What Impact will Proteomics, Including CSF Analysis, have on the Near-term Development of Biomarkers for Nervous System Diseases?
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1 What Impact will Proteomics, Including CSF Analysis, have on the Near-term Development of Biomarkers for Nervous System Diseases? Daumier 1 Howard Schulman (howard.schulman@menlo.ppdi.com)
2 Biomarker Discovery, Validation, Implementation Progress Toward Biomarker Test Specific fit-for-use Biomarker Tests Clinical Trial Samples (plasma/csf) -Multiplexed Immunoassays -MRM -Ab-based quantitative MS Clinically Validated Candidates Replication/Validation Studies (fluid) -Multiple Reaction Monitoring (MRM) -Proteomic/Peptidomic/Metabolomic profiling -Multiplexed Immunoassays, Ab-based quantitative MALDI-MS, protein chip (paucity of neuro-related Ab assays kits in catalogues) Scientific Literature Gene expression (tissue) Sub-proteomic profiling (fluid and/or tissue) Proteomics/Peptidomics/Metabolomics profiling (fluid and/or tissue) Source and Number of Biomarker Candidates 2
3 Proteomic technologies for neuroscience applications More proximal to disease expression than gene expression, but incomplete depth of coverage Sample processing Separation Differential quantification Identification 2-D gels [DIGE (Differential in-gel electrophoresis)] with MS ID SELDI (MALDI-based) 1D LC/MS and Multi-dimensional LC-LC/MS itraq (isobaric tags) for differential quantification Label-free (software-based) differential quantification Enhance functional sensitivity for discovery by compartment phenotyping (e.g. CSF) Enhance functional sensitivity for discovery by sub-proteome capture (e.g. glycated proteins, ubiquitylated proteins) 3
4 Differential Quantification: Proteins & Peptides Peptidome LOW MW Biological Fluid Proteome HIGH MW -Abundant Proteins Proteolytic Digestion Size Exclusion DeepLook TM MS Cation Exchange 4 Fractions 4/8 Fractions 2D HPLC ESI-MS HPLC ESI-MS 1D HPLC ESI-MS 2D >8,000 Components >6000 >30,000 Label free differential quantification and molecular identification (MS/MS) 4
5 Pharmacodynamic profiling (~400 of 20,000 at p<0.001) RIA (1) vs Mass Spec (20,000) RIA of Adiponectin (ACRP30) Mass spec: Mean of 3 adiponectin peptides from profiling data of 20,000 components RIA (mg/ml) Normalized Intensity Normalized intensity C D E Vehicle Dose 1 Dose 2 C D E Vehicle Dose 1 Dose 2 5
6 CSF vs plasma Smaller dynamic range of CSF facilitates depth of analysis Can detect intracellular proteins due to cell death/degeneration More proximal to disease pathology and physiological response CSF (200 ml) vs Blood (5000 ml) Discover in CSF and convert to plasma test CSF can be a barrier to adoption; not for population screen ADNI planned 25% CSF but increased to 50% with motivation More common in Europe/Scandinavia. Low adverse effects Standard of care in lymphoma metastasis to CNS despite poor diagnostic cytological test Adoption will be based on value of biomarker for clinical or trial decision 6
7 From Leigh Anderson 7
8 Dynamic Range of Proteins in CSF Hühmer et al
9 CSF Proteome: proteins profiled from 1 ml Biological Processes Represented in the CSF Proteome The percentage indicates the % of proteins identified in our CSF library corresponding to a particular biological process. Biological Process % Biological Process % CELLULAR PHYSIOLOGICAL PROCESS 65% BIOPOLYMER CATABOLISM 5% MACROMOLECULE METABOLISM 40% RESPONSE TO WOUNDING 4% CATABOLISM 27% NEUROGENESIS 4% CELL COMMUNICATION 25% CELL-CELL SIGNALING 4% ORGANOGENESIS 20% CELL MOTILITY 3% REGULATION OF CELLULAR PROCESS 19% WOUND HEALING 2% DEVELOPMENT 18% REGULATION OF CELL PROLIFERATION 2% RESPONSE TO EXTERNAL STIMULUS MORPHOGENESIS CELL ADHESION RESPONSE TO STRESS CELL PROLIFERATION PROGRAMMED CELL DEATH 16% 12% 10% 9% 9% 8% REGULATION OF BODY FLUIDS HEMOSTASIS GROWTH COAGULATION MUSCLE CONTRACTION 2% 2% 3% 2% 1% PROTEOLYSIS AND PEPTIDOLYSIS 5% COMPLEMENT ACTIVATION, CLASSICAL PATHWAY 1% PROTEIN CATABOLISM 5% CELL MIGRATION 1% 9
10 APP Sequences Identified in CSF Library 1 mlpglallll aawtaralev ptdgnaglla epqiamfcgr lnmhmnvqng kwdsdpsgtk 61 tcidtkegil qycqevypel qitnvveanq pvtiqnwckr grkqckthph fvipyrclvg 121 efvsdallvp dkckflhqer mdvcethlhw htvaketcse kstnlhdygm llpcgidkfr 181 gvefvccpla eesdnvdsad aeeddsdvww ggadtdyadg sedkvvevae eeevaeveee NH2 241 eadddedded gdeveeeaee pyeeatertt siattttttt esveevvrev cseqaetgpc 301 ramisrwyfd vtegkcapff yggcggnrnn fdteeycmav cgsamsqsll kttqeplard 361 pvklpttaas tpdavdkyle tpgdenehah fqkakerlea khrermsqvm reweeaerqa 421 knlpkadkka viqhfqekve sleqeaaner qqlvethmar veamlndrrr lalenyital 481 qavpprprhv fnmlkkyvra eqkdrqhtlk hfehvrmvdp kkaaqirsqv mthlrviyer 541 mnqslsllyn vpavaeeiqd evdellqkeq nysddvlanm iseprisygn dalmpsltet 601 kttvellpvn gefslddlqp whsfgadsvp antenevepv darpaadrgl ttrpgsgltn 661 ikteeisevk mdaefrhdsg yevhhqklvf faedvgsnkg aiiglmvggv viatvivitl 721 vmlkkkqyts ihhgvvevda avtpeerhls kmqqngyenp tykffeqmqn Peptides identified in our 1D CSF library Aβ42 Aβ42 COOH β-secretase α-secretase γ-secretase 10
11 Compartment Phenotyping in CSF to Detect Markers Closer to the Source of Pathology Monitoring Metastasis of Cancer to Brain With James Rubenstein, UCSF Metastasis to CNS occurs in ~ 30% of lymphoma; 25,000 NHL deaths annually, primarily due to CNS complications Diagnosis of primary CNS lymphoma currently calls for cytological analysis of CSF and brain biopsy that often involve complications and diagnostic delays, leading to worse outcomes. Diagnostic test would replace brain biopsy and enable monitoring of therapeutic intervention. 11
12 Diagnostic and prognostic info from single marker ELISA-based marker (ng/ml) 12
13 Stratification based on a single marker Survival Distribution Function Low marker level High marker level with James Rubenstein, UCSF 13
14 Biomarker Opportunities in Neuroscience Comprehensive Phenotyping (Discovery- and hypothesis-based approches; protein, genes, imaging.. ) Antecedent markers in schizophrenia Patient responder/non-responder stratification in depression Stratification of autism spectrum disorders Biomarkers of placebo effect (in depression) (Antecedent markers in AD) 14
15 Biomarker-enabled Drug Development Daumier 15
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