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A Chemo-Sensitivity Assay: Personalized Cancer Therapy and Drug Discovery
A Chemo-Sensitivity Assay   Personalized Cancer Therapy and Drug Discovery
Background – Current State of Technology •HTCA (Human Tumor Clonogenic Assay) - 1st assay used to predict chemo effectiveness – fell out of favor due to low efficiency, 20 day turn around, cells were grown for up to 2 weeks •MTT (Dimethylthiazole 5-Diphenyl Tetrazolium Bromide) - Yellow MTT is reduced to purple formazan in mito. Colored precipitate dissolved in DMSO and measured, determine living cells, false positives. •ATP-TCA - ATP levels are measured via Luciferase assay. Depletion of ATP is thought to correlate to loss of cell viability •DiSC (Differential Staining Cytotoxicity Assay) preferential membrane stain for dead/dying cells •SRB (Sulforhodamine B) - binds to cellular protein as an indirect marker for cell viability •Fluoroscein Diacetate Assay - complicated mechanism for turn over of FDA •All are indirect measures of cell viability •All use total fluorescent measurements •NONE offer ability to distinguish between effected and unaffected populations •NONE combine multiple and corroborating novel cellular markers directly associated with DNA damage
Background     Current State of Technology    HTCA  Human Tumor Clonogenic Assay  - 1st assay used to predict chemo effect...
Background & Market § Precision Therapeutics, Pittsburgh PA, www.precisiontherapeutics.com § ChemoFx® - plate based assay, measures loss of adherent cells following drug treatment § Remission Labs, Inc. New York, NY, http://remissionlabs.com § Variety of diagnostic testing, chemoresistance and chemosensativity assays § OncoTech, Tustin CA, www.oncotech.com § EDR (Extreme Drug Resistance) and DiSC § Xenometrix – Allschwill, Switzerland, www.xenometrix.ch/ § Various Cytotoxicity assay § Patient driven market, however MUCH PSUEDO SCIENCE, LOW DEGREE OF QUANTIFICATION. MOST ARE +/§ Most do not have, nor need, FDA as they provide reference diagnostics
Background   Market    Precision Therapeutics, Pittsburgh PA, www.precisiontherapeutics.com    ChemoFx   - plate based ass...
Scenarios For Chemo-Sensitivity Assay Biopsy MD Pharma & Academia Sample Clinical Laboratory Black Box •Hardware Sample •Hardware + •Software •Reference •Ease of Use Black Box •KIT •Reagents •Protocols Sample •Software •Blue Image •Reference •Analytical Feedback + •KIT •Reagents •Protocols
Scenarios For Chemo-Sensitivity Assay Biopsy  MD  Pharma   Academia  Sample Clinical Laboratory Black Box     Hardware Sam...
Optimized Cell Based Sample Prep #1 - Cell Plating àe.g. Biopsy (clinical) immortalized (drug discovery) Day 0 #2 – Treatment Series à e.g. #3 – Fixation à e.g. pre-determined time point of interest #4 – Fluorescent Staining à p-ATM, ɤH2AX & PI Pulse, static, titrations, drugs/IR and combinatorial approaches Day 1 #5 – Scan à High Throughput and Custom Analysis Day 2 Methanol
Optimized Cell Based Sample Prep  1 - Cell Plating   e.g. Biopsy  clinical  immortalized  drug discovery   Day 0   2     T...
Distillation of Data LEVEL 0 LEVEL 1 LEVEL 2 LEVEL 3 LEVEL 4 LEVEL 5 Raw Data Collection Individual Cell Profiling Cellular Data Integration Extract Time Dependence Drug & Dose β* Personalized dose Academic Research, Pharma and Drug Discovery MD
Distillation of Data LEVEL 0 LEVEL 1 LEVEL 2 LEVEL 3 LEVEL 4 LEVEL 5  Raw Data Collection Individual Cell Profiling Cellul...
Automated Data Process (cont’) LEVEL ZERO LEVEL ONE Raw Data Individual Cell Profiling • p-ATM signal SA(c,t,d,dd) •ɤ-H2AX signal SH(c,t,d,dd) •# Cells N(t,d,dd) A =p-ATM, H =ɤH2AX, S =Signal, c =Cells, t =Time, d=Drug, dd=Drug Dose, N=Population, +=Positive, < > = Average
Automated Data Process  cont     LEVEL ZERO  LEVEL ONE  Raw Data  Individual Cell Profiling      p-ATM signal SA c,t,d,dd ...
Distillation Process Level Two Cellular Data Integration Single wellà at every time point, drug and drug dose Threshold # Of Cells Average signal all cells Average signal positive cells Number positive cells (t,d,dd) NH+(t,d,dd) NH+(t,d,dd)/all cells Percent affected ɤH2AX or p-ATM Signal of Individual Cells A =p-ATM, H =ɤH2AX, S =Signal, c =Cells, t =Time, d=Drug, dd=Drug Dose, N=Population, +=Positive, < > = Average
Distillation Process Level Two Cellular Data Integration  Single well   at every time point, drug and drug dose  Threshold...
Distillation Process Level Three Extraction of Time Dependence Cell Numbers ɤH2AX + (t) NH+d,dd(t) Nd,dd(t) Time A =p-ATM, H =ɤH2AX, S =Signal, c =Cells, t =Time, d=Drug, dd=Drug Dose, N=Population, +=Positive, < > = Average Cell Number Signal (a.u.)
Distillation Process Level Three  Extraction of Time Dependence  Cell Numbers   H2AX     Sad,dd  t  NH d,dd t  Nd,dd t   T...
Distillation Process Level Four Extraction of Dose & Drug Dependence Cell Number dd2 dd3 Cell Number dd1 ddoptimal dd4,5,6 Nd1(t,dd) toptimal Time Ntoptimal,d1(dd) Drug Dose A =p-ATM, H =ɤH2AX, S =Signal, c =Cells, t =Time, d=Drug, dd=Drug Dose, N=Population, +=Positive, < > = Average
Distillation Process Level Four  Extraction of Dose   Drug Dependence  Cell Number  dd2  dd3  Cell Number  dd1  ddoptimal ...
Distillate Level Five – Personalized Dosing β* = ddoptimal x correction Factor (e.g. BMI) A =p-ATM, H =ɤH2AX, S =Signal, c =Cells, t =Time, d=Drug, dd=Drug Dose, N=Population, +=Positive, < > = Average
Distillate Level Five     Personalized Dosing        ddoptimal x correction Factor  e.g. BMI   A  p-ATM, H    H2AX, S  Sig...
Distillation of Data LEVEL 0 LEVEL 1 LEVEL 2 LEVEL 3 LEVEL 4 LEVEL 5 Raw Data Collection Individual Cell Profiling Cellular Data Integration Extract Time Dependence Drug & Dose β* Personalized dose Academic Research, Pharma and Drug Discovery MD
Distillation of Data LEVEL 0 LEVEL 1 LEVEL 2 LEVEL 3 LEVEL 4 LEVEL 5  Raw Data Collection Individual Cell Profiling Cellul...
Level 3 Distillation of Taxol Treated MCF7 Cells gH2AX positive cells fraction positive cells 1.2 •Maximum appears to be same for all titrations 1 0.8 •Lowest recorded level 5uM, next is 10nM? 0.6 0.4 10nM 100nM 1uM 5uM 0.2 NH+d,dd(t) 0 0 5 10 15 time (hours) 20 25 30
Level 3 Distillation of Taxol Treated MCF7 Cells gH2AX positive cells  fraction positive cells  1.2     Maximum appears to...
Level 3 Distillation of Taxol Treated MCF7 Cells • 5uM signal reaches low at ~4h, no change •All others similar • Coincides with decreased cell count (t) •All dd show varying levels of increase at 12h, function
Level 3 Distillation of Taxol Treated MCF7 Cells     5uM signal reaches low at  4h, no change    All others similar     Co...
Level 4 Distillation of Taxol Treated MCF7 Cells Nd1(t,dd) •All dd kill • Titration dependent Kill level toptimal •Titration should be optimized, between 1 and 5uM
Level 4 Distillation of Taxol Treated MCF7 Cells Nd1 t,dd      All dd kill     Titration dependent Kill level  toptimal   ...
Level 3 Distillation of Etoposide Treated HeLa Cells •Good separation •No Decrease in Etoposide: 0.08uM Etoposide: 0.4uM Etoposide: 2uM pATM Mean Intensity •No Decrease 20000 15000 Etoposide 0.8 uM Etoposide 0.4 uM Etoposide 2uM H2AX Mean Intensity 8000 10000 Mean Intensity - Background Mean Intensity - background •No Kill 5000 6000 4000 2000 0 0 0.1 1 10 Time of Treatment (hr) 100 0.1 1 10 Time of Treatment (hours) 100
Level 3 Distillation of Etoposide Treated HeLa Cells    Good separation  SA     No Decrease in  SA   Etoposide  0.08uM Eto...
Summary MCF7 & HeLa § MCF7 § No information prior to Taxol treatment § Dose, treatment, timing unknown § loss viability § 6 hours § , NH+ saturated, little separation § RESULT – decrease dose § HeLa § No information prior to Etoposide treatment § Same as for Taxol § titration dependent increases § No decrease § similar levels damage, exception 2uM persistent signal with no decrease § RESULT – Increase dose NO KILL Locate , NH+optimal (Below Saturation) at Effective Kill Nd1(t,dd)
Summary MCF7   HeLa    MCF7    No information prior to Taxol treatment    Dose, treatment, timing unknown    loss viabilit...
Drug Discovery & Clinical Application § Rapid assessment of unknown drug efficacy § Within 2 days (48h) à Drug Discovery § Reference library of all clinically used drugs § 6-8 weeks à Drug discovery and Clinical § Direct drug comparisons and Synergy § D1 saturates with low kill, D2 rapid kill with saturation § Synergy, increase efficacy § 2nd phase of Reference library, additional 90 days § à Drug Discovery and Clinical § Create a reference library using cells lines, xenographed mice and Human biopsy § Compare data to clinical journals, are we consistent? Do we provide new information? Is our data more informative, yes. § 12 months – ongoing as we develop a data base of tested primary biopsy, xenographs and if necessary additional immortalized cell lines àDrug Discovery and clinical NO FDA § Screen sensitivity in an individual, normal vs. cancerous biopsy § service under as a new organization, providing reference data for clinicians , public à Clinical
Drug Discovery   Clinical Application    Rapid assessment of unknown drug efficacy    Within 2 days  48h     Drug Discover...