PERSONALIZED CANCER CARE

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1 Translational Research Promises to Advance Molecular Medicine for Cancer Patients Molecular Data Diagnosis / Therapy PERSONALIZED CANCER CARE Biospecimen Distribution Biospecimen Collection Biospecimen Processing processing and banking Banking

2 Molecular Research and Analyte Variation Pitfalls for translational research using human specimens: Varying methods of collection, processing, and storage can alter the physical/biologic state of the specimen Varying associated specimen data elements alter what the scientist knows about the character/nature of the specimen Variable clinical information alters what the scientist knows about the patient (biologic context of the specimen) Variable restrictions (patient consent; other ethical, legal, and policy issues) alter what the scientist may do with the specimen and/or data

3 Workflow for Tissue Biospecimens for R&D Acquisition, Handling & Processing Distribution Medical/Surgical Procedures QA/QC Patient Consent Data Compilation And Management Experimental Outcomes

4 Infiltration Clearing Dehydration Post-Fixation Variability in Needle Biopsy Tissue Processing Protocols Harvard/DFCI Institution I FHCRC/U Institution Wash II Institution JHU III Institution Umich IV Institution MDACC V Mayo Institution Clinic VI Institution MSKCC VII Condition Time Temp Time Temp Time Temp Time Temp Time Temp Time Temp Time Temp 10% Formalin ? % Formalin RT Penfix % formalin in 95% etoh RT % ethanol 5 RT RT % ethanol % ethanol 15 RT ? % ethanol 15 RT ? % ethanol 15 RT RT % ethanol 15 RT ? % ethanol 15 RT RT 5? % ethanol 15 RT RT 15? % ethanol RT % etoh/ 17%eosin % etohl/ 50% Xylene Xylene 15 RT RT 5? Xylene 15 RT RT 5? Xylene 20 RT Paraffin 20 RT ? Paraffin 20 RT ? Paraffin Paraffin Total Time (minutes) among 11 prostate cancer SPOREs; Angelo M. De Marzo, Samson Fine, Bruce J. Trock

5 Does it Matter? Effect of Processing SOPs on IHC results p27 staining intensity: with minimum fixation time (p=0.039); with dehydration time (p=0.011) 34βE12 staining intensity: with maximum fixation time (p=0.015) AMACR staining intensity : with minimum fixation time (p=0.001); with dehydration time (p=0.0002) Ki-67 ln(%) cells staining: with infiltration temperature (p=0.035) Angelo M. De Marzo, Samson Fine, Bruce J. Trock

6 Does it Matter? Effect of Processing SOPs on IHC results ER testing in Breast Cancer Patients Intermountain Healthcare Study All ER testing done in one location 27 hospitals where breast cancers could be removed Processes vary by site Question: If all testing done in standard way, would outcomes be related to pre analytic variables at site? Outcome to test: ER negative rate Elizabeth Hammond

7 Frequency of ER negative test results by hospital Hospital Cases ER positive ER negative % ER negative Hosp A % Hosp B % Hosp C % Hosp D % Ref Hosp* % Hosp F % Hosp G % ALL % *Surgical specimens removed and tested in house at the reference laboratory Sample from 27 hospitals processed at 6) Elizabeth Hammond

8 Data Analysis ER negative was significantly higher in some facilities and in most facilities on weekends (Friday/Saturday excisions) 5077 pts over 7 years during which the assay has been stable. Data was controlled for variation due to stage of disease, age of patient and tumor size. Concluded that this increased ER negative rate was likely due to the more variable preanalytic variable handling on weekends and at remote sites. Elizabeth Hammond

9 Workflow for Blood Collection and Plasma Processing Collection Tubes and Order of draw Processing Procedure, Temperature and Time Blood Draw Procedure Distribution & Storage Patient Consent and Preparation Molecular Analysis

10 Plasma collection protocol variations among 5 institutions Procedure Venipuncture (Needle gauge, details of blood collection set) Phlebotomy (tourniquet technique, patient position, tube order, blood source, volume collected) Variations Needle gauge and priming volumes differed Patient position varied from seated to lying down, variable tube orders, variable venipuncture sites Collection device Blood derivative and processing (anticoagulant type, processing time and protocols) Different types of tubes Different anticoagulants, different temperatures, different centrifugation temperatures and speeds Amount of elapsed time between collection and storage Storage (temperature, elapsed time for storage, storage duration, storage material, shipping temperature) Variations between institutions Different elapsed times before storage, different storage temperatures

11 How Might Different Collection and Processing Protocols Result in Different Molecular Profiles? Potential sample heterogeneity due to biological processes: Ex-vivo activation of signaling pathways Degradation of proteins and key enzymes Activation of platelets etc. Not always a question of an ideal protocols But can define caveats for experimental design and analysis IF we: consistently use standardized procedures annotate samples with SOP used and deviations from SOP

12 Urgently Needed RESEARCH to better understand how pre-analytical variables affect the molecular integrity of the biospecimen The NCI Biospecimen Research Network BEST PRACTICES: state-of-the-science guidance for biobanking to harmonize procedures for collection, processing, storage and distribution of biospecimens The NCI Best Practices for Biospecimen Resources INFORMATION about how biospecimens are collected, processed and stored What information? How to get it?

13 The Recorded Life History of a Biospecimen: A Story in Progress Patient Medical/ Surgical Procedures Acquisition Handling/ Processing Storage Distribution Scientific Analysis Restocking Unused Sample Compilation and Analysis of Biospecimen Information Compilation and Analysis of Scientific Data Clinical and Research Outcomes

14 Annotated Variables are Key Pieces to the Puzzle Key Questions: Pre-acquisition: Patient consented? Biospecimen available for distribution? Treatments administered? Surgical variables? Post-acquisition: SOPs for specimen processing? Preservation and processing variables? Storage and shipping conditions? QA/QC measures taken for specimen processing and processing reagents used?

15 Variables of Biospecimen Acquisition Pre-acquisition variables: Antibiotics Other drugs Type of anesthesia Duration of anesthesia Arterial clamp time Blood pressure variations Intra-op blood loss Intra-op blood administration Intra-op fluid administration Pre-existing medical conditions Patient gender Post-acquisition variables: Time at room temperature Temperature of room Type of fixative Time in fixative Rate of freezing Size of aliquots Type of collection container Biomolecule extraction method Storage temperature Storage duration Storage in vacuum

16 Strawman Data Capture Model for Annotation of Tissue Acquisition Variables Event Patient Considerations Consent level Distribution authorization Patient information Patient therapeutic history Medical/Surgical Procedures Type and duration of anesthesia Other drugs administered Time and duration of other events Blood or fluids given Inter-operative ischemia time Acquisition Handling & Processing Preservative type Preservative time Preservative temp Time at RT Rate of freezing Post-operative ischemia time Storage time and condition Type of container used Size of aliquot Distribution Method of transport Temperature of transport Time of transport QA/QC SOPs for specimen processing QA/QC QA/QC of reagents used Recorded History

17 Strawman Data Capture Model for Annotation of Blood Collection and Processing Variables Event Recorded History Patient Preparation Consent level Distribution authorization Patient information Patient therapeutic history Blood Draw Preparation Type and duration of patient position Blood Draw Procedure Additives Volume collected Numbers and types of tubes collected Handling and Processing Temperature and duration before processing Spin conditions Total duration prior to freezing Temperature of storage Freeze-thaw events Distribution Date and time specimen sent Date and time received Condition of specimen upon receipt QA/QC SOPs for specimen processing QA/QC QA/QC of reagents used

18 Collection of Normal Tissues for Research: What s Normal? Genotype-Tissue Expression (GTEx) Project NIH Roadmap Project Identify Expression QTLs in 160 individuals, looking at 30 or more different tissues per individual Logistical challenges Tissues from deceased individuals; follow on from organ donation programs and/or targeted rapid autopsy programs Good RNA quality/low PMI needed It s all about the data! Clinical data: subjective inclusion/exclusion criteria one investigator s normal is another s diseased (e.g. mental health status) Family history Issues of access to EHR Still need the biospecimen collection, processing, and storage data, record of informed consent, etc.

19 Clinical Variability in Human Biospecimens: Information Challenges for R&D and Patient Care Helen M. Moore, Ph.D. October 15, 2009