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Next generation image analysis
for immunohistochemistry
quantitation
Ben Vainer
Department of Pathology, Rigshospitalet
University of Copenhagen Medical Center
”
Men are only so good as their technical
developments allows them to be.
George Orwell
Visiopharm’s User Group Meeting 2015
Rigshospitalet, University of Copenhagen
Inter-laboratory variation
Eight North American and European
laboratories.
“At a hypothetical 13.5% cutoff, there are
32.3% cases that Laboratory D would call high
Ki67 but Laboratory B would call low Ki67.”
”Our data suggest that even if a common
Ki67 cutoff is agreed upon, lack of
interlaboratory reproducibility in Ki67
measurements represents a major obstacle
to confident use of Ki67 for clinical
decisions.”
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Intralaboratory variation
Same 50 breast cancer cases (two 1 mm-cores from each) assessed
on three consequtive days.
Numerical differences.
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Three pathologists, using their
own scoring method
Interval < 2 months
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Manual counting
Image analysis
Eyeballing
Laboratory variation - Ki67 in breast cancer
2nd NordiQC Conference on Applied Immunohistochemistry
Andre metoder
Hot spots
Vyberg & Røge, NordiQC
Overall estimate
Hvor tælles?
Rigshospitalet, Københavns Universitet
Explanations…?
Counting method
Experience
Vyberg & Røge, NordiQC
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
How can we improve the assessment?
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
A: Same pathologist, 2 weeks interval
B: Digital assessment, 2 weeks interval
Visiopharms software
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Neuroendocrine tumours in the
gastrointestinal tract
Cutoff-values in neuroendocrine tumours
• G1: <2 mitoses per HPF and Ki67 2%
• G2: 2-20 mitoses per HPF and/or Ki67 2.1-20%
• G3: >20 mitoses per HPF and/or Ki67>20%
• Mitosis count in 50 HPF (2 mm2)
• Ki67 proliferation index in hot spots (500-2000 cells)
ENETS Consensus Guidelines. Neuroendocrinology 2012; 95.
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Digital photos
ImmunoRatio software
Original proliferation indices (PI): From pathology reports
Re-assessment PI: One ”qualified” pathologist
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
22 cases from Memorial Sloan-Kettering Cancer Center
Eyeball-estimate (EE)
Manual counting (MC) of 2000 cells (!)
Digital image analysis (Aperio’s QIA-algorithm)
Manual selection of hot-spots
Manuel exclusion of stained non-tumour cells
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
HER2-CONNECT in breast cancer
Breast Cancer Res Treat 2012; 132: 41-9.
99.5 % konkordans med FISH
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
The Rigshospitalet study
Holten-Rossing H, Talman MLM, Kristensson M, Vainer B: Optimizing HER2 assessment in breast cancer application of automated image analysis. Breast Cancer Research and Treatment 2015
• 462 breast cancer samples
• TMA with automated
dearraying
• Zeiss Axio Scan Z.1
• Visiopharm’s cloud-based
software
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Results
HER2 IHC assessment
Classification
Image Analysis
0/1+
2+
3+
Total
Manual
reading
673
68
0
41
0
18
673
127
0
741
0
41
104
122
104
904
90.5%
0.741
0/1+
2+
3+
Total
Agreement
Cohen´s kappa
HER2 FISH assessment
Classification
Image Analysis
Negative
Equivocal
Amplification
Total
Manual
Reading
Negative
Equivocal
Amplification
738
0
3
40
0
1
14
0
108
792
0
112
Total
741
41
122
904
93.6%
0.758
Agreement
Cohen´s kappa
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Results
HER2 IHC assessment
Classificatio
n
Manual
0/1+
reading
2+
3+
Total
Agreement
Cohen´s kappa
Image Analysis
0/1+
2+
3+
Total
673
68
0
741
0
18
104
122
673
127
104
904
90.5%
0.741
Amplification
Total
0
41
0
41
HER2 FISH assessment
Image Analysis
Classification Negative
Equivocal
Manual
Reading
Negative
Inconclusive
Amplification
738
0
3
40
0
1
14
0
108
792
0
112
Total
741
41
122
904
93.6%
0.758
Agreement
Cohen´s kappa
67% reduced need for reflex testing (FISH)
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Results
HER2 IHC assessment
Classificatio
n
Manual
0/1+
reading
2+
3+
Total
Agreement
Cohen´s kappa
Image Analysis
0/1+
2+
3+
Total
673
68
0
741
0
18
104
122
673
127
104
904
90.5%
0.741
Amplification
Total
0
41
0
41
HER2 FISH assessment
Image Analysis
Classification Negative
Equivocal
Manual
Reading
Negative
Equivocal
Amplification
738
0
3
40
0
1
14
0
108
792
0
112
Total
741
41
122
904
93.6%
0.758
Agreement
Cohen´s kappa
67% reduceret behov for reflex-testing (FISH)
When adjusting the build-in cutoff to optimal values:
Equivocal: 1.4%
Reduced need for FISH: 90%
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Other applications
• Ki67 proliferation index in other tumour types
• PHH3 as a substitute for mitoses count
• Other diagnostic, prognostic or predictive markers
• Unambiguous staining / cell marking
• ”Translation” to clinically relevant information
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Malignant melanoma
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
HPV-positive orophangyal cancer
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Using automated image analysis to prognosticate
HPV+ oropharyngeal carcinomas
Virtual double staining with serial sections of CK5 and marker of
interest (Visiopharm)
• Ki67
• PHH3
• PD-L1 (T-cell modulator)
Hani Channir, Rigshospitalet, UCPH
Ki67
CK5
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Challenges
Visiopharm’s User Group Meeting 2015
Rigshospitalet, University of Copenhagen
Selection of invasive areas in breast cancer –
automated elimination of DCIS
Torben Steiniche, Aarhus
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Virtual double staining
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Alignment and analysis
Ki67
CK
Analysis: Identification
of positive/negative
cells. Exclusion of CKnegative cells.
Michael Grunkin, Visiopharm
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Example: Ki67 in breast cancer
Ki67
Visiopharm (Novartis Cambridge)
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Automated detection of hot spots
(Visiopharm)
Heatmaps
8/20 = 40%
Positive
cells
4/4 = 100%
Visually more dense…!
Negative
cells
Thomas Ebstrup, Visiopharm
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Digital hot spot-detektion – example
(Visiopharm)
Ki67
Negative heatmap
CK
Positive
heatmap
Index-heatmap
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
Next generation immunoassessment - Why?
• Minimal inter- and intraobserver variation
• Same result every time
• Likelyhood for a given treatment is identical for all
patients
• Increased patient safety
•
•
•
•
Correct and exact algorithms
Stable laboratory work-up
Possibly adjustment of treatment protocols
Integration with existing reporting systems
and databases
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen
”
When the winds of change blows, some
people build walls, while others build
windmills.
Chinese proverb
2nd NordiQC Conference on Applied Immunohistochemistry
Rigshospitalet, University of Copenhagen