Patient Biospecimen Preanalytics: Even the Perfect Test Can Give the Wrong Result If the Analyte Is Compromised Carolyn Compton, MD, PhD Professor Life Sciences, Arizona State University Professor Laboratory Medicine and Pathology, Mayo Clinic Adjunct Professor of Pathology, Johns Hopkins CMO, National Biomarker Development Alliance CMO, Complex Adaptive Systems Institute Scottsdale, AZ National Academies of Sciences, Engineering, and Medicine Return of Research Results Workshop Washington, DC September 6, 2017

Bias and Biospecimens Sources of Bias in Molecular Marker Research in Cancer - David F. Ransohoff and Margaret L. Gourlay, 2010

Translational Research Biospecimen Quality Affects Both Molecular Medicine and Translational Research Analysis Data Decision Making DETERMINES QUALITY HERE Clinical Medicine or Translational Research QUALITY HERE Biospecimen Analysis Biospecimen Collection Biospecimen processing and ba Preanalytic Biospecimen Handling and Processing

Pre-analytical Factors Affect Both Molecular Composition and Molecular Quality Specimen is viable and biologically reactive Molecular composition subject to further alteration/degradation Factors (examples): Antibiotics Other drugs Type of anesthesia Duration of anesthesia Arterial clamp time Factors (examples): Time at room temperature Temperature of room Type of fixative Time in fixative Rate of freezing Size of aliquots Time 0 ANALYS I S STORAGE Patient Pre-Op Acquisition Handling/ Processing Storage Distribution Scientific Analysis Medical/ Surgical Procedures Restocking Unused Sample Pre-acquisition Post-acquisition

pMAPK IHC of Colon Cancer : HER2 IHC and FISH in Breast Cancer: Does It Matter? Cold Ischemia Time and Molecular Assay Results pMAPK IHC of Colon Cancer : HER2 IHC and FISH in Breast Cancer: Loss of Biomarker Signal Gain of Biomarker Signal 30 min 10 min HER2/CEP17 = 0.98 a 20 min 4 hr delay 2 hr 60 min c Khoury T, et al., Mod Pathol. 2009 Nov;22(11):1457-67 Hartmut Juhl, Indivumed GmbH, BRN

Blood Collection and Plasma Processing: Biomarkers and Circulating Tumor Cells Procedure, Temperature and Time Blood Draw Procedure Collection Tubes and Order of draw Distribution & Storage Molecular Analysis Patient Consent and Preparation Blood draw: peripheral vein vs. central line Specimen type: plasma vs. serum Tube type Tube fill level Tube inversions Time to processing Centrifuge speed Temperature variation before and during processing Storage conditions and freeze-thaw cycles Transport conditions

Analysis in the Clinical Lab: How Is It Assured? Place where test is done CLIA/CAP laboratory accreditation People doing the test Education Proficiency testing Licensure Platforms used for testing CDRH approved devices Processes followed for testing SOPs Quality management Patient samples to be tested Variably variable: uncontrolled More is known about the quality of beef in the supermarket than is known about the quality of human biospecimens in research

Impact of Biospecimen Quality on Analysis Data Quality Effects on Clinical Care Potential for incorrect diagnosis Potential for incorrect treatment Therapy linked to diagnostic test on a biospecimen Effects on Research Irreproducible results Variation in mutation data Variation in gene expression data Misinterpretation of artifacts as biomarkers Return of bad data to patients?

The National Academies of Sciences, Engineering and Medicine (IOM) 2016 Biomarker Tests for Molecularly Targeted Therapies: Key to Unlocking Precision Medicine  National Academies Press, Jun 30, 2016  One of the 10 stated goals is to specifically address biospecimen quality. Goal 9: Enhance specimen handling and documentation to ensure patient safety and the accuracy of biomarker test results.  “The reliability of biomarker test results depends on the quality of the patient specimens.” “Professional organizations and health care institutions should develop and implement standards for obtaining adequate specimens.”

Powerful Tools: Powerful Risks Analysis technologies are powerful than ever. The technological capacity exists to produce low-quality data from low- quality analytes with unprecedented efficiency. It is now possible to get the wrong answer with unprecedented speed. Even the best technology cannot spin straw into gold – you must begin with gold. If garbage has gone in, results must not go out - to either patients or physicians

Patient Biospecimen Preanalytics: Even the Perfect Test Can Give the Wrong Result If the Analyte Is Compromised Carolyn Compton, MD, PhD Professor Life Sciences, Arizona State University Professor Laboratory Medicine and Pathology, Mayo Clinic Adjunct Professor of Pathology, Johns Hopkins CMO, National Biomarker Development Alliance CMO, Complex Adaptive Systems Institute Scottsdale, AZ National Academies of Sciences, Engineering, and Medicine Return of Research Results Workshop Washington, DC September 6, 2017