WEDNESDAY, JANUARY 28
8:00-8:30 Sponsored Presentation (Opportunity Available) or Morning Coffee
Contact Ilana Schwartz, Manager, Business Development, 781-972-5457 or ischwartz@healthtech.com.
Assay Development for ‘Omic’ Biomarkers
8:30-9:00 Genomics Technologies for Research and Clinical Applications
Don A. Baldwin, Ph.D., Director, Penn Genomics Facility, University of Pennsylvania, School of Medicine
As technologies for highly parallel molecular biology assays have matured, their use in development and implementation of clinical tests has launched the field of translational genomics. A range of microarray research methods, including new RNA assays, genotyping, enriched DNA mapping, and deep sequencing, will be reviewed along with illustrations of biomarker or other clinical applications. Just as functional genomics will require the integration of multiple data types, translational genomics must integrate a variety of molecular assays with other clinical data to advance personalized medicine.
9:00-9:30 Low Abundance Protein Biomarker Quantitation Using Immunoaffinity Enrichment And Nanoflow LC-MS/MS
Hendrik Neubert, Ph.D., Clinical, Quantitative and Innovative Medicine (CQIM), Pfizer Global Research and Development
Hyphenating immunochemistry with LC/MRM detection is a promising area of bioanalytical development. Advances in immunoaffinity extraction strategies at peptide or protein level, together with robust nanoflow chromatography and nanospray ionization techniques, offer significant sensitivity increases required for low-abundance protein biomarker quantitation from biological matrices such as serum/plasma or saliva. A popular approach is the targeted detection of diagnostic peptide fragments originating from the protein biomarker of interest, primarily through enzymatic digestion. This presentation will describe these concepts, highlight pitfalls during assay development, and demonstrate with case studies that mass spectrometry based approaches can be considered as viable alternatives to immunoassays offering complementary assay options for protein biomarker quantitation.
9:30-10:00 Utilizing a LC/MS/MS (MRM) Quantitative Targeted Biomarker Panel to Elucidate Multiple Biological Changes Simultaneously
Jon Butler, Ph.D., Assistant Senior Biochemist, Integrative Biology, Biomarkers, Eli Lilly and Company
Biomarkers are commonly used to characterize a disease and/or follow disease state progression. Biological processes can be complex and have multiple feedback mechanisms to keep them in a regulated state. When these systems begin to fail, a single biomarker may not reflect that the process is in an uncontrolled state. Biomarker panels can be a tool used to help elucidate these complex biological systems, such as musculoskeletal diseases. A protein biomarker panel can show processes involved in disease and/or disease states, thus allowing the striation of patients for tailored therapeutics. Musculoskeletal diseases in humans result in extended medical costs but, more importantly, reduce the quality of patient life. Muscle physiology has been characterized at the message level and here we extend that to the protein level via tissue extracts from mouse atrophy models as compared to controls. Global proteome profiling is used along with in silico protein digestion followed by MRM development to create a panel of proteins involved in muscle physiology. These processes narrow down proteins amenable to detection with this technology. LC/MS/MS/MRM is used to quantitate changes due to perturbations in these animal models. This panel can be used to access early changes in muscle physiology which currently is measured at a much later point in time by size and/or strength. This talk will show: global proteome profiling for development of panel candidates; in silico MRM development for panel candidates; formation of a targeted biomarker panel; and uses of targeted protein biomarker panels.
10:00-10:15 Sponsored by
.JPG "Monarch")
Multiplex Measurement of Target Proteins in Serum/ Plasma Using Mass Spectrometry
Marci Copeland, Research Associate, Monarch LifeSciences
Animal models are commonly used in pre-clinical studies for various areas of disease research, including cardiovascular disease, osteoporosis, chronic liver disease, and many others prior to translation to human disease research and drug therapies. The development of biomarker panels for disease diagnosis requires ongoing studies and validations of disease biomarkers, including their predictive consistency across different ethnic groups, gender, and other sources of patient population variability. The use of a multiplex panel of proteins to evaluate and set disease parameters is a useful and necessary part of forming diagnostic capabilities of protein markers. Greater understanding of disease characteristics will lead to earlier diagnosis and treatment. Evaluation of a panel of apolipoproteins in cardiovascular disease helps to understand the underlying mechanisms in lipid metabolism beyond traditional cholesterol measurements. Multiplex measurement of procollagen type-I and type-III aminoterminal propeptide (PINP, PIIINP) represents a direct measurement of alterations in the metabolism of collagen type I and type III. These markers have been used to assess collagen incorporation into bone or the presence of fibrosis, for example, and have been used in many specific areas of research including osteoporosis and other bone degeneration disorders, liver fibrosis, and muscle anabolism research. We have developed and implemented the use of multiplex mass spectrometry-based assays to quantitatively measure PINP and PIIINP in a single measurement and a panel of apolipoproteins without antibody enrichment of the target proteins. These multiplexed assays have been optimized for a high-throughput format to handle hundreds of samples simultaneously and result in absolute quantification of the target proteins. With the measurement of target protein biomarkers in pre-clinical rodent studies or human patients, serum protein biomarkers allow continual non-invasive patient monitoring, resulting in a clearer representation of disease status, progression, or regression in a patient’s body in a native disease state or response to drug treatments.
10:30-11:30 Coffee Break with Poster and Exhibit Viewing
Flow Cytometry and Circulating Cell Assays
11:30-12:00 Validation and Implementation of Flow Cytometric Assay to Evaluate Pharmacodynamic Biomarkers in Clinical Study
Dianna Wu, Ph.D., Principal Scientist, Clinical Biomarker, Bristol-Myers Squibb Co.
Flow cytometry technology is being widely used in pharmacodynamic studies to examine target engagement, drug efficacy, and possible toxicities. This is because blood, as one of the most easily accessible tissues, can be extensively analyzed in flow cytometry-based biomarker assays. More importantly, analyzing the samples using validated biomarker assays in early stage human clinical trials would gain valuable information and time to guide the clinical development. The current talk provides examples of using the fit-for-purpose approach to validate and implement flow cytometry assay to evaluate pharmacodynamic biomarkers in clinical study.
12:00-12:30 Validating Cell-Based Biomarker Assays
Iman Jilani, M.S., CLS, MT (ASCP), Biomarker Assay Specialist, Manager, Clinical Assay Group, Pfizer
With the emergence of biomarkers and their role in the development of effective and safe drugs, it is crucial to standardize these assays to be compliant with Good Laboratory Practice guidelines. To date, cell-based assays have not been standardized even though they are a crucial part of biomarker discovery and development. In this presentation we will review challenges that pharmaceutical and biotechnology companies face and progress made towards standardizing cell based assays including Flow Cytometry, Circulating Tumor Cells (CTC), and Circulating Endothelial Cells (CEC).
12:30-2:00 Lunch on your own
Translation from In-House Development to Clinical Laboratory
(Shared session with Translational Cancer Medicine meeting)
2:00-2:30 Development and Integration of Biomarkers into Clinically Useful Diagnostics
Anne-Marie Martin, Ph.D., Director, Oncology, MDC, GlaxoSmithKline
With the advancement of understanding in the molecular basis of disease we have increased our ability to identify genomic biomarkers relevant to disease pathogenesis and widened opportunities to develop genomics-based tools to tailor treatment options and assess treatment response. Thus, genomic biomarker research promises to provide more precise predictors of outcome to treatment not previously attainable with traditional biomarkers. However, before genomic biomarker tests become commonplace in clinical practice, several issues need to be addressed in order to generate the essential levels of evidence to demonstrate analytical and clinical validity and utility. Assay validation is required not only in the context of in-house developed assays (i.e. "homebrew" tests) but also prior to the use of commercially available Food and Drug Administration (FDA)-approved diagnostic tests. This presentation will illustrate some the steps required to achieve clinical utility of a diagnostic test.
2:30-3:00 Molecular Diagnostics Laboratories (MoDEL): A Program to Support Cancer Biomarker Clinical Assay Development
J. Milburn Jessup, M.D., Chief, Diagnostics Evaluation Branch, Cancer Diagnosis Program, DCTD/National Cancer Institute
Although biomarkers will become increasingly important as targeted and personalized therapies dominate the care of the cancer patient, the development and validation of useful biomarkers is often stopped by inadequate assay development. The Cancer Diagnosis Program (CDP) of NCI issued a Request for Information that defined significant needs for assay development as: specimen acquisition, access to standards and reagents, guidance on assay development for academia to facilitate transfer to industry, and support for improving assay performance, evaluation and validation for clinical use. As a result, CDP will present its plans for developing MoDEL as a suite of services that 1) will assist the utilization of biomarkers in late phase clinical trials in oncology and 2) will be available to both academia and industry.
3:00-4:00 Networking Refreshment Break
Closing Plenary Session:
Circulating Tumor Cells
(Shared session with Translational Cancer Medicine meeting)
3:30-4:00 Circulating Tumor Cell Assays: A Prognostic and Predictive Factor for Breast, Prostate and Colon Cancer
Herbert A. Fritsche, Ph.D., Professor and Chief, Clinical Chemistry, The University of Texas M. D. Anderson Cancer Center
The current hypothesis of cancer metastasis proposes that tumor cells escape into the blood and circulate until they are either eliminated by host response mechanisms or until they find an environment in which to reside in a dormant condition and to proliferate at a later time. Thus, the detection of circulating tumor cells may represent an early indication of micro-metastasis or of aggressive tumors which are able to shed tumor cells into the blood. The circulating tumor cells can be captured using antibody labeled magnetic beads, either in positive or negative selection schema. After the circulating tumor cells are isolated, they may be characterized by immunohistochemistry and counted. Alternatively, these cells may be characterized by gene expression analysis using RT-PCR. One of the CTC detection methods (Veridex Inc, Cell Search Assay) has been cleared by the US FDA for use as a prognostic test in patients with metastatic cancers of the breast, prostate and colon. In these cases of metastatic cancer, the pre-treatment presence of tumor cells is prognostic of a poor outcome, at any time during the course of the disease. Furthermore, in metastatic breast cancer patients, the presence of tumor cells at the end of the first course of chemotherapy is predictive of treatment failure. Thus, the CTC test may permit the oncologist to make an early decision to discontinue first line therapy for metastatic breast cancer and pursue more aggressive alternative treatments. We have addressed the practical aspects of routine testing for CTC’s using the Veridex Cell Search method. We have also evaluated the Adnagen Breast Cancer Cell Select and Detect assay for CTC detection. The method uses multi-antigen cell capture with antibody labeled magnetic beads, followed by RT-PCR characterization of selected genes. This assay may compliment the Cell Search assay for tumor cell detection in blood. Other CTC assays based on new capture technologies are currently in development.
4:00-4:30 Biomarker Assay Development and Validation: An Epiphany for Drug Development and the Management of Patients with Cancer
Martin Fleisher, Ph.D., Chair, Department of Clinical Laboratories, Memorial Sloan-Kettering Cancer Center
Biomarkers can be used to predict whether or not a treatment modality is effective in early-stage clinical trials, judge the response to therapy, identify which cancer patients are at high risk of tumor recurrence and predict how effective an investigational drug is against a specific type of cancer. This information is valuable when decisions must be made on stratifying patients based on likely response to the therapy. We have initiated a biomarkers development and validation program that focuses on targeted therapy in patients with prostate and ovarian cancer and mesothelioma. The enumeration of circulating tumor cells (CTC) in patients with metastatic prostate cancer (PC) receiving targeted therapy for androgen receptor over expression has demonstrated impressive clinical sensitivity when compared with PSA response. As new targeted therapies enter the pipeline, predicting the effectiveness of the drug on the target by isolating and characterizing CTC in patients with metastatic cancer is clinically essential. In patients with ovarian cancer, biomarkers validated in our laboratory, such as YKL-40, HE4 and Mesothelin have been shown to be more effective than CA125 in monitoring Stage 1 and 2 ovarian disease and in detecting tumors with mucinous histology. Two biomarkers, Osteopontin and Mesothelin, have been validated and measured in patients with mesothelioma before and after chemo- and radiation therapy. Preliminary data suggest that these two biomarkers will be clinically effective in staging patients for future therapy and in monitoring recurrence of disease. Our CLIA certified laboratory has developed a rigorous validation protocol that assesses the
pre-analytical, analytical and post-analytical assay performance characteristics of biomarkers under clinical evaluation. This validation protocol optimizes biomarker effectiveness essential for pharmacodynamic and clinical outcome studies.
4:30 Close of Conference