Using Mass Spectrometry as a Tool. Emergencies

Transcription

1 United States Department of Health & Human Services Office of the Assistant Secretary for Preparedness and Response Rapid, Sensitive Dose Assessment Using Mass Spectrometry as a Tool to Identify Markers for Public Health Emergencies Lynne K. Wathen, PhD Division of CBRN Countermeasures Biomedical Advanced Research and Development e e Authority ty April 19, th Annual Oak Ridge Conference Emerging Technologies for 21 st Century Diagnostics San José, California

2 Biomedical Advanced Research and Development Authority (BARDA) Mission Ensuring the availability of medical countermeasures and diagnostic tools to address public health emergencies Three threat areas: Chem/Bio/Rad/Nuc, Pandemic Influenza, Emerging Infectious Diseases Comprehensive portfolio approach to development and acquisition of products Unique niche in USG biomedical R&D Bridge the Valley of Death Mid- to late-stage product development Work with industry to progress product candidates through the pipeline ASPR: Resilient People. Healthy Communities. A Nation Prepared.

3 Public Health Emergency Medical Countermeasures Enterprise National Biodefense Science Board NIH BARDA BARDA and CDC BARDA and CDC CDC and ASPR/OPEO Research and Development Advanced Development Acquisition FDA Storage/ Maintenance Deployment Utilization PHEMCE COORDINATED PLANNING & EXECUTION Ex Officio Members: ASPR: Resilient People. Healthy Communities. A Nation Prepared. 3

4 Spectrum of Radiation Health Effects ASPR: Resilient People. Healthy Communities. A Nation Prepared. 4

5 Biodosimetry Concepts of Operation Event Days Million People to Screen <2 Gy Testing with POC Device > 2 Gy? 2 Gy High Through- Put Dosimetry and Additional Screening Therapeutic Administrations Follow-on Care Evacuate Area ASPR: Resilient People. Healthy Communities. A Nation Prepared. 5

6 Current Biodosimetry Capabilities Radiation Dose Assessment Tools Victim s location relative to incident Signs and Symptoms Blood Cell Counts Laboratory-based Cytogenetics Deficiency Provides only a rough estimate false positive rate Requires two blood draws separated by days and the ability to connect data Dicentric Chromosome Assay takes 2-3 days for result and few laboratories offer the test ASPR: Resilient People. Healthy Communities. A Nation Prepared. 6

7 Current BARDA Biodosimetry Program Contractors Point of Care TECHNOLOGY (POC) or High Through-put (HT) Protein DNA mrna Volatile Organic Compounds Ocular Electron Paramagnetic Resonance Contractor 1 Both Contractor 2 HT Contractor 3 POC Contractor 4 HT Contractor 5 HT Contractor 6 POC Contractor 7 POC Contractor 8 POC ASPR: Resilient People. Healthy Communities. A Nation Prepared.

8 Example of One Contractor s Approach Using Mass Spectrometry as a Tool for Identifying Biodosimetry Markers of Interest ASPR: Resilient People. Healthy Communities. A Nation Prepared. 8

9 Example Project Methodology Animal Studies Mouse in vivo IR Human RT Patients Sample Analysis LC-MS/MS Analysis Protein Change List ELISA Analysis Data Analysis Regression Models Cluster Analysis Linear Disc. Analysis Tree Based Classifiers Candidate Biomarkers Candidate Markers (Mouse) Protein 1 Protein 2 Protein 3 Selection Criteria Fold Change Natural & CF variability Predictive Sensitive & specific assays Different pathways Candidate Markers (Human) Protein 4 Protein 5 Protein 6 Final Biomarkers Validated Markers Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Protein 6 ASPR: Resilient People. Healthy Communities. A Nation Prepared. 9

10 Complex Mixture of Proteins Example LC-MS/MS Approach for Protein Identification digestion A L E L F R H H K P N D M A A K G A S E D I P V K D H L K L K F G G H P E T L E D E S E M K H K A F D K K D K H L K K S A L F K V Y K Q E L F K L Q I P V K A L D V A GM HM K G I G W GG V Q G A A L E L F R E E G V E L G V Q F Q G L N G M I I S E D E M K K M R L L S Separation G L V S D G E W Q L F D F K K A I K N D M A A K L T A E A S E D L K V H T H G H E A E L T P L A Q S H A T S K by HPLC N G I E L G F Q G Y L E F G H P E T L E K Peptide digest K Y K F K H P G D F G A D A Q G A M S K V E A D V A G H G Q E V L I R Y L E F I S E A I I Q V L Q S K G H H E A E L T P A Q S H A T K M G L S D G E W Q L V L N V W G K On-line analysis by OrbiTrap MS Spectrum Database search Protein ID ASPR: Resilient People. Healthy Communities. A Nation Prepared. MS/MS Spectrum 10

11 Protein 1 LC-MS/MS Results E E L E L L V S S L R A Q V E S F R 6 Gy, Day 5 6 Gy, Day 5 0 Gy, Day 5 0 Gy, Day 5 E F W L G L D N L S K 6 Gy, Day 5 0 Gy, Day 5 centration (ng/ml) Protein 1 Con ELISA Results 0 & 6 Gy, Day Gy 6 Gy Dose (Gy) Demonstrated Correlation Between LC-MS/MS Analysis on Pooled Samples ASPR: and ELISA Resilient Analysis People. Healthy on Individual Communities. Samples A Nation Prepared. 11

12 Example Project Plan Focused on Product Development Initial Year Second Year Third Year Fourth Year Product Development 180 candidate mass peaks 36 identified proteins 6 marker panel Human panel Mass peaks Key radiation Predictive Predictive e Complete human only no protein ID Good classifiers with 5-10 peaks responsive proteins identified ELISA confirmation of mouse marker panel Tested against 4 potentially confounding marker panel Alpha Instrument Development validation in irradiated patients Beta Testing Complete mass spec. conditions FDA filing data ASPR: Resilient People. Healthy Communities. A Nation Prepared. 12

13 Scatter Plots to Separate Irradiated versus Non-irradiated Mice Non Irradiated Irradiated (ng/ml) Protein 1 Conc false negatives out of 298 samples (0.67% Fn) 1 false positive out of 298 samples (0.34% Fp) Protein 2 Conc (ng/ml) Protein 1 alone results in 99% Correct classification of non-irradiated from irradiated ASPR: Resilient People. Healthy Communities. A Nation Prepared. 13

14 Additional Proteins are Necessary for < and 6 Gy in Mice Protein 1 Conc (ng/ml) <6 Gy >6 Gy Scatter plot of Protein 1 and Protein 2 results in 87% correct classification <6 Gy from 6 Gy 0 false negatives out of 298 samples(00%fn) (0.0% 38 false positive out of 298 samples (12.8% Fp) All false positive samples are 4 Gy classified as 6Gy Protein 2 Conc (ng/ml) Additional Proteins and More Sophisticated Models are Needed to Separate and < 6 Gy ASPR: Resilient People. Healthy Communities. A Nation Prepared. 14

15 Human Radiotherapy Patient Panel The three most important radiation responsive proteins in the panel include Protein 4 (up-regulated) Protein 5 (down-regulated) Protein 6 (up-regulated in TBI patients) Two of these proteins are used in the mouse panel Tree classifiers using these three proteins can distinguish irradiated patients from controls with 96% accuracy Data provides positive indication that plasma protein biodosimetry is feasible in humans requires NHP study to expand panel and confirm 0 tion (ng/ml) Plasma Concentrat Plasma Co oncentration (ng/ml) Concentration (ng/ml) Plasma Protein 4 Controls Infection Trauma TBI(d0) TBI(d1) TBI(d2) TBI(d3) Sample Group Protein 6 Controls RT(d0) RT(d1) RT(d2) RT(d3) Sample Group Protein 5 Controls RT(d0) RT(d1) RT(d2) RT(d3) Sample Group ASPR: Resilient People. Healthy Communities. A Nation Prepared. 15

16 Example Summary and Conclusions Using LC-MS/MS techniques identified 180 mass peaks for interigation The LC-MS/MS Approach identified 36 Radiation-responsive proteins in the mouse model A subset of these proteins have been confirmed in mouse plasma using ELISA Using 6 proteins from this panel correctly classifies all large-scale l mouse data sets Two groups (< 6 Gy or 6 Gy) with low false negative (FN) and false positive (FP) rates The LC-MS/MS Approach identified 8 Radiation-responsive proteins in human radiotherapy patient samples Preliminary panels of 3 proteins can easily distinguish between irradiated patients and controls ASPR: Resilient People. Healthy Communities. A Nation Prepared. 16

17 Areas of Interest for Contractors in Diagnostic Development A pre-proposal conference is planned to on Thursday, May 3, 2012, from 2:00pm to 4:00pm provide details regarding the following potential Broad Agency Announcements (BAAs): BARDA-CBRN-BAA SOL-00011, entitled Advanced Research and Development of Chemical, Biological, Radiological, and Nuclear Medical Countermeasures ; BARDA-SST-BAA SOL-00013, entitled Science and Technology Platforms Applied to Medical Countermeasure (MCM) Development, and; BARDA-FLU-BAA , entitled Advanced Development of Medical Countermeasures for Pandemic Influenza. To register for the conference, please the name of your company, as well as the names and *citizenship of registrants to AMCG-Opportunities@hhs.gov by 3:00 PM EDT Tuesday, May 1, ASPR: Resilient People. Healthy Communities. A Nation Prepared. 17

18 Interfacing with BARDA Portal to BARDA Register, request a meeting Tech Watch presentation Official announcements and detailed information about all government contract solicitations See presolicitation announcement: a1eaed051a0223ffd180b5cbe&tab=core&_cview=1 ASPR: Resilient People. Healthy Communities. A Nation Prepared.