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Digital Pathology in Clinical Trials

Arnold B. Gelb, M.D.Senior Staff Pathologist

Quest Diagnostics Clinical Trials

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Potential Conflict of Interest Statement

Employee of Quest Diagnostics Owner of stock in Quest DiagnosticsNo financial interest in any imaging company

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Clinical Trials

Image Analysis

Image Analysis

Digital Archiving

Digital Archiving

AdjudicationAdjudication

Introduction Introduction

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Quest Diagnostics Clinical Trials: Overview

One of the world's largest Central Laboratories, working to accelerate clinical drug development

Scientific Expertise– > 900 in-house scientists and pathologists covering all therapeutic areas– Extensive test menu and consultative capabilities– Full service Biomarkers and Anatomical Pathology capabilities

Laboratory Management– GCP/ICH operating philosophy– Extensive resources, knowledge and experience in handling and

managing clinical specimens, logistics, and data in regulatory compliant environment

Global Reach– Quest owned central lab facilities in North America, Europe, and India– Affiliate laboratories in China, Singapore, and Australia– >4,000 cities in 60 countries– >99% assays performed in-house.

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Spectrumweb application

RAID

Internet

Site B

ScanScopeXT’s

DSRanalysis

algorithms

slidescanners

slidestorage

RAID

Site A

ScanScopeXT’s

DSRanalysis

algorithms

slidestorage

slidescanners

PCPC Viewing Stationsrunning ImageScope

(unlimited)

Spectrumweb application

DatacenterRAID central

database

centralservers

network connections

digital slidesfirewallsinternal networks

Redundant Fail-Safe

Server

RedundantFail-Safe Server

Digital Pathology Systems and Networks

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InScape™

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Layout

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Case Details Page

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The Digital Slide Image

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•There is a toolbar at the top.

• Slide information such as the slide barcode or the slide ID can be seen in the title bar.

•Zoom tool is present

• The thumbnail and label can be turned off in the View toolbar.

Digital Slide Viewing Software

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Image Analysis Window

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Clinical Trials

Image Analysis

Image Analysis

Digital Archiving

Digital Archiving

AdjudicationAdjudication

Introduction Introduction

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IHC Biomarkers

Over 50 assays developed and validated, including esoteric and novel markers, e.g.– Apoptosis– Cell cycle control– mTOR pathway– erbB receptor inhibition– IGF pathway– Angiogenesis– CD antigens

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Quantitative Analysis of IHC Biomarkers

Identify and quantify morphology and intensity Subcellular localization – algorithm selection

– Nuclear, membranous or cytoplasmic Tissue (tumor) specific classification

– Manual selection of representative tumor regions by pathologist

– Automatic feature extraction (pattern recognition)

Enables analysis of invasive tumor from entire image

(LANL)

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Image Analysis Algorithms

May need to adjust intensity of staining – Scanning typically based on % transmittance

Customize or “tune” algorithm to a laboratory’s unique staining process– Controls– Future: batch to batch “tuning”

Validation– Sufficient number of (de-identified) slides across

percentage range and intensity scores– 3 blind evaluation arms

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“Tuning” Nuclear Segmentation

Original Image Normal Segmentation

Over Segmented Under Segmented

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Control Slide

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Validation

Image Analysis

Digital Slide Scoring

Manual Slide Scoring

Validate Algorithm Performance

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pHH3

Histone H3 has a key role in the folding and inter-association of the chromatin fiber.

Phosphorylation of Histone H3, at serine-10 and -28, coincides with the induction of mitotic chromosome condensation.

H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation.

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pHH3

Digital assessment of the nuclear stain:

– Index = 13.7%

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RXRA

Members of the RXR family, including RXRα, β and γ, are activated by 9-cis retinoic acid, that is expressed in both liver and kidney.

RXRα is an orphan nuclear receptor encoded on chromosome 9q34.2.

It plays roles in a variety of processes including embryonic patterning and organogenesis, cell proliferation and differentiation.

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RXRA

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pFOX03a

The forkhead family of transcription factors mediate signaling via a pathway involving IGF-1R, PI3K and Akt.

The gene is located on chromosome 6q21. Spliced transcript variants encoding the same protein have

been observed. In the presence of survival factors, Akt phosphorylates

FOXO3a, leading to its retention in the cytoplasm. Survival factor withdrawal leads to its dephosphorylation,

nuclear translocation, and target gene activation. Within the nucleus, it triggers apoptosis most likely by

inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

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pFOX03a

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pLIMK

LIMK1 and LIMK2 are serine/threonine kinases that are involved in actin cytoskeletal reorganization, cell growth regulation and tumor suppression via cofilin phosphorylation downstream of distinct Rho-dependent protein kinase signaling pathways.

Both are activated by various extracellular stimuli, including stromal cell-derived factor-1a and insulin, through activation of small GTPases and their downstream kinases, ROCK and PAK1.

Both LIMK 1 and 2 have been related to tumorigenesis and, thus, are studied in conjunction with anti-cancer therapies that target this pathway.

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pLIMK

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Integrins

Heterodimers composed of noncovalently associated transmembrane α and β subunits.

16 subunits yield over 20 receptors.Roles in cell adhesion, cytoskeletal

organization, and signaling.Involved in cell growth, differentiation,

migration and apoptosis.

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An Integrin

Digital assessment of membrane staining component:

– 3+ cells = 54.7%– 2+ cells = 23.4%– 1+ cells = 21.9%

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Clinical Trials

Image Analysis

Image Analysis

Digital Archiving

Digital Archiving

AdjudicationAdjudication

Introduction Introduction

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Adjudication

Advantages of digital pathology for global clinical trials– Avoids time delays and costs of shipping slides

Read slides from different locations

– Education of satellite groups in trial host countries– Ensure high level of agreement/ consistency– Conferencing between central lab pathologist(s) and

sponsor’s study pathologist(s) or experts Fairer resolution than a simple majority or expert rule

Digital substitute for glass slides– When a clinical trial host country has export limitations – Primary diagnosis H&E (breast cancer) in progress

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UNITED STATES

ARGENTINA

BRAZIL

ENGLAND

AUSTRALIA

BELGIUM

INDIA

CHINA

SINGAPORE

Clinical Trials Laboratories & Offices Affiliate Laboratories

Adjudication: Global Clinical Trials Laboratories

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Adjudication - Validation

FDA– Validate as much as one can ahead of time– Awaiting completion of FDA approval process

Validation– Ideally study and tissue specific validation– Compare performance of digital to glass slides

Can compute intra- and inter-observer kappa coefficients

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Clinical Trials

Image Analysis

Image Analysis

Digital Archiving

Digital Archiving

AdjudicationAdjudication

Introduction Introduction

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Digital Archiving

Static images → whole slides– Third party multi-format image support– Third party information systems

Scan Types– Bright field: dry, oil– Fluorescence

Advantages– Circumvents problems of slide breakage or stain fading– Real-time access possible

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Digital Archiving

Length of storage– Duration of study

On server– Long-term

Meet minimum retention requirements FDA – 5 years post application (21 CFR §58.195)

• But when is it submitted by sponsor? CAP – 10 years for glass slides Default – 15 years

Removable media/ hard drives Costs of storage tend to decrease over time

Other– Use in submission of data to FDA

Digital sides more compelling evidence than static images e.g., hepatitis C

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Key Points

Image Analysis– Customize staining and algorithms– Validation– Software advances → entire-slide analysis

Adjudication– Decrease TAT– Harmonization– Conferencing

Digital Archiving– Real-time access– Long term storage– Regulatory submission

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Acknowledgements

Stephanie AstrowMarc EdwardsVicki FreemanRon LuffMaribeth Raines

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Thank you very much for your time and attention

Arnold B. Gelb, M.D.arnold.b.gelb@questdiagnostics.com

(661) 799-6079