Translational Research Studies Research Articles

Improving CKD Therapies and Care

The Kidney Research National Dialogue, supported by the National Institute of Diabetes and Digestive and Kidney Diseases, asked the research and clinical communities to formulate and prioritize research objectives that would improve our understanding of kidney function and diseases. This commentary outlines the high-priority research objectives for CKD. The goal of these research objectives is to enhance knowledge to improve outcomes in people with CKD. Basic and translational research, longitudinal observations, and epidemiologic studies can each point to targets for intervention. Future interventions must be informed by data from well designed, large representative observational studies that include collection of genetic and phenotypic data as well as biospecimens. Interaction of genetic and environmental factors must be part of the analysis, including the influence of diet, comorbid conditions, and medication. The focus should be not only on slowing or preventing progression of CKD, but also on regression of disease to the greatest extent possible.

The CTSA Consortium's Catalog of Assets for Translational and Clinical Health Research (CATCHR)

The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. Catalog of Assets for Translational and Clinical Health Research (CATCHR) is the Consortium's effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open-access, searchable database of consortium resources to support multisite clinical and translational research studies. Data are collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable Web-based tool. Additional easy-to-use Web tools assist resource owners in validating and updating resource information over time. In this paper, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources.

The Armadillo as a Model for Peripheral Neuropathy in Leprosy

Leprosy (also known as Hansen's Disease) is a chronic infectious disease caused by Mycobacterium leprae that primarily targets the peripheral nervous system; skin, muscle, and other tissues are also affected. Other than humans, nine-banded armadillos (Dasypus novemcinctus) are the only natural hosts of M. leprae, and they are the only laboratory animals that develop extensive neurological involvement with this bacterium. Infection in the armadillo closely recapitulates many of the structural, physiological, and functional aspects of leprosy seen in humans. Armadillos can be useful models of leprosy for basic scientific investigations into the pathogenesis of leprosy neuropathy and its associated myopathies, as well as for translational research studies in piloting new diagnostic methods or therapeutic interventions. Practical and ethical constraints often limit investigation into human neuropathies, but armadillos are an abundant source of leprotic neurologic fibers. Studies with these animals may provide new insights into the mechanisms involved in leprosy that also might benefit the understanding of other demyelinating neuropathies. Although there is only a limited supply of armadillo-specific reagents, the armadillo whole genomic sequence has been completed, and gene expression studies can be employed. Clinical procedures, such as electrophysiological nerve conduction testing, provide a functional assessment of armadillo nerves. A variety of standard histopathological and immunopathological procedures including Epidermal Nerve Fiber Density (ENFD) analysis, Schwann Cell Density, and analysis for other conserved cellular markers can be used effectively with armadillos and will be briefly reviewed in this text.

Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes

As an emerging research direction, nanomedicine has been increasingly utilized to treat inflammatory diseases. In this head-to-head comparison study, four established nanomedicine formulations of dexamethasone, including liposomes (L-Dex), core-cross-linked micelles (M-Dex), slow releasing polymeric prodrugs (P-Dex-slow), and fast releasing polymeric prodrugs (P-Dex-fast), were evaluated in an adjuvant-induced arthritis rat model with an equivalent dose treatment design. It was found that after a single i.v. injection, the formulations with the slower drug release kinetics (i.e., M-Dex and P-Dex-slow) maintained longer duration of therapeutic activity than those with relatively faster drug release kinetics, resulting in better joint protection. This finding will be instructional in the future development and optimization of nanomedicines for the clinical management of rheumatoid arthritis. The outcome of this study also illustrates the value of such head-to-head comparison studies in translational nanomedicine research.

Characteristics Of Chronic Lymphocytic Leukemia (CLL) In Senegal. Clinical Features, Cytology, Immunophenotype, Cytogenetic Abnormalities and Altered Expression Of Micro-RNA

The incidence of CLL in Africa is not known for several reasons, among them the difficulty to access the diagnosis since there is only clinical features and cytology which are available in several countries in francophone Africa, especially in Senegal. The WHO criteria which have been published since 2001 from the first revised WHO classification of tumours of haematopoitic and lymphoid tissues cannot be applied since there is no facility for immunophenotyping and cytogenetic. Moreover, no research in place can be set up without any precise diagnosis.In this study, we have developed the flow cytometry technique in the laboratory of Immunology in CHU Le Dantec in Dakar to obtain the immunophenotype looking at the expression of Kappa/lambda light chains, CD19, CD22, CD20, FMC7, CD5, CD10, CD23, CD38. Peripheral blood smears were fixed according to the technique for FISH to detect the following cytogenetic abnormalities: trisomy12, deletion 13q14, deletion 11q22-23, deletion 17p. RNA to test by qRT-PCR the expression of microRNA mir15a, mir16-1, mir181a, mir181b, and mir 34a and 34b was obtained from peripheral blood lymphocytes.Twelve cases of CCL aged ranging from 50 to 80 year old were characterized at clinical level according to the Binet's system: 1 stage A, 5 stage B, 6 stages C. In most of cases (9 cases) hyperlymphocytosis of small lymphocytes with clumped chromatin and scanty cytoplasm was very high (122.000 to 336 000/mm3). The “Matutes” score obtained by immunophenotyping was 5 or 4 for 11 patients in favor of typical CLL. The expression of CD38 which is a marker of unfavorable prognosis is positive in 9 patients. Nine patients had cytogenetic abnormalities: 3 trisomy 12, 6 del 13q14, 2 del 11q22-23, 1 del17p. Four patients had two simultaneous cytogenetic abnormalities (tri 12, tri 13 ; del13q14, del11q22-23 ; del13q24, del17p ; del13q24, tri12).The analysis of mi-RNA showed high expression of mir15a and mir 16-1 and low value of mir181a and mir 181b which were described in aggressive CLL ; however the expression of mir 34a and mir34b was also high.This study shows the aggressiveness of CLL in Senegal, probably due to the delay of the diagnosis. We have also demonstrated the possibility to set up immunophenotype and the possibility of a precise diagnosis of lymphoproliferative disorder in place, in western Africa and the series will be extended. Moreover we have also demonstrated the possibility to construct translational research project from samples characterized in the University Hospital in Dakar gathering the cases from different sanitary structures in the country.Such experiment would be a model for epidemiologic, clinico-biologic and translational research studies in order to set up in the country the capacity building for diagnosis and research. Disclosures:No relevant conflicts of interest to declare.

T3 Translational Science in Gastroenterology: Getting to Best Outcomes

Achieving the best possible outcomes requires the reliable implementation of best practices for every patient. Specifically, optimizing outcomes requires a spectrum of research spanning basic science, drug development, clinical efficacy and effectiveness, health services, quality improvement, and implementation research. However, our rapid increase in understanding the mechanisms of health and disease and their treatment has far outpaced our ability to reliably provide that care, resulting in poor reliability and enormous variation in care. T3 translational research studies attempt to answer questions surrounding reliable implementation of interventions, decreasing variations in care, and spreading effective therapies. To answer these questions, T3 research may use traditional research methodology such as randomized controlled trials (RCTs); however, various other approaches such as quasiexperimental designs (eg, time-series analysis) are often used. Although uncommon, T3 research has shown promise in not only improving process measures such as correct dosing of medications, but also outcome measures such as improved remission rates in patients with IBD. A more complete integration of T3 translational research into the more traditional research continuum is necessary if we are to achieve the best possible outcomes for our patients.

Integrating the myocardial matrix into heart failure recognition and management.

In contrast to public perception, the morbidity and mortality and the resultant healthcare costs associated with chronic heart failure (HF) are increasing and arguably reaching epidemic proportions. Although basic research efforts have provided unique insights into fundamental processes that govern myocardial extracellular matrix (ECM) growth and function, the translation of these findings to improved diagnostics and therapeutics for HF has not been as forthcoming. The factors that contribute to this relative paucity of new clinical tools for HF are multifactorial but likely include the need to recognize and differentiate HF phenotypes and to couple the use of biomarkers and multimodality imaging in early translational research studies. Recognizing the classification scheme of HF with a reduced ejection fraction (EF) to that of HF with a preserved EF and incorporating unique and differential measurements of ECM remodeling to these specific disease processes are warranted. For example, profiling pathways of ECM degradation such as the matrix metalloproteinases in patients with ischemic heart disease and HF with a reduced EF can provide prognostic information in terms of risk of progression to HF. In patients with chronic hypertensive disease and HF with a preserved EF, plasma profiling indexes of ECM synthesis and turnover, as well as advances in ECM imaging, have been shown to provide diagnostic and prognostic use. In terms of therapeutics, strategies to stabilize the ECM in HF with a reduced EF hold potential, whereas in contradistinction, selective antifibrotic agents may hold promise for HF with a preserved EF.

Translational research on esophageal adenocarcinoma: from cell line to clinic

Human esophageal adenocarcinoma (EAC) cell lines have made a substantial contribution to elucidating mechanisms of carcinogenesis and drug discovery. Model research on EAC relies almost entirely on a relatively small set of established tumor cell lines because appropriate animal models are lacking. Nowadays, more than 20% of all fundamental translational research studies regarding EAC are partially or entirely based on these cell lines. The ready availability of these cell lines to investigators worldwide have resulted in more than 250 publications, including many examples of important biomedical discoveries. The high genomic similarities (but certainly not completely identical) between the EAC cell lines and their original tumors provide rational for their use. Recently, in a collaborative effort all available EAC cell lines have been verified resulting in the establishment of a reliable panel of 10 EAC cell lines. It could be expected that the value of these cell lines increases as unlimited source of tumor material because new biomedical techniques require more tumor cells and the supply of viable tumor cells is diminishing because of neoadjuvant chemo(radio)therapy of patients with EAC. Here, we review the history of the EAC cell lines and their utility in translational research and biomedical discovery.

Ohio CTSAs Implement a Reliant IRB Model for Investigator‐Initiated Multicenter Clinical Trials

Effective clinical and translational research is an essential component of improving human health as delineated in the National Institutes of Health (NIH) roadmap, 1,2 yet efficiently conducting that research remains problematic. 3,4 Challenges include the high costs of conducting research, a shortage of qualified investigators, 5 low subject enrollment rates, 6,7 slow dissemination of results, 8 reduced funding, 3 lagging IT systems, 9 and increased regulatory burden. 10,11 A priority for the National Center for the Advancement of Translational Science (NCATS), and its Clinical Translational Science Award (CTSA) Consortium, is to address this last concern regarding regulatory burden. Specifically the focus is on the improvement of Institutional Review Board (IRB) processes; especially novel models for regulatory review of multicenter clinical and translational research studies. The three Ohio CTSA sites, housed at Case Western Reserve University (CWRU) in Cleveland, The Ohio State University (OSU) in Columbus, and the University of Cincinnati (UC), have collaborated to create a novel IRB review concept with the hopes of addressing this concern and achieving a more effective and efficient IRB review of multicentered clinical research studies.

Effective incorporation of biospecimen collection for translational studies into a phase III trial: The NeoALTTO experience (BIG 01-06).

3012 Background: Translational research studies in the context of clinical trials using targeted therapies are essential for the identification of biomarkers of response or resistance. However, many challenges (e.g. logistic, technical) can impede proper biological sample collection. The NEOALTTO study provided an excellent opportunity to address these issues. Methods: In this large neoadjuvant phase III multicenter, multinational clinical trial, serial samples (plasma, serum, frozen and FFPE specimens) from patients with breast cancer were collected in 86 sites (23 countries) at several time points (baseline, week 2 and surgery). Standardized operating procedures were made available to sites together with pre-assembled kits for sample collection, processing, storage, and shipment to reduce the variability and increase compliance with the study protocol. All biospecimens were centralized for long term storage. Baseline fresh/frozen samples were processed for downstream analyses according to a pre-defined workflow. Evaluable sample population (ESP) was determined based on the following criteria: tumor cellularity, RNA integrity (RIN), and yield. Results: A total of 12,193 serial samples from 449 of 455 randomized patients was collected. The overall sample missing rate was 9.5% (includes samples not collected by site, withdrawn patients and pCR). Missing rates were 9%, 8%, 13%, and 9% for plasma, serum, frozen and FFPE specimens, respectively. At least 1 frozen tumor tissue was available for 423 (94%), 431 (96%), and 334 (74%) patients at baseline, week 2, and surgery, respectively. After 2 extraction cycles, 629 and 516 baseline frozen samples were processed for RNA and DNA extraction, respectively. The ESP was 71% (58% when RIN considered) and 80% for downstream RNA- and DNA-based analyses, respectively. Incorrect material, inappropriate sample processing and low or absent tumor cellularity were some of the factors affecting the ESP. Conclusions: These results show that careful upfront logistic and technical planning in NEOALTTO had a significant impact on the compliance and the quality of collected material, which may ultimately result in valuable research data. Clinical trial information: NCT00553358.

Abstract 3208: Development of applications for acoustic membrane microparticle assay technology for translational medicine.

Abstract The detection and study of proteins and their interactions utilizing small samples of cells or tissues is an ongoing problem in cancer research. Recent advances in the early detection of many cancers have resulted in smaller sized tumors for biopsy and evaluation. This has created a need for sensitive and robust assays for pathway related proteins. One mission for the Antibody and Protein Purification Unit (APPU) of the National Cancer Institute has been to evaluate emerging technologies and create new applications for these technologies. Using Acoustic Membrane MicroParticle (AMMP) assays on the ViBE Workstation (Bioscale, Lexington, MA) for solution phase immunoassays, the APPU has developed several applications which support the great utility and breadth of use for this technology in translational research. AMMP assays employ a homogenous, sandwich assay format with paired antibodies, one coupled to a magnetic bead and the second tagged for capture on the sensor, and the analyte forming a complex which interacts with a vibrating membrane sensor. The assay measures the concentration of the analyte by a resonant frequency shift of the membrane caused by the protein interaction – without matrix interference or signal scatter seen in optical techniques. A highly sensitive assay for Prolactin showed no serum interference, which is a common problem in optical immunoassays for circulating peptide hormones. An assay for the important marker C-Met required less than 125 nanograms of cell lysate protein for detection and quantitation. An AMMP assay was further able to quantitate C-Met on the surface of Hep2 cells using an antibody capture method. In an application for detection and quantitation of a member of the MAPK kinase family, an ultrasensitive assay for the detection of MEK1 was developed as well as a highly specific solution phase assay for pMEK1 (S218/S222) as shown by cognate phosphopeptide inhibition. And finally in an application which we believe to be truly unique, a solution heterodimer assay for the ERK1/MEK1 interaction was shown to be susceptible to inhibitor treatment. In conclusion, the AMMP technology was able to perform a wide range of applications required by translational research studies. The assays showed superior lower limit of detection and were reproducible, only requiring nanogram quantities of lysate proteins. The ability to measure protein/protein interactions in solution phase is especially useful for drug development and evaluation of drug effects in targeted therapies. Citation Format: Christopher McAndrew, Christopher Heger, Ashley Saab, W Matthew Dickerson, Paul K. Goldsmith. Development of applications for acoustic membrane microparticle assay technology for translational medicine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3208. doi:10.1158/1538-7445.AM2013-3208

HLA-DR expression as a biomarker of inflammation for multicenter clinical trials of ocular surface disease

There are currently no validated minimally invasive objective metrics for the classification and evaluation of ocular surface diseases and/or for evaluating treatment efficacy. We thus sought to establish a standardized methodology for determining the relative amount of the inflammatory biomarker HLA-DR on the ocular surface and to evaluate the precision, reliability and repeatability of its use for large multicenter clinical trials and translational research studies of ocular surface disease. Multiple studies were conducted to establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a minimally invasive, objective, ocular surface inflammatory biomarker. The established SOPs provide specific guidelines for HLA-DR collection and analysis, in order to incorporate it reliably into multicenter clinical trials and/or translational research. Duplicate cell samples from impression cytology (IC) samples of both normal and dry eye individuals were collected and split to assess repeatability (between the splits and between the duplicate samples). To determine storage capability, one duplicate was stained immediately and the other after 30 days cold storage. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians out-of-state were trained to collect IC samples, and the samples shipped to our Biomarker Laboratory, logged, processed and analyzed. Demonstration of the ability to incorporate of IC into a randomized double masked clinical trial of dry eye disease (DED) was performed. In all cases, processing and analyses were performed by a masked independent observer. The validity/viability of the SOPs was established by demonstrating that: 1) sufficient numbers of cells can be collected via IC; 2) the precision/repeatability of the relative biomarker expression quantified in samples; 3) personnel at distant sites can be taught to collect, store and ship samples successfully; 4) samples can be stored for up to 30 days (refrigeration) before processing without affecting results; 5) IC can be incorporated into a double blind randomized clinical trial (RCT) of DED; and 6) the Biomarker Laboratory can track a large number of masked samples reliably. In conclusion, our standard operating procedure for impression cytology analysis of HLA-DR expression appears to be repeatable and reproducible for use in multicenter clinical trials, providing a minimally invasive objective biomarker of inflammation of the ocular surface.

Quality assessment of tissue specimens for studies of diabetic foot ulcers

Diabetic foot ulcers (DFUs) represent an important clinical problem resulting in significant morbidity and mortality. Ongoing translational research studies strive to better understand molecular/cellular basis of DFU pathology that may lead to identification of novel treatment protocols. Tissue at the non-healing wound edge has been identified as one of major contributors to the DFU pathophysiology that provides important tool for translational and clinical investigations. To evaluate quality of tissue specimens and their potential use, we obtained 81 DFU specimens from 25 patients and performed histological analyses, immunohistochemistry and RNA quality assessments. We found that depth of the collected specimen is important determinant of research utility, and only specimens containing a full-thickness epidermis could be utilized for immunohistochemistry and RNA isolation. We showed that only two-thirds of collected specimens could be utilized in translational studies. This attrition rate is important for designs of future studies involving tissue specimen collection from DFU.

Establishment of a biorepository for migraine research: the experience of Interinstitutional Multidisciplinary BioBank (BioBIM)

The development of Biobanks and recent advances in molecular biology have enhanced the possibility to accelerate translational research studies. The Interinstitutional Multidisciplinary BioBank (BioBIM) is organized in a large healthy donors collection and pathology-based biobanks with the aim to provide a service for development of interdisciplinary studies. A new pathology-based biobank has been organized to specifically collect biospecimen from patients affected by migraine, with the final goal to centralize data, collect blood, plasma, serum, DNA and RNA of patients with this disease. The BioBIM is fully equipped for the automation of sampling/processing, storage and tracking of biospecimens. Standard Operating Procedures have been developed for processing and storage phases as well as archive of clinical data. The availability of biospecimens and clinical data will constitute a resource for various research projects.

A strategic approach to diabetes prevention: how much evidence is required before we act?

1 ISSN 1758-1907 10.2217/DMT.12.79 © 2013 Future Medicine Ltd Diabetes Manage. (2013) 3(1), 1–4 The global diabetes epidemic continues unabated with the number of people with diabetes predicted to increase from 366 million in 2011 to 552 million by 2030 [1]. In Australia, the burden of Type 2 diabetes is increasing and diabetes is expected to become the leading cause of disease burden by 2023 [2]. It is now just over a decade since the landmark US Diabetes Prevention Program (USDPP) and Finnish Diabetes Prevention Study both found that the risk of developing Type 2 diabetes could be reduced by 58% by lifestyle modification that focused on nutrition, physical activity and modest weight loss, and, more recently, that it could be sustained [3,4]. Cost–effectiveness analyses of different strategies for screening and prevention of Type 2 diabetes suggest that screening for Type 2 diabetes and impaired glucose tolerance, followed by interventions, is cost effective [5]. Furthermore, in the USDPP, lifestyle intervention was more cost effective than pharmacological intervention [6]. The potential to prevent or delay Type 2 diabetes in high-risk individuals by lifestyle intervention has been unequivocally established (proof of concept); however, most landmark studies, conducted exclusively in primary care settings, used intensive, individualized interventions that would be difficult to roll out as population-wide programs [7]. The challenge is to translate this evidence into feasible and effective communitybased programs that can be scaled up and rolled out more broadly in existing health and community systems [8,9]. In response to the challenge, a promising range of translational research studies have emerged over the past decade. In addition to the primary care setting, several studies have demonstrated the feasibility of screening and recruiting in workplaces and community settings, together with the effectiveness of group-based interventions [10–12]. Moving from scientific discovery to population-wide implementation requires a series of steps, each building upon the achievements of the previous step, from basic research to diffusion of interventions [13]. Over the last decade the USA, Finland and other parts of Europe have been leading the way in exploring policy efficiency, availability and distribution, as well as diffusion of diabetes interventions. In these countries, governments are showing

Abstract P1-07-08: Effect of sample preservation method and transportation duration on tumor gene expression profiling in breast cancer.

Abstract Background: Translational research studies in the context of international clinical trials may incur prolonged transportation time of tumor specimens. The effect of prolonged transportation time and sample preservation method on RNA quality and on reliability of derived gene expression profiling (GEP) is unknown. Methods: Tumor material from surgical specimens of patients with primary breast cancer (BC) was obtained with cold ischemia time (CIT) recorded for each patient. Each sample was divided into 8 aliquots 4 that were placed in RNAlater and 4 that were frozen. To simulate prolonged transportation duration, samples were kept 1 week in a bucket of dry ice replenished daily, with temperature monitoring. The bucket was stored at room temperature. RNA extraction and GEP using Affymetrix HG-U133 Plus2 were performed at baseline and at day 2, 4 and 7. Linear mixed effect models were used to explore effects of transportation time and preservation method on RNA quality (RNA Integrity Number-RIN). Pre-defined single genes (n = 12), gene modules (n = 25) and molecular subtypes (PAM50) were evaluated. P < 0.01 was considered for significance. Results: Samples were collected from 13 patients with an average CIT of 37 minutes (range: 20–85). During the simulation period, the mean temperature at which samples were stored was −77.8°C (SD+/−1.6°C). RIN values decreased significantly over time (1-day change −0.08, CI = −0.12 to −0.04, p < 0.001) and by RNAlater (−0.29, CI = −0.07 to −0.51, p < .01), but remained always above 6.5. RNA degradation evaluated with actin 3/5 and gapdh 3/5 ratios was significantly higher over time for samples preserved in RNAlater compared with frozen (difference in changes 0.68, CI=0.49 to 0.87, p < 0.001 and 0.33,0.25 to 0.4, p < 0.001, respectively), and for 5 RNAlater (38%) samples these ratios exceeded the Affymetrix thresholds (3 and 1.25, respectively) at least at 1 time-point. Comparing tissue preservation methods, only the expression of PTEN gene and CASP3 module changed significantly more over time in RNAlater compared with frozen samples (interaction p < 0.01). Genes and gene modules that were significantly differently expressed over time, independent of preservation method, were AURKA, PTEN, CASP3 and WOUND (p < 0.01, range of 1-day changes in log2 expression:-0.05 to 0.10), while the expression of ESR1 and ERBB2 was unaffected. Those that were significantly influenced by sample preservation method, adjusted for time, were genes ESR1, PLAU, VEGF, PIK3CA, PTEN and gene modules GENE21 and GENE70 was (P < 0.01, range: −0.61 to 0.16). Using PAM50, 8 (61%) samples were classified at least once as a different subtype over time compared to baseline, and 4 (30%) samples fell in a different Genomic Grade Index (GGI) class risk (high/low) at least once over time. Conclusions: In this study, frozen samples were significantly less degraded over time compared with samples stored in RNAlater. Preservation method had a stronger influence on genes and gene modules values compared with prolonged transport duration. In international clinical trials we recommend freezing samples to assure more stable gene expression analysis and limit transport duration to less than 3 days. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-08.

Abstract P3-05-07: Poor prognosis early breast cancer: pathological characteristics of the Unicancer-PACS08 trial including patients treated with docetaxel or ixabepilone in adjuvant setting

Abstract Background: The PACS08 trial aimed to compare adjuvant FEC100-Docetaxel regimen to FEC100-Ixabepilone (Ixa) in poor prognosis early breast cancer (BC). The study population included BC patients presenting with triple-negative (TN) [i.e. estrogen receptor (ER)−/progesterone receptor (PR)−/HER2−] or ER+/PR−/HER2− tumor, which are subgroups significantly associated with worse prognosis. Central review was performed and detailed pathological characteristics of the cohort are reported herein. Patients and method: Between 2007 and 2010, 762 patients with unilateral TNBC (n = 592, 78%) or ER+/PR−/HER2− BC (n = 170, 22%) were enrolled. Recruitment was interrupted due to BMS decision to stop Ixa development in adjuvant setting. As defined by inclusion criteria, TNBC were either node+ or node-, and ER+/PR−/HER2− BC only node+. Following the validation of ER, PR and HER2 status on whole sections prior to inclusion, paraffin blocks (n = 754) were sent for central pathology review, tissue microarray (TMA) construction and constitution of the trial collection for translational research studies. Review of the cases (n = 754) was performed by a board of expert breast pathologists on a one-week working session with discussion of the difficult cases under a multihead microscope. Tumor characteristics were assessed on whole tissue sections. Immunohistochemical detection of Ki67, EGFR, cytokeratins (CK)5/6 and 14, was performed on TMAs. Results: TNBC were significantly associated with younger age at diagnosis (median age 51yr vs 57.5yr in the ER+/PR- subgroup, p < 0.001). Most tumors were invasive ductal carcinomas (72%). Distribution of special histological subtypes was significantly different in the TNBC and ER+/PR−/HER2− subgroups, with the former comprising medullary (n = 16/17) and metaplastic (n = 34/34) subtypes while the latter was enriched in invasive lobular carcinomas (n = 27/35, p < 0.0001). TNBC were significantly associated with higher histological grade as compared to ER+/PR−/HER2− subgroup (Table 1). Accordingly, TNBC displayed significantly higher proliferative activity as shown by mitotic count and Ki67 index (p < 0.001). As compared to ER+/PR−/HER2− subgroup, TNBC showed distinct characteristics, and displayed a so-called basal-like phenotype in 80%. Among the ER+/PR−/HER2− subgroup, most tumors were classified as luminal B (64%). Interestingly, the presence of tumor lymphocytic infiltrate was more frequently observed in luminal B (59%) as compared to luminal A (30%) subtype (p < 0.001). Conclusion: The Unicancer-PACS08 patient cohort is mainly composed of TNBC that harbour distinct pathological features. Description of the PACS08 collection provides a solid basis for translational research projects, which have been initiated with regards to genomic instability and DNA damage repair, immune system, and biomarker studies to identify new therapeutic targets. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-07.

An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic, it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study, we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary, the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies.

Novel Alginate-Enclosed Chitosan–Calcium Phosphate-Loaded Iron-Saturated Bovine Lactoferrin Nanocarriers for Oral Delivery in Colon Cancer Therapy

To develop polymeric-ceramic nanocarriers (NCs) in order to achieve oral delivery of the anticancer neutraceutical iron-saturated bovine lactoferrin (Fe-bLf) protein. Fe-bLf or paclitaxel (Taxol®) were adsorbed onto calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate. The Fe-bLf or Taxol-loaded NCs indicated as AEC-CP-Fe-bLf or AEC-CP-Taxol NCs, respectively, were made by combination of ionic gelation and nanoprecipitation. Size distribution, morphology, internalization and release profiles of the NCs were studied along with evaluation of in vitro and in vivo anticancer activities and compared with paclitaxel. AEC-CP-Fe-bLf NCs obtained spherical morphology and showed enhanced endocytosis, transcytosis and anticancer activity in Caco-2 cells in vitro. AEC-CP-Fe-bLf NCs were supplemented in an AIN 93G diet and fed to mice in both prevention and treatment human xenograft colon cancer models. AEC-CP-Fe-bLf NCs were found to be highly significantly effective when given orally, as a pretreatment, 1 week before Caco-2 cell injections. None of the mice from the AEC-CP-Fe-bLf NC-fed group developed tumors or showed any signs of toxicity, while the mice fed the control AIN 93G diet showed normal tumor growth. Fe-bLf or Taxol, when given orally in a diet as nanoformulations post-tumor development, showed a significant regression in the tumor size with complete inhibition of tumor growth later, while intratumoral injection of Taxol just delayed the growth of tumors. The pharmacokinetic and bioavailability studies indicated that nanoformulated Fe-bLf was predominantly present on tumor cells compared to non-nanoformulated Fe-bLf. Fe-bLf-loaded NCs were found to help in absorption of iron and thus may have utility in enhancing the iron uptake during iron deficiency without interfering with the absorption of calcium. With the promising results of our study, the future potential of NC-loaded Fe-bLf in chemoprevention and in the treatment of human colon cancer, deserves further investigation for translational research and preclinical studies of other malignancies.

115IN - Addressing Clinical Challenges in Targeted Therapies with Advanced Imaging

ABSTRACT Advanced imaging techniques are being developed to provide information relevant for patient management beyond changes in tumor size. Treatment with novel targeted therapies showed that conventional RECIST measurements are insufficient to assess response as changes in tumor size occur relatively late, if at all. It has been shown that molecular imaging techniques provide predictive and prognostic information at baseline or by comparing baseline scans to scans obtained very early after the start of treatment. It aims at the (very) early identification of responders before tumor shrinkage occurs, but also indicating a high likelihood of relapse even before tumor growth is apparent. Furthermore, molecular imaging with radiopharmaceuticals may aid in identifying receptor overexpression or pathway activation in tumors before treatment with targeted therapies is initiated. The ability of FDG-PET to predict response of metastatic GIST to imatinib became the role model for the potential of molecular imaging to provide clinically relevant answers within days after the start of treatment. Since the introduction of integrated PET/CT the acceptance of the technology enormously increased. A large number of PET/CT studies was performed in patients treated with various targeted therapies (both antibodies and TKIs) in a wide variety of tumor types with both FDG and other radiopharmaceuticals such as FLT, a marker of proliferation. Similary, advanced CT (perfusion) and MRI (dynamic MRI, DWI, MRS) techniques have taken advantage of the development of technology. Following the introduction of therapeutic monoclonal antibodies such as bevacuzimab and trastuzumab, studies with the radiolabeled derivatives have been used to image the tumor-associated factors and receptors, targeted by these antibodies (immunoPET and immunoSPECT). It has been shown that this approach could successful visualize the antibody targets. More recently, small molecules (e.g. paclitaxel, lapatinib) have been radiolabeled and used in clinical studies. Following the numerous proof of principle and translational research studies, the time has come time to develop the role of the advances towards evidence-based imaging in clinical practice by systematically positioning advanced imaging for treatment selection in clinical trials. Disclosure The author has declared no conflicts of interest.