes, and the final percent of epithelial cells present 40%). Following this initial evaluation, 565 tissue samples were selected and further processed into 10 micron thick sections (cryostat) and reevaluated (H&E stained sections) prior to final processing for RNA and DNA extraction. Of the 565, 524 samples have been selected for RNA and DNA extraction, which is now in progress and should be completed shortly., The original document contains color images.

The purpose of this study was to determine the effect of rapidly induced hypothermia on neurological outcomes following fluid percussion injury in swine. A second objective was to test the safety and efficacy of inducing hypothermia through augmented heat extraction from the lungs using heliox and perfluorocarbon mist ventilation. The hypothermia device was built and bench tested. Brain temperatures in all hypothermic animals reached target (32-33 C) within 60 minutes. We standardized the anatomical location and fluid force necessary to create consistent severe brain injury in pigs. Seven of the eight hypothermic and four of the eight normothermic animals survived to the end of the five-day study. Hypothermia effectively prevented the gradual rise in intracranial pressure. Neurological and behavioral scores were better in the surviving normothermic animals on day #3; however these differences disappeared by day #5. The results of serum biomarkers for brain injury and histopathological scores are being processed. The study was extended by six months due to the additional safety and feasibility requirements placed by Mayo IACUC and the inclusion of serum biomarkers into the protocol. If additional funding becomes available, the investigators will add a third arm to the study consisting of blast injury plus hemorrhagic shock., The original document contains color images.

The U.S. military is currently fitting amputees with state-of-the-art prosthetic devices. In addition to walking and changing direction on a variety of surfaces, amputees must be able to manage uneven terrain, crowded environments, stairs, ramps, and hills. The largest problem for a lower extremity amputee is falls. Among individuals with a lower extremity amputation, 52% reported having fallen in the previous 12 months, 49% reported being fearful of falling, and 65% had low balance confidence scores. Falling history and balance confidence are associated with reduced mobility capability and social activity. As a result, lower extremity amputees with limited balance and stability are at risk for diminished quality of life. The goal of this research effort is to rehabilitate lower extremity amputees to reduce falls using a novel training method. In this first year of the research effort, we have developed a novel experimental method to induce unexpected trips to amputees under controlled laboratory conditions. We have identified objective biomechanical variables that are causally related to the success or failure in avoiding a fall after a trip, objective tests to quantify functional outcomes, and a subject-specific questionnaire to determine if the training method is effective. Enrollment has begun. Our target is to deliver a quantitatively derived, deployment-ready, advanced gait rehabilitation system that can improve functional outcomes and/or shorten the time required for injured servicemen and women to return to active duty or to a productive civilian life., The original document contains color images.

Polytrauma is most often caused from explosive devices and accounts for about 65% of injuries to our military personnel. People with polytrauma are at increased risk of developing either bleeding and/or a clot in their veins which cause a life-threatening event known as venous thromboembolism (VTE). We began enrollment of patients into the study on 2 February, 2011. As of 21 September, 2011, we have successfully enrolled and collected blood samples on 279 patients and 64 healthy volunteers. We also began analyzing plasma samples to assess their potential for thrombin generation., The original document contains color images.

Lymphedema is a common, chronic, and potentially devastating complication of primary breast cancer therapy. Radiation increases patients lymphedema risk up to 36% as conventional fields irradiate vital lymphatic tissues. Fusion imaging technologies that combine anatomical and physiological data, e.g. SPECT/CT, may identify lymphatics critical for arm drainage and allow the creation of conformal radiation treatment fields that minimize the exposure of lymph nodes (LNs) and vessels while delivering therapeutic doses to target tissues. This study uses SPECT/CT scanning to localize lymphatics critical for arm drainage, and has established the feasibility of fusing SPECT/CT images with the CT scans used for radiation planning, thereby creating the opportunity to spare essential LNs needless radiation. The research for this project was conducted in two phases. The first involved estimating the levels of incidental, non-therapeutic radiation delivered to the lymph nodes essential for arm drainage following axillary surgery for breast cancer. The second involved estimating the dose reduction in levels of incidental radiation to critical arm-draining lymph nodes that could be achieved by integrating SPECT/CT images into the radiation planning process. In the second study, among 28 patients the mean lymph node radiation exposure was 23.6 Gy (SD 18.2) with the standard and 7.7 Gy (SD 11.3) with the SPECT/CT modiifed plans (p 0.001). These studies realized the BCRP goals by elucidating a novel means of refining breast cancer treatment to minimize patients' risk of developing the most prevalent and dreaded complication of conventional therapy, lymphedema., The original document contains color images.

Rheumatoid arthritis (RA) and multiple sclerosis (MS) are chronic inflammatory autoimmune diseases affecting millions of people. Here we are proposing a novel approach to cure MS, by administration of a specific strain of human commensal-bacteria. Recent studies have shown that intestinal microflora plays an important role in the health of the host and posses probiotics like qualities. We hypothesize that Gram-negative commensal bacteria from human gut have the potential to be used as a therapeutic agent. We have used collagen induced arthritis (CIA) in HLA-DR4DQ8 mice and PLP91-110 induced experimental autoimmune encephalomyelitis (EAE) HLA-DR3DQ8 mice to test our hypothesis that treatment with commensal bacteria Prevotella histicola can modulate disease. First using various doses of bacteria, we have identified the optimal dose to be used for treatment of CIA as well as EAE. Treatment of mice with P histicola as probiotics is ongoing. Our study showed that treatment of mice with 3-4 doses of P. histicola in collagen/PLP91-110-immunized mice led to suppression of antigen-specific immune response in-vitro in both CIA as well as EAE model. Our data indicates that P histicola induced immune responses in the gut cause induction of immune tolerance in periphery leading to suppression of antigen specific response., The original document contains color images.

We received funding from the Department of Defense Breast Cancer Program to study a long stress-responsive non-coding transcript which is called LSINCT5. There were three Specific Aims to our research proposal whose overall goal was to better understand the structure and function of this long stress-responsive non-coding transcript which was found to be frequently over-expressed in breast cancer as compared to matched normal breast epithelial tissue. Our three Specific Aims were to (1) Characterize the LSINCT5 transcript to determine its full length, which RNA polymerase transcribes it, and its sub-cellular localization: (2) To analyze the phenotype effect of modulating the expression of LSINCT5 in a normal breast epithelial cell line and in breast cancer cell lines that over-express it; and (3) To determine what other genes and non-coding transcripts have altered expression in direct response to altering the expression of LSINCT5. In this annual progress report we summarize our work on the analysis of LSINCT5, and we have completed most of the proposed work that we originally outlined. However, the most important goal is to determine what is the function of LSINCT5 in normal cells and how its over-expression in breast cancers contributes to breast cancer development. We have, therefore, initiated experiments aimed at determining what are the genes and non-coding transcripts that interact with LSINCT5 so that we can unravel the precise cellular functions of this interesting non-coding transcript., The original document contains color images.

Individuals with germline mutations in BRCA1 have an elevated but incomplete risk of developing ovarian cancer suggesting the presence of genetic modifiers of ovarian cancer in this population. A genome wide association study (GWAS) for ovarian cancer in BRCA1 mutation carriers was initiated in an effort to identify common genetic variants that modify ovarian cancer risk. The discovery phase of the study has been completed. A replication phase of the 6,000 most significantly associated variants is underway. In a separate study, variants in a 19p13.1 locus have been identified as modifiers of both breast and ovarian cancer in BRCA1 and BRCA2 mutation carriers.

Ovarian cancer affects approximately 22,000 women and kills approximately 16,000 women each year in the United States. Resistance to chemotherapy is the major contributing factor in ovarian cancer-related death. The primary objective of this study is to gain mechanistic insights into the development of chemotherapy resistance in ovarian cancer. We expect that advances in this study will contribute to our understanding of chemotherapy resistance in ovarian cancer and will facilitate the development of more effective therapies to overcome the problems of chemotherapy resistance., The original document contains color images.

Prostate cancer (CaP) is characterized by unique prostate-associated antigens; hence, it has been considered a prime candidate for immunotherapy. Despite numerous laboratory advances, clinical outcomes have been partial and transient. The overall goal of the proposed studies is to optimize the effectiveness of therapeutic whole-cell CaP vaccines by taking into consideration tumor-associated hypoxia as a relevant determinant of tumor antigenicity. Transcriptome studies revealed that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. Transcripts of hypoxia-associated genes DLG7, CCNB1 and HMMR were associated with Gleason score and with disease prognosis suggesting their potential as CaP biomarkers with prognostic value. By 2D-gel electrophoresis, we screened patient sera and detected novel hypoxic-cell reactive autoantibodies (under validation). Our data suggest that hypoxically cultured CaP cells are more akin to tumor cells in situ than are cells grown normoxically. We have identified hypoxia-reactive proteins, pathways and autoantigens with potential value as biomarkers or therapeutic targets. Introduction of pO2 as a variable can constitute a tool for the development of more effective immunotheraphy for CaP., The original document contains color images.

The availability of hepatitis B immune globulin (HBIG) and several oral antiviral therapies has reduced but not eliminated hepatitis B virus (HBV) recurrence. We aimed to determine the rate of HBV recurrence after orthotopic liver transplantation (OLT) in relation to virologic breakthrough pre-OLT and HBIG regimens post-OLT. Data from the NIH HBV-OLT database were analyzed. A total of 183 patients transplanted between 2001 and 2007 followed for a median of 42 months (range 1???81) post-OLT were studied. At transplant, 29% were hepatitis B e antigen (HBeAg) (+), 38.5% had HBV DNA > 5 log 10 copies/mL, 74% were receiving antiviral therapy. Twenty-five patients experienced virologic breakthrough before OLT. Post-OLT, 26%, 22%, 40% and 12% of patients received intravenous (IV) high-dose, IV low-dose, intramuscular low-dose and a finite duration of HBIG, respectively as maintenance prophylaxis. All but two patients also received antiviral therapy. Cumulative rates of HBV recurrence at 1 and 5 years were 3% and 9%, respectively. Multivariate analysis showed that listing HBeAg status and HBV DNA level at OLT were the only factors associated with HBV recurrence. In conclusion, low rates of HBV recurrence can be accomplished with all the HBIG regimens used when combined with antiviral therapy including patients with breakthrough pre-OLT as long as rescue therapy is administered pre- and post-OLT.

Lymphedema is a common, chronic, and potentially devastating complication of primary breast cancer therapy. Radiation increases patients lymphedema risk up to 36% as conventional fields irradiate vital lymphatic tissues. Fusion imaging technologies that combine anatomical and physiological data, e.g. SPECT/CT, may identify lymphatics critical for arm drainage and allow the creation of conformal radiation treatment fields that minimize the exposure of lymph nodes (LNs) and vessels while delivering therapeutic doses to target tissues. This study uses SPECT/CT scanning to localize lymphatics critical for arm drainage, and has established the feasibility of fusing SPECT/CT images with the CT scans used for radiation planning, thereby creating the opportunity to spare essential LNs needless radiation. Further, precise quantification of the dosimetry delivered to LNs draining the arm has revealed harmful levels of incidental irradiation with tangent beam configurations and subtherapeutic exposure with 4-field configurations. Data collection is complete, however interpretation and analysis of follow up SPECT/CT scans is ongoing. Data analysis will address the hypothesis that increased arm volume correlates with high levels of radiation dosimetry delivered to the LNs draining the arm. Additionally, data analysis will determine whether the radiation dose delivered to specific LNs is inversely correlated with radiolabeled tracer uptake, a surrogate measure for functional status, on follow up scans. The proposed study realizes the BCRP goals by elucidating a novel means of refining breast cancer treatment to minimize patients' risk of developing the most prevalent and dreaded complication of conventional therapy, lymphedema.

Optimal early detection and prevention strategies for breast cancer are predicated on our ability to identify individuals at significantly increased risk for this disease. The purpose of this Center was to bring molecular risk prediction for breast cancer into the clinical arena. The three areas of scientific inquiry included:(i) Establishment of a tissue repository of benign breast disease; (ii) Assessment of potential biomarkers of risk in this tissue set and (iii) Discovery of new, potentially relevant biomarkers of risk. We developed a cohort of 9,376 women, 758 (8%) of whom have been diagnosed with breast cancer since the time of their benign biopsy. We established our tissue repository of benign breast tissue and have collected the subsequent breast cancer tissue. We assessed the significance of benign histology in predicting the risk of future breast cancer, examining in detail the role of proliferative disease, atypia, papillomas, radial scars and involution. We explored the link between centrosome amplification, COX-2 expression, ER, Ki67 and breast cancer outcomes. Lastly, we characterized the histopathology in a cohort of African-American women that we established with Wayne State University.

The sequencing of the human genome has revealed that only 2% of the genome actually codes for protein. However, the remainder of the genome is not "junk" and it has recently been revealed that most of the genome is transcriptionally active. We utilized a tiling array approach to examine the entire genome for transcriptional activity and found a large number of non-coding transcripts. When we originally proposed the work in this Concept Award, we proposed to study a group of long, highly conserved, non-coding transcripts which had altered expression and sometimes mutations in breast cancer. We have subsequently found that many of these highly conserved transcripts are either part of known genes or highly homologous to known genes. However, we've now identified two new groups of novel non-coding transcripts which are not part of genes. The first group is noncoding transcripts that have increased expression in response to the DNA damage induced by the carcinogen NNK. The second group is identified by analyzing breast cancer cell lines with tiling arrays searching for non-coding transcripts that had consistently altered expression. We chose one of the NNK-induced transcripts, NIT 5, and one of the breast cancer altered transcripts, bcNCT 28, and began to characterize them further. Here we describe our final report on the work done with these two transcripts to determine their role in breast cancer., The original document contains color images.

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for postexposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAc) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously, we reported a serotype-selective, small-molecule BoNTAc inhibitor with a K1pp value of 3.8 0.8 M. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC50 value of 100 m., The original document contains color images. Published in PLoS ONE, v4 n11 p1-18, Nov 2009. Sponsored in part by the Army Research Office, contract W911NF-09-1-0095, and the Defense Threat Reduction Agency, 3.10023_07_RD_B and 3.1001_08_WR_B.

The objectives of this study are to test whether CTLA 4 blockade + AA therapy canenhance clinical treatment responses in advanced prostate cancer patients relative to AA therapy alone. Study patients are randomized to 3 months of combined AA therapy + MDX-010 or versus AA therapy alone. To date, 46 patients have been enrolled and 42 randomized per protocol. An additional 84 patients have been screened and deemed ineligible for study. 29 patients now have sufficient follow-up to assess if any treatment effects may be occurring. In general, patients receiving combined AA + MDX-010 have exhibited greater PSA responses than patients receiving AA therapy alone. We have also observed that MDX-010 does not likely affect initial testosterone production but may delay testosterone recovery. Our preliminary studies also indicate atypical and favorable responses to combined MDX-010 + AA therapy including reversal of rising PSA, rapid resolution of obstructive urinary pathology and dramatic tumor downstaging resulting in unexpected 1 year disease-free patient status. Based on these preliminary observations, we believe that combined AA + MDX-010 treatment may encompass a promising approach to improve advanced prostate cancer treatment.

No abstract.

The objective of this pilot study was to identify if androgen levels are adversely affected by adjuvant chemotherapy for breast cancer and whether low androgen levels are correlated with the frequency and severity of fatigue, weight gain, psychological symptoms, vasomotor symptoms and libido. A longitudinal, descriptive design was used with questionnaires completed and blood drawn from 20 premenopausal women at 4 time periods: before treatment, mid-treatment, immediate post-treatment and 6 months later. Questionnaires included the Female Sexual Function Index, Greeene Climacteric Scale, Profile of Mood States, Schwartz Fatigue Scale and a menses diary. Data analysis involved descriptive statistics, plots of hormone levels over time, t-tests to examine changes in hormone levels, and correlational analysis to determine relationships between hormone levels and symptoms. Preliminary data indicate that women > 40 stopped menstruating, had FSH levels > 40 IU/l and androgen function decreased by at least 35%. Women < 40 retained ovarian function. Sexual function, vasomotor symptoms, vigor and fatigue became progressively worse through treatment. These data provide evidence that chemotherapy impacts androgens., The original document contains color images.

The current gold standard for repair of a nerve injury is the interposition of an autologous nerve graft. This has several disadvantages, such as donor site morbidity, limited availability, size mismatch and painful neuroma formation. In addition, functional recovery rarely returns to pre-injury level. Therefore, the development and optimization of nerve conduits is an increasingly important focus in the field of peripheral nerve regeneration. One promising approach that may enhance nerve regeneration is the long term delivery of growth factors. However, accurate delivery and concentration maintenance of growth factors within the nerve conduit still pose problems. We therefore studied the long term release of nerve growth factor (NGF) using a microsphere delivery system. First, we evaluated the release of NGF from five different types of polymer microspheres both in vitro and in vivo. Next, we assessed the effect of both NGF and glial cellline derived neurotrophic factor (GDNF) releasing microspheres on functional regeneration using polymer nerve conduits., See also ADM002187. Proceedings of the Army Science Conference (26th) Held in Orlando, Florida on 1-4 December 2008, The original document contains color images.

The AURORA-A/BTAK/STK15 gene encodes a centrosome-associated kinase that causes centrosome amplification, failure of cytokinesis, and aneuploidy when amplified and/or overexpressed in breast tumors. A number of gene association studies using matching breast cancer cases and controls have shown that the F31I polymorphism in STK15 increases risk of breast. We hypothesized that the F31I polymorphism is associated with increased risk of breast cancer in BRCA1 and BRCA2 mutation carriers. Using over 7,000 carriers of BRCA1 and BRCA2 deleterious mutations we showed that F31I has no influence on breast cancer risk. In parallel, we completed an association study of 2400 polymorphisms in other cell division regulatory genes in 800 breast cancer cases and 800 controls collected at the Mayo Clinic. A total of 52 of these polymorphisms along with 332 other polymorphisms displaying associations with breast cancer risk were evaluated for associations with breast cancer risk in approximately 5,400 BRCA1 and BRCA2 mutation carriers. Several polymorphisms displaying significant associations (p<10-4) with risk of breast cancer in BRCA2 mutation carriers were observed.

The sequencing of the human genome has revealed that only 2% of the genome actually codes for protein. However, the remainder of the genome is not junk and it has recently been revealed that most of the genome is transcriptionally active. We utilized a tiling array approach to examine the entire genome for transcriptional activity and found a large number of non-coding transcripts. When we originally proposed the work in this Concept Award, we were proposing to study a group of long, highly conserved, non-coding transcripts which had altered expression and sometimes mutations in breast cancer. We have subsequently found that many of these highly conserved transcripts are either part of known genes or highly homologous to known genes. However, we ve now identified two new groups of novel non-coding transcripts which are not part of genes. The first group are non-coding transcripts that have increased expression in response to the DNA damage induced by the carcinogen NNK. The second group were identified by analyzing breast cancer cell lines with tiling arrays searching for non-coding transcripts that had consistently altered expression. We have requested and obtained a no-cost extension for this work and will begin to characterize these transcripts exactly as proposed in our original Concept Award to determine the role they play in the development of breast cancer., The original document contains color images.

Optimal early detection and prevention strategies for breast cancer are predicated on our ability to identify individuals at significantly increased risk for this disease. The purpose of this Center is to bring molecular risk prediction for breast cancer into the clinical area. This will require progress on three fronts of scientific endeavor:(i) Establishment of a tissue repository of benign breast disease; (ii) Assessment of potential biomarkers of risk in this tissue set and (iii) Discovery of new, potentially relevant biomarkers of risk. We have made significant progress on these aims. Our cohort is comprised of 9,376 women, 758 (8%) of whom have been diagnosed with breast cancer since the time of their benign biopsy. We established our tissue repository of benign breast tissue and have collected the subsequent breast cancer tissue. We assessed the significance of benign histology in predicting risk of future breast cancer, examining in detail the role of proliferative disease, atypia, papillomas, radial scars and involution. We explored the link between centrosome amplification, COX-2 expression and breast cancer outcomes and are currently exploring the significance of p16, ER and Ki67. We are working with Wayne State to characterize the histopathology in a cohort of African American women. Our focus in 2008-2009 will be on the Wayne State cohort., The original document contains color images.

The objectives of this study are to test whether CTLA 4 blockade + AA therapy canenhance clinical treatment responses in advanced prostate cancer patients relative to AA therapy alone. Study patients are randomized to 3 months of combined AA therapy + MDX-010 or versus AA therapy alone. To date, 46 patients have been enrolled and 42 randomized per protocol. An additional 84 patients have been screened and deemed ineligible for study. 29 patients now have sufficient follow-up to assess if any treatment effects may be occurring. In general, patients receiving combined AA + MDX-010 have exhibited greater PSA responses than patients receiving AA therapy alone. We have also observed that MDX-010 does not likely affect initial testosterone production but may delay testosterone recovery. Our preliminary studies also indicate atypical and favorable responses to combined MDX-010 + AA therapy including reversal of rising PSA, rapid resolution of obstructive urinary pathology and dramatic tumor downstaging resulting in unexpected 1 year disease-free patient status. Based on these preliminary observations, we believe that combined AA + MDX-010 treatment may encompass a promising approach to improve advanced prostate cancer treatment., The original document contains color images.

PARK2 (Parkin) is a common fragile site (CFS) gene. We examined Parkin in primary ovarian tumors and found that this gene was frequently inactivated. We also found that re-introduction of Parkin is associated with greater sensitivity to apoptotic induction in ovarian cancer cell lines. We also discovered an entire family of very large common fragile site genes. We measured the expression of Parkin and 13 other CFS genes in panels of different cancers. This revealed non-random inactivation of these genes and greater inactivation in cancers that have a poorer clinical prognosis. We then utilized whole genome tiling arrays to characterize all transcripts (not just coding transcripts) and their response to stress. These studies revealed non-coding transcripts within Parkin and other large CFS genes. We discovered a new class of large non-coding transcripts. A sub-set of these were highly evolutionarily conserved; when we examined these transcripts in ovarian and other cancers, we found alterations in their expression. We also found that some of these non-coding transcripts were mutational targets in ovarian and other cancers. Thus, this work has discovered a new group of targets that are altered during the development of ovarian cancer., The original document contains color images.

Prostate cancer is a major cause of cancer-related mortality for men in the United States. The established treatment protocol for the advanced stage of the disease is by surgical castration and or chemotherapy. Although androgen ablation therapy is initially effective in inhibiting tumor growth in most patients, however with time, the tumors recurs with a more aggressive and metastatic phenotype, ultimately causing death. Thus limitation of treatment underscores the need of development of an alternative treatment approach. Several reports suggest that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) induced angiogenesis is essential for the growth and metastasis of prostate cancer. As our recent reports indicate that the neurotransmitter dopamine (DA) can specifically and significantly inhibit VPF/VEGF induced angiogenesis, therefore, we investigated whether DA can inhibit angiogenesis, and thereby growth of prostate cancer. We here in this report demonstrate that DA can significantly inhibit PC3 human prostate cancer growth by inhibiting VPF/VEGF mediated angiogenesis by suppressing VEGFR-2 phosphorylation in tumor endothelial cells. Thus our results are significant because DA is already in clinical use with an established safety record; therefore our study may be rapidly translated to the clinics as a new and more effective therapy for prostate cancer., The original document contains color images.

Emerging data suggest that the hepatitis B virus (HBV) genotype and the precore and core promoter variants impact the outcome of orthotopic liver transplantation (OLT) for hepatitis B. The aim of this study was to determine if there is a correlation between HBV genotype, precore and core promoter variants, and pre- and post-OLT outcomes. Serum samples from patients participating in the National Institutes of Health HBV-OLT study were tested for HBV genotype and precore and core promoter variants. A total of 123 patients were studied: 43% were Asians, 46% were Caucasians, and 8% were African Americans. HBV genotypes A (35%) and C (35%) were the most prevalent, followed by genotypes D and B. Precore and core promoter variants were detectable in 44% and 90% of patients. Patients with genotype C were more likely to have hepatocellular carcinoma (HCC) at listing ( P < 0.001). Waitlist mortality was highest among patients with genotype D, while posttransplant mortality was highest among patients with genotype C. Precore or core promoter variants did not correlate with pre- or post-OLT survival. In conclusion, in this US patient population, patients with genotype C were more likely to have HCC at the time of transplant listing and to die after transplant than patients with non-C genotypes. Patients with genotype D had the highest posttransplant survival, but this was offset by higher waitlist mortality. Our study suggests that HBV genotypes but not precore or core promoter variants may have an impact on pre- and post-OLT outcomes of hepatitis B patients. Liver Transpl 14:1420???1427, 2008. ?? 2008 AASLD.