© LD True (copyright pending)

Larry True, MDProfessor of Pathology

Co-Director of UW-FHCRC Prostate Cancer Specimen BankUniversity of Washington

Seattle, WA

Assessing the Quality of Tissue

NCI Biospecimen Best Practices Forums Seattle, WA, January 28, 2008

I. Standards for assessing preservation of macromolecules (RNA, protein) in tissue

II. A specification for tissue localization experiments (MISFISHIE)

Patient Handling/Processing Storage

Medical/Surgical

Procedures

Lifecycle of a BiospecimenFactors potentially having significant effect on gene expression

Post-acquisitionPre-acquisition

PatientAge Diet

SurgeryIschemiaAnesthetic

Time 0

Time 0

Controllable variablesUncontrollable variables

Tissue Acquisition

Prostate biopsies

Diagnosis: Cancer

Design of experiments to evaluate effect of variables on gene expression

External factorsTemperatureTime of day

PatientAge Diet

SurgeryTissue ischemia

Anesthetic

Tissue handlingTime on bench

StorageLaser capture

Prostate biopsies

Gene expression

profile

Prostate biopsies

Gene expression

profile

Expression profilingLab of Peter Nelson, MD

Comparative gene expression analysis

Time 0

Patient Acquisition Handling/Processing

StorageMedical/Surgical

Procedures

Post-acquisitionPre-acquisition

Effect of 6 weeks of high-caloric diet

Lin, D. et al. Cancer Epidemiol Biomarkers Prev 2007;16:2150Time 0

PatientAge Diet

Uncontrollable variables

Time 0

Patient Acquisition Handling/Processing

StorageMedical/Surgical

Procedures

Post-acquisitionPre-acquisition

SurgeryIschemiaAnesthetic

Effect of ischemia during surgery on tissue

0.00

6.00

12.00

18.00

DUSP1-pre DUSP1-post KLF6-pre KLF6-post AMACR-pre AMACR-post

Thre

shold

Cycle Q1

MIN

MEDIAN

MAX

Q3

p=0.001

p=0.05

p=0.94

AMACRKLF-6DUSP-1

pre post pre post pre post

Lin, Coleman, Hawley..JCO ’06; 10;24(23):3763-70

Time 0

Time 0

Uncontrollable variables

Time 0

Patient Acquisition Handling/Processing

StorageMedical/Surgical

Procedures

Lifecycle of a Biospecimen

Post-acquisitionPre-acquisition

PatientDietMMP7IGF2R

SurgeryIschemia

DUSP1KLF6

Record the data & develop a molecular “fingerprint”

Optimize handling & processing procedures

Controllable variablesUncontrollable variables

Time 0

Patient Acquisition Handling/Processing

StorageMedical/Surgical

Procedures

Lifecycle of a Biospecimen

Post-acquisitionPre-acquisition

Factors affecting immunohistochemical stains

antigenDAB + H2O2

tissue ABC complex

1o AbBiotin-2o Ab

Tissue microarray

Antigen retrieval

Tissue handling, storage

Primary antibody

Specificity, affinity,

saturation of binding sites Secondary

antibodySpecificity,

affinity, saturation of binding sites

ChromogenIncubation

time, temp., [H2O2), [DAB]

Immuno-stains/FISH

images

Controllable variables

Avidin-biotin

complexAffinity,

saturation biotin sites

True LD, Am J Clin Path 1988;90:324

MISFISHIE – report all data in your experiments

• Minimum Information Specification For In SituHybridization and Immunohistochemistry Experiments (MISFISHIE)

• The purpose is to "Insure that the minimum information that a researcher at a different lab needs to reproduce or evaluate the experiment is provided."

• MISFISHIE does not specify the data format, merely whatinformation must be communicated

• Based on principles of MIAME (specification for reporting expression microarray data)

Deutsch E, et. al. OMICS. 2006;10(2):205-8 (PMID: 16901227 ); Nature Biotechnology (in press)

Major Sections:1. Experiment Design2. Specimens, Treatments3. Probes or Antibodies4. Staining

Protocols/Parameters5. Imaging Data and

Parameters6. Image Characterizations

University of Washington

School of Medicine

32 articles from:

Am J Clin PathAm J Surg PathCancer ResJ Clin PathLab InvestModern PathMolecules & CellsNatureScience

Section 3. Reporters (Antibodies, Probes)

Information not supplied (25% of articles):Antibody clone numbers and/or catalogue numbersConditions of antigen retrieval (buffer, heat duration)

Other important attributes (e.g., mono- or polyclonal, organism in which antibody generated)

Protocol for obtaining exact reporter (purchase from..., create, etc.)Full sequence or clone id of the reportersUnambiguous reporter identification, ideally genomic

Section 4. Staining

Information not supplied (25% of articles):Was nonspecific protein binding blocked?Was endogenous peroxidase inhibited?Negative controls?

Details about positive and negative controlsStaining protocol (enough to reproduce?)Detection Method (number of reporters, detection reagent & systems)

http://scgap.systemsbiology.net/resources/protocols.php

Section 5. Imaging Data

Information not supplied (85+ % of articles):Images of all specimens (for reader to evaluate for her/himself)(for example) Was “nonspecific staining” expression of an antigen by cells not expected to stain?

The digital images for each assay (can the images be downloaded to your computer and evaluated ?)

Conclusions

• To determine if a molecular profile of experimental tissue is abnormal:– For uncontrollable variables (diet, anesthesia, patient age,

duration of surgery, tissue ischemia) we need to develop a molecular fingerprint and subtract that fingerprint profile from the experimental gene expression profile

– For controllable variables (rapidity of processing, freezing, storage), we need to optimize handling of specimens to minimize the effect of steps that most affect gene expression profiles

• To achieve these goals, we need to– Obtain all relevant information from all experimental data

sets (MISFISHIE specification)

Eric Deutsch, PhD: Bioinformatics

Laura Pascal, PhD:Post-doc, Stem cell biology

NIDDK U01 Stem Cell Consortium (Prostate & Bladder)

Alvin Liu, PhD: PI, Molecular Biologist

Questions ??

University of Washington

School of Medicine

Is there a set of “housekeeping” genes/proteins that can be used as a metric for preservation?

Housekeeping genes

02000400060008000

100001200014000160001800020000

LNCaP C4-2 DU145 PC3 CL1 CL1.1 CL1.31

Cell lines

mRN

A le

vels

GAPDHa-tubulinb-actin

Range of mRNA:β-actin: 5,500 – 19,000α tubulin: 4,000 – 15,500GAPDH: 8,000 – 18,000

There is no single “housekeeping gene” and probably not a generic set of genes that can be used as a general metric for RNA preservation.