HORIZON DISCOVERY
Standardizing EML4-ALK and BRAF V600E Immunohistochemistry
testing using defined Reference Standards
Farah Patell-Socha, Vicky Spivey, Jonathan Frampton, Regan Fulton, John Garrett, Blake Gilks, Keith Miller, Paul Morrill, Karin Schmitt
Introduction
There is a growing concern regarding relatively poor intra- and inter-laboratory reproducibility of Immunohistochemistry (IHC) on Formalin-Fixed Paraffin Embedded (FFPE) tissue sections, particularly the subjective
interpretation and scoring of positive immunostaining. The availability of genetically defined reference materials offers an industry standard for development and quality control of IHC assays, directly improving their accuracy
and reproducibility. Drawing upon the proprietary genome editing platform by Horizon Discovery, we reconstitutes clinically relevant cancer genes in human cell lines. We have developed precisely defined IHC HDx™ Reference
Standards for EML4-ALK and BRAF V600E that have been validated externally.
Development of EML4-ALK & BRAF V600E IHC Reference Standard
Slides
Horizon’s precision gene engineering platform allows the building of cell lines carrying rare genetic
abnormalities. Using our technology we have engineered both SNP and translocation lines. A BRAF V600E
cell line was created by using precision gene engineering technology. Additionally, in a novel application of
the gene engineering technology, we report for the first time the creation of an ALK-EML4 translocation
cell line. Both cell lines were extensively characterized using IHC, FISH, sequencing, gene expression
analysis and digital PCR. IHC HDx Reference Standard slides were then generated (Figure 1). Wild Type and
engineered cell lines were pelleted and processed into a homogenous 3 core FFPE cell block.
BRAF V600E external validation study
Three engineered and characterized BRAF V600E cell lines were pelleted and processed into TMAs for
BRAF V600E testing by IHC in partnership with the Canadian Immunohistochemistry Quality Control
(cIQc). The purpose of the challenge was to ensure optimized IHC protocols in laboratories and to
establish “best practice” in BRAF V600E IHC. The cIQc challenge consisted of a colorectal carcinoma tissue
microarray and a microarray encapsulating Horizon cell lines. Slides were sent to 14 laboratories, 10
laboratories participated and returned their slides for evaluation. Laboratories performed BRAF V600E
immunohistochemical staining according to usual laboratory protocol. Participating laboratories were
asked to stain a colorectal carcinoma tissue microarray enriched for MLH1-deficient cases that have been
subjected to BRAF V600E mutational analysis. Available slides from all other participating labs were
blindly reviewed by cIQc assessors.
BRAF V600E external validation study
• Horizon cell line standards results were in concordance with on-slide tissue controls.
• A range of protein expression levels were detected in Horizon cell line standards – negative (core 1),
intermediate (cores 2 &3) and strong (core 4).
• Lab A had clearly superior staining compared to all other labs, in that the positive cores were more
intensely positive, with no significant increase in background staining.
• Lab B showed suboptimal staining. There were three false positives, as a result of high background
staining, with weak cytoplasmic immunoreactivity seen in mutation negative cases. True positive tissue
cores showed relatively weak staining (core 16, low signal to background ratio).
Lab A results: Optimal
BRAF V600E staining
Lab B results: Suboptimal
BRAF V600E staining
Core 1. BRAF
V600E Negative
Cell line
Core 2. BRAF V600E
Intermediate Cell line
Cell line growth & FFPE Block production
•
SNP 6.0
•
Cell growth
•
Sanger Sequencing
•
Stringent biobanking
•
Digital PCR
•
Fixing and pelleting
•
RT-PCR
•
FFPE block production
•
Western blot
•
H&E, IHC & FISH QC
IHC HDx™
Reference
Standard
Cell Line Validation
A
B
C
‘Your’Tissue
sample
Core 3. BRAF V600E
Intermediate Cell line
Fully validated Genetic Defined FFPE
Reference slides with controlled
protein expression levels
Figure 1. Cell engineering and IHC HDx Reference Slide production. EML4-ALK translocation and BRAF V600E mutation were
introduced into the wild type cell line using gene engineering technology. Cell lines undergo extensive cell line validation and
quality control. Fully validated IHC HDx Reference Standard slides are then produced.
Core 4. BRAF V600E
Strong Cell line
EML4-ALK IHC external validation study
Two engineered EML4-ALK and Wild-Type (WT) IHC cell lines were pelleted and processed into
homogenous FFPE cell blocks for ALK testing. The development and testing of the cell lines were done in
partnership with United Kingdom National External Quality Assessment Service (UK-NEQAS). EML4-ALK
IHC HDx Reference Standards were sent to 15 laboratories for external evaluation. Laboratories
performed ALK immunohistochemical staining and Fluorescence In-Situ Hybridization (FISH) according to
usual laboratory protocol.
Core 15. BRAF
V600E ( TMA –ve
by ddPCR)
Results
• Horizon’s cell line standards results were in concordance with on-slide tissue controls.
• Positive and negative ALK protein expression were detected in the cell line standards from Horizon
Ventana ALK detection kit: Signal in Core B and C was stronger (scored 3+; 99% of cells positive for ALK)
• DAKO staining platform: Signal in Core B and C was intermediate to strong (scored 2-3+; 99% of cells
positive for ALK). This was also observed when using the Leica Bond staining platform (data not shown).
• FISH results were in concordance with what was expected. Core A was negative for the translocation (2
fusion signals) and cores B and C were both positive for the translocation (1 fusion, 1 red + 1 green
signal).
Ventana
Benchmark XT
DAKO
Autostainer Link
ALK breakapart
FISH
Core 16. BRAF V600E
( TMA +ve by ddPCR)
Figure 3. Reported IHC staining pattern in BRAF V600E cell lines and control tissue
Conclusion
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•
•
•
•
EML4-ALK Variant 1 with protein expression levels (A: Negative, B: Positive and C: Positive) on one slide
BRAF V600E IHC HDx Reference standards with a range of protein expression levels
FISH compatible: Negative and positive reference standards on the same slide
Provides a consistent and renewable source of precisely defined IHC HDx Reference Standards
Fully characterized using IHC, FISH, sequencing, gene expression analysis using digital PCR and western blot
Core A
Applications
•
•
•
•
•
•
•
•
E
Core B
Core C
ALK +ve
Lung tissue
control
Figure 2. Reported IHC and FISH staining pattern
in EML4-ALK IHC HDx Reference Standards
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e [email protected]
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Standardization and verification of IHC workflows
Validating new antibodies for IHC and probes for FISH assays
Validation of new SOPs and staining kits to ensure test accuracy, consistency and reproducibility
Compare assay performance between platforms, assays and users from a common reference point
Potentially use as a same slide/on slide control to test your sample and provide confidence to your IHC assay
Routinely calibrate and monitor the sensitivity of your assays
Providing materials for staff training and proficiency schemes
Common reference point : Clinical Trials and Ring Studies