+ Development of a serum protein assay for organ confined prostate cancer 15th th June 2014 Steve Pennington UCD Conway Institute, UCD, Dublin
+ Protein Biomarker Discovery and Development Confirmation Validation/ Approval & Discovery Assay development Qualification Adoption Sample accrual Antibody based Regulatory Authorities Western blotting Additional clinical Protein Discovery Clinician Adoption ELISA samples Impact Mass Spectrometry Large Multicentre Cohorts measurement based Protein Identification and Large Scale Clinical Trials Multiple Reaction Characterisation Monitoring (MRM) Clinical ‘Robust‘ high - assays throughput assays Other analytes Multi-analyte assays (anything measurable) Sample Numbers
+ Protein Biomarker Discovery and Development Confirmation Validation/ Approval & Discovery Assay development Qualification Adoption QUALIFICATION Sample accrual Antibody based VALIDATION Regulatory Authorities CANDIDATES Western blotting DISCOVERY Additional clinical Protein Discovery Clinician Adoption ELISA samples Impact Mass Spectrometry Large Multicentre Cohorts measurement based PANEL Protein Identification and Large Scale Clinical Trials Multiple Reaction Characterisation Monitoring (MRM) Clinical ‘Robust‘ high - assays throughput assays Other analytes Multi-analyte assays (anything measurable) Statistical Methods
+ Biomarker Futility Specimens Fragmented approach
+ Clinical Utility 2006
+ Clinical Utility: 8 years on 2014 How will we discover them? How will we discover them? How will we measure them? How will we measure them? How will we validate them? How will we validate them? Will the protein biomarkers we discover be useful? How will we proceed to them gaining utility?
+ From Biomarkers to Diagnostics Tests must have analytical validity, clinical value and financial value.
+ From Biomarkers to Diagnostics Biomarkers should be fit for purpose and their purpose known 1. Reform regulatory review 2. Increase re-imbursement of tumour tests with clinical utility 3. Increase investment in research (cf. therapeutics) 4. Increase rigour for assessment - publication 5. Adhere to high-level evidence based recommendations for use Tests must have analytical validity as well as clinical and financial value.
+ Can we identify and develop protein biomarkers of clinical value in prostate cancer ? Tests to guide treatment decisions
+ Imagine this scene …..
+ Imagine the screen Blood – FBC, Hb & Fe, cholesterol, glucose, liver & kidney function Urine Heart Hearing Vision http://www.thewell.ie/executive_ medicals_men.asp
+ Imagine the screen http://www.thewell.ie/executive_ medicals_men.asp
+ Imagine the screen http://www.thewell.ie/executive_ medicals_men.asp
+ All clear doc? ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ PSA
+ All clear doc? ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ PSA 14.2ng/ml
+ DRE
+ DRE
+ TRUS Biopsy
+ Gleason Scoring of Biopsy 3 4 5
+ So, t he result….. Gleason 3 + 4 DRE – abnormal PSA 14.2ng/ml “What now?”
+ Decis ions…. The patient's treatment decision is a momentous one. He must gather all the reliable information he can so he can participate in the diagnostic process, then ultimately select the therapy most reasonable under the circumstances. As the patient confronts his condition - and he must do so - he should take into account his personal goals regarding the available therapies and their peculiar morbidities. In his decision process he may get differing medical opinions http://www.pccnc.org/patient_resources/ Prostate Cancer Coalition understanding_diagnosis/
+ NCI Statistics In Ireland: about 3000 men are diagnosed with prostate cancer very year (UK >25,000) In the UK: one man dies of prostate cancer every hour http://seer.cancer.gov/statfacts/html/prost.html
+ Personalised - Population All 7’s aren’t equal 3+4 ≠ 4+3
+ NCI Statistics Over-diagnosis and over-treatment is a major problem Most men die with rather than of prostate cancer But, there is currently no effective treatment In Ireland: about 3000 men are diagnosed with for metastatic prostate cancer prostate cancer very year (UK >25,000) In the UK: one man dies of prostate cancer every hour http://seer.cancer.gov/statfacts/html/prost.html
+ Decisions, Decisions, Decisions Radical Prostatectomy (RP) Radiation (with hormones) No treatment (Active Surveillance)
+ 26 Diagnosis and Treatment Diagnostic Test Diagnosis Treatment PSA Normal Active Surveillance BPH DRE RP Confined Prostate cancer Surgery no Non Biopsy Confined RP Prostate cancer Radiation
+ Can we identify and develop protein biomarkers of clinical value in prostate cancer ? To guide treatment decisions Accessible, Repeatable, Reliable
PCa Multidisciplinary Teams UCD Conway Teams Prostate Cancer Research Consortium National Prostate Cancer Research Group
+ Define the Clinical Question First RP No RP
+ Biomarker Panel Development PCRC Serum Sample Bioresource Biomarker discovery Label-free 2D-DIGE LC-MS/MS Biomarker Candidate list
+ Discovery: 2D-DIGE 50 age matched serum samples from PCRC 14 BPH, 36 PCa patients (Organ Confined and Non Organ Confined) 14 BPH and 36PCa patients
+ 2D-DIGE candidates
+ Discovery: Label free LC-MS/MS Mars 14 column Trypsin digestion Trans- Proteomic Create reference Pipeline pool sample from each pool depleted sample Affinity Progenesis, Depletion In-solution database search using MARS GS5 (n = 10) digestion and result filtering 14 column Depleted Label-free Peptide/protei GS7 OC (n = 10) serum LC-MS/MS n expression samples on Q-TOF profile GS7 NOC (n = 10) TPP and Skyline Serum samples Protein assay and 1D gel Public In-house Protein MS/MS MS/MS concentration spectral spectral normalization library library
+ Label free LC-MS/MS data Feature Selection Feature Alignment (a) (b) • >90,000 features (c) • Ion counting for quantification • Alignment using Progenesis Feature Quantification Protein Expression Changes • Mascot search for protein id 1.E-08 • Mascot Score > 34 (FDR = 3.08%) 1.E-07 1.E-06 • Remove non-unique mapping peptides 1.E-05 ANOVA p-value 1.E-04 1.E-03 1.E-02 • MS/MS library construction 1.E-01 1.E+00 2 0.1 0.5 1 10 Fold change ratio - drug treated/vehicle control • Trans-Proteomic Pipeline (TPP) Principle Component Analysis • Peptide to protein roll up • Analysis of differential protein expression • 59 Proteins differentially expressed (p-value<0.05)
+ PCRC OC Biomarker Candidates PCRC Serum Sample Bioresource Biomarker discovery Literatur Label-free 2D-DIGE LC-MS/MS e review 64 Candidate Proteins Biomarker Candidate list
+ PCRC OC Biomarker Candidates PCRC Serum Sample Bioresource Biomarker discovery Literatur Label-free 2D-DIGE LC-MS/MS e review MRM 64 Candidate Proteins Biomarker Biomarker Validation Candidate list
+ MRM Targeted approach for measuring multiple proteins simultaneously Features: Dynamic range of >4 orders of magnitude Up to 50 proteins per assay (more) Can be quantitative: moles of protein of interest/g of protein sample Very robust: CV’s of less than 10% NOT as sensitive as ELISA in most cases Identify and measure peptide which is unique to the protein of interest and measure it (mass/charge ratio) and fragments of it generated in the MS
+ Multiplexed quantification 16 Cytochrome P450’s
+ Another protein panel assembly
+ MRM development pipeline Initial SRM method Proteins - 57 Survey run – determine Refined method Peptides - 174 detectability of peptides Proteins - 52 Transitions - 1681 Peptides - 119 15 injections of pooled sample - 8-10 transitions per peptide Transitions - 609 (~ 13 hours instrument time) - 1-5 peptides per protein - 5 transitions per peptide - 1-5 peptides per protein Technical variance measurement Collision energy optimisation 16 injections of pooled sample 10 injections pooled sample (~14 hours of instrument time) (~17 hours instrument time) Mean CV = 5.7 % Final SRM method Proteins - 48 Measurement in 30 individual Peptides - 109 samples Transitions - 545 (~51 hours instrument time) - 5 transitions per peptide - drug treated or vehicle control) - 1-5 peptides per protein
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