HER2 Immunohistochemistry, in-situ hybridization and...
Transcript of HER2 Immunohistochemistry, in-situ hybridization and...
![Page 1: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/1.jpg)
HER2
HER2 Immunohistochemistry,
in-situ hybridization and
quality assurance
Søren Nielsen
Scheme Manager & Project coordinator
NordiQC
Aalborg Hospital, Denmark
![Page 2: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/2.jpg)
HER2
I have nothing to declare
![Page 3: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/3.jpg)
3
Nordic immunohistochemical Quality Control
Founded 2003 by Nordic pathologists
Independent, scientific, not-for-profit organisation
Institute of Pathology, Aalborg University Hospital
General module: 3 runs/year
15-18 different markers
Breast cancer IHC module: 2 runs
HER-2, ER/PR, Ki67/E-Cad,….
HER-2 ISH module: 2 runs/year
BRISH, FISH
HER2 – NordiQC perspective
![Page 4: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/4.jpg)
4
www.nordiqc.org HER2
![Page 5: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/5.jpg)
0
50
100
150
200
250
300
350
400
450
500
1 5 9 16 20 24 28 32 36 40
Generel Breast
HER2 – NordiQC perspective
HER-2 ISH
2000 2004 2006 2008 2010 2012 2014
App. 700 laboratories in total
> 70 countries
![Page 6: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/6.jpg)
HER2 – NordiQC perspective
Semiquantitative IHC/ISH test – number/localization/intensity will define class
![Page 8: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/8.jpg)
Decalcification
Preparation Pre-
analytic
Analytic
Post-
analytic
Tissue
Type, Dimension,
Laser resection,
De-differentiation
Fixation
Time, Type, Volume
Section
Thickness
Storage
Drying Visualization
Sensitivity, Specificity
Primary antibody
Clone, Dilution
Buffer, Time, Temp
Development
Sensitivity,
Localization
Interpretation
Localization
Positive/Negative - cut-off level Quantification
Reporting
Controlment
Pre-treatment
With 3 choices for 5
variables in each phase = >
4 million protocols….
Manual
Stainer
IHC – The Technical Test Approach
… The biomarker protocol trap – Caution: not for faint-hearted lab personel !!!!!
![Page 9: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/9.jpg)
HER2 – NordiQC perspective
60°C 1h. HER-2: 3+ 80°C 16h. HER-2: 1+
![Page 10: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/10.jpg)
Bogen et al.; Arch Pathol Lab Med - Vol 132, February 2008
CAP 2006B proficiency scheme 18.3% Error rate (109 labs)
Jefferson T et al; Appl Immuno Mol Morphol Vol 19, N 05, ’2009
CIQC / CAP < 3% Error rate (18 labs)
HER2 – NordiQC perspective
Rhodes A et al; Am J Clin Pathol, vol 118, 408-417 2002
UK NEQAS 38% Error rate (93 labs)
IHC/ISH HER2 testing…
Primarily focusing on the analytical phase !
Wolff AC et al.; Journal of Clinical Oncology 25:118-145 2007
ASCO/ CAP up to 20% Error rate
Kaufman A P et al; Cancer Vol 120, Issue 17, 2014
VIRGO study 4% Error rate (552 patients)
![Page 11: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/11.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
2. Analytical method used – robustness and calibration
3. Control material – precision of information
4. Interpretation – experience, consistency, cut-off value
![Page 12: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/12.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
A. Cold ischemic time – as short as possible < 1 hour
B. 10% Neutral buffered formalin
C. Fixation time 6-72 hours
Consistenly questionned and challenged….
![Page 13: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/13.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
Concl.: HER2 assay not affected by none of the 3 variables…….
Am J Clin Pathol 2011;136:754-761
Material: one tumour, 3+, highly amplified
![Page 14: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/14.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
Cold ischemic time – as short as possible < 1 hour
Material: 9 tumours, 3+, low-high.
HercepTest
Oracle
PATHWAY
PathVision
![Page 15: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/15.jpg)
HER2 – NordiQC perspective
15 H-score: intensity (0-3) x proportion (%)
4°C
20°C/RT
![Page 16: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/16.jpg)
HER2 – NordiQC perspective
VS 4C - 1h VS 4C - 20h
VS 4C - 92h VS RT - 92h
VS 4C - 1h VS 4C - 44h
VS RT - 44h
Concl.: Cooling preserved specimens, whereas vacuum sealing added no effect
(IHC and molecular assays)
VS RT - 92h
![Page 17: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/17.jpg)
HER2 – NordiQC perspective
Pre-analytical
variable
Published
guidelines
ASCO/CAP - CLSI
Litterature based
guidelines*
Fixation delay 1 hour < 12 hours (3-4 hours)
Transportation temp. No data 4°C better than RT
Vacuum sealing No data (Not recommended)
* Engel and. Moore (2011) Effects of Preanalytical Variables on the Detection of Proteins by Immunohistochemistry in Formalin-Fixed, Paraffin-Embedded Tissue. Archives of Pathology & Laboratory Medicine: May 2011, Vol. 135, No. 5, pp. 537-543.
![Page 18: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/18.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
Fixation time in 10% NBF, 6-72 hours
30 min fixation in NBF adequate….
![Page 19: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/19.jpg)
HER2 – NordiQC perspective
Internal
IHC validation 4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 1+ 1+ 1+ 1+
Tumour 2 3+ 3+ 3+ 3+
Tumour 3 0 0 0 0
Tumour 4 1+ 1+ 1+ 1+
Tumour 5 0 0 0 0
Tumour 6 3+ 3+ 3+ 3+
Tumour 7 0 0 0 0
Tumour 8 0 0 0 0
Tumour 9 0 0 0 0
Breast carcinomas, HER-2 PATHWAY, rmAb 4B5
(CC1 Mild, Ab inc. 20 min. 36°C, UltraView DAB)
![Page 20: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/20.jpg)
HER2 – NordiQC perspective
4 h
48 h
24 h
168 h
Breast carcinomas 1+, 3+, HER2 PATHWAY, rmAb 4B5
![Page 21: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/21.jpg)
HER2 – NordiQC perspective
Internal
IHC
validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 + + + +
Tumour 2 - - - -
Tumour 3 + + + +
Tumour 4 + + + +
Tumour 5 + + + +
Tumour 6 + + + +
Tumour 7 - - - -
Tumour 8 + + + +
Tumour 9 + + + +
Internal
IHC
validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 1+ 1+ 1+ 1+
Tumour 2 3+ 3+ 3+ 3+ 3+
Tumour 3 0 0 0 0
Tumour 4 1+ 1+ 1+ 1+
Tumour 5 0 0 0 0
Tumour 6 3+ 3+ 3+ 3+ 3+
Tumour 7 0 0 0 0
Tumour 8 0 0 0 0
Tumour 9 0 0 0 0
Internal
IHC
validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 + + + +
Tumour 2 - - - -
Tumour 3 + + + +
Tumour 4 + + + +
Tumour 5 + + + +
Tumour 6+ + + + +
Tumour 7 - - - -
Tumour 8 + + + +
Tumour 9 + + + +
Internal
IHC
validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 + + + +
Tumour 2 + + + +
Tumour 3 + + + +
Tumour 4 + + + +
Tumour 5 + + + +
Tumour 6 + + + +
Tumour 7 + + + +
Tumour 8 + + + +
Tumour 9 + + + +
HER2
rmAb
4B5
ER
rmAb
SP1
PR
rmAb
1E2
ECAD
mAb
NCH-36
Conclusion: IHC biomarkers not affected by NBF fixation time and patient material and control material can be fixed from 4 - 168h in 10% NBF …. but
![Page 22: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/22.jpg)
HER2 – NordiQC perspective
Internal
SISH validation 4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 - - - FN
Tumour 2 Amp + + + +
Tumour 3 (?) - FN FN
Tumour 4 - - FN FN
Tumour 5 - - - -
Tumour 6 Amp + + + +
Tumour 7 - - - FN
Tumour 8 poly. - - - FN
Tumour 9 poly. - - - FN
HER-2 ISH: 8/36 cores could not be assessed..!
Breast carcinomas, Dual SISH CCrb ext, P3. 8 m
![Page 23: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/23.jpg)
HER2 – NordiQC perspective
4 h
48 h
24 h
168 h
Breast carcinoma, 1+ Dual SISH CCrb ext, P3. 8 m
![Page 24: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/24.jpg)
HER2 – NordiQC perspective
Pre-analytical
variable
Published
guidelines
ASCO/CAP - CLSI
Litterature based
guidelines*
Fixative 10 % NBF** 10 % NBF
Fixation time 6 – 72 hours 24 hours
Fixative – tissue ratio 1:10 1:1 – 1:20
MW assisted fixation Pre-fixation 6 hours Pre-fixation 0 – 24 h.
* Engel and. Moore (2011) Effects of Preanalytical Variables on the Detection of Proteins by Immunohistochemistry in Formalin-Fixed, Paraffin-Embedded Tissue. Archives of Pathology & Laboratory Medicine: May 2011, Vol. 135, No. 5, pp. 537-543. ** 10 % Neutral buffered formalin = 4 % Neutral buffered formaldehyde
![Page 25: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/25.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
2. Analytical method used – robustness and calibration
3. Control material – precision of information
4. Interpretation – experience, consistency, cut-off value
![Page 26: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/26.jpg)
26 26
HER2 – NordiQC perspective
CK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 1+ Unampl. 1+ Unampl. 0
Poor
> 6.0 2.3 – 2.6 1.3 – 1.7 1.1 – 1.4
10-20% HER2+
found in 2+ cat.
![Page 27: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/27.jpg)
27 27
HER2 – NordiQC perspective
CK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 2+ Unampl. 3+ Unampl. 1
Poor
> 6.0 2.3 – 2.6 1.3 – 1.7 1.1 – 1.4
![Page 28: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/28.jpg)
HER2 – NordiQC perspective
Material processed
according to ASCO/CAP
![Page 29: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/29.jpg)
29
HER2 – NordiQC perspective
App 90 % of insuff. results are FN and seen both by FDA / CE-IVD kits and laboratory developed assays. FP results have virtually only been seen by laboratory developed assays.
Pass rate of 19 HER2 IHC assessments in NordiQC
![Page 30: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/30.jpg)
30
HER2 – NordiQC perspective
Typical causes for insufficient results in the NordiQC HER2 IHC breast module:
FDA / CE-IVD HER2 IHC kits:
PATHWAY®, Ventana: Too short HIER (<24M) and/or too short incubation of primary Ab (<12M)
HercepTest™, Dako: Too short HIER (<40M) and/or too short incubation of primary & secondary Ab (<30M)
Oracle™, Leica: No single or combination of causes have been identified
Laboratory developed assays: Inappropriate titre of primary Ab, less successful primary Ab, insufficient HIER, etc…..
![Page 31: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/31.jpg)
![Page 32: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/32.jpg)
32 32
CK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 2+
Good
HER2 – NordiQC perspective
ISH: 2.5-2.8
ISH: 2.5-
2.8
ISH: 1.3-1.7
Increased need for
additional ISH
![Page 33: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/33.jpg)
33
HER2 – NordiQC perspective
![Page 34: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/34.jpg)
34
HER2 – NordiQC perspective
![Page 35: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/35.jpg)
35
HER2 – NordiQC perspective
Technically optimal results in the NordiQC HER2 ISH breast module:
INFORM™ HER2 Dual ISH, Ventana ZytoDot® 2C, ZytoVision HER2 black – chr17 red HER2 green – chr17 red
U
A
![Page 36: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/36.jpg)
36
HER2 – NordiQC perspective
Typical causes for insufficient results in the NordiQC HER2 ISH breast module:
FDA / CE-IVD HER2 BRISH (CISH/DDISH/etc) kits: INFORM™ HER2 Dual ISH, Ventana: Excessive proteolysis (>16M), HIER in CC1. DuoCISH™ pharmDx™, Dako: Insufficient proteolysis, inappropriate handling of chromogen. ZytoDot® 2C, ZytoVision: Excessive proteolysis. In 90% of insufficient results, no single or combination of causes could be identified
![Page 37: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/37.jpg)
37
HER2 – NordiQC perspective
90% of ins.
results used
approriate
protocol.
![Page 38: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/38.jpg)
38
HER2 – NordiQC perspective
Technically insufficient results in the NordiQC HER2 ISH breast module:
INFORM™ HER2 Dual ISH, Ventana HER2 black – chr17 red
U
Excessive protelysis
Neg areas >25% Silver precip.
U
![Page 39: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/39.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
2. Analytical method used – robustness and calibration
3. Control material – precision of information
4. Interpretation – experience, consistency, cut-off value
![Page 40: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/40.jpg)
HER2 – NordiQC perspective
Central issues to adress for control material of HER2 IHC test
1. External control – no useful internal HER2 expression
2. Must be processed under similar conditions as patient
material – otherwise documented that identical results are
generated by other methods (fixative, duration, etc)
3. Must reflect the range of HER2 expression seen for
diagnostics
4. Consistent and stable HER2 expression throughout the
control material
![Page 41: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/41.jpg)
HER2 – NordiQC perspective
Central issues to adress for control material of HER2 IHC test
1. Control material for initial calibration and validation
e.g 100 samples ranging 0, 1+, 2+, 3+.
Optimally all samples confirmed by ISH
Metrix can be generated and test implemented.
2. Control material to monitor consistent and right level of
sensitivity as identified by calibration – transfer of method –
is obtained in each test performed.
Focus: The issue to identify and use proper control material
to monitor consistency of test
![Page 42: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/42.jpg)
HER2 – NordiQC perspective
Central issues to adress for control material of HER2 IHC test
In NordiQC app. 60-70% of laboratories use a 3+ tumour
as routine positive control for HER2 IHC
Question:
Is this reliable to
monitor a
consistent level
of HER2 assay ?
![Page 43: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/43.jpg)
43 43
HER2 – NordiQC perspective
CK7
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Optim
al
Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0
Poor F
N
3+ (> 6.0) 2+ (2.3–2.6) 2+ (1.3–1.7) 0/1+ (1.1– 1.4)
Poor F
P
![Page 44: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/44.jpg)
PATHWAY 1 PATHWAY 2 PATHWAY 3
2+ Unamp
2+ Amp
3+ Amp
CC1_64/32M/OP CC1_64/16M/UV CC1_24/12M/UV
![Page 45: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/45.jpg)
HER2 – NordiQC perspective
Central issues to adress for control material of HER2 IHC test
In NordiQC app. 60-70% of laboratories use a 3+ tumour
as positive control for HER2 IHC
Optimally:
Use small TMA
with 1+, 2+ & 3+
mounted on
same slide as pt
material for daily
control of HER2
IHC assay
![Page 46: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/46.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Pre-analytics – mainly time-to and time-in NBF fixation
2. Analytical method used – robustness and calibration
3. Control material – precision of information
4. Interpretation – experience, consistency, cut-off value
![Page 47: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/47.jpg)
HER2 – NordiQC perspective
Lab 1; scored 1+ Lab 2; scored as 1+
![Page 48: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/48.jpg)
HER2 – NordiQC perspective
Lab 1; scored 3+ Lab 2; scored as 3+
![Page 49: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/49.jpg)
HER2 – NordiQC perspective
Lab 1; scored 2+ Lab 2; scored as 2+
![Page 50: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/50.jpg)
50
HER2 – NordiQC perspective
B11 B12 B13 B14 B15 B16 B17 B18 B19
Sufficient staining and consensus: average 87% Insufficient staining and consensus: average 47%
Sufficient result + consensus
Insufficient result + consensus
![Page 51: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/51.jpg)
HER2 – NordiQC perspective
What is faint ?
What is weak ?
Up to 20-40% HER2 IHC tests are reflexed to ISH
due to expanded criteria for 2+ (internal data)
![Page 52: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/52.jpg)
HER2 – NordiQC perspective
Digital computer
assisted analysis
to be integrated.
![Page 53: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/53.jpg)
HER2 – NordiQC perspective
![Page 54: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/54.jpg)
HER2 – NordiQC perspective
To improve consistency
To reduce cohort of 2+
To serve as internal QC
![Page 55: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/55.jpg)
HER2 – NordiQC perspective
PATHWAY
HercepTest
Oracle
Pt HER2
Cell line HER2
FN Optimal FP
CL1 < 0.20 0.20-0.30 > 0.30
CL2 < 0.40 0.40-0.50 > 0.50
![Page 56: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/56.jpg)
PATHWAY 1 PATHWAY 2 PATHWAY 3
1+ Unamp
2+ Amp
3+ Amp
CC1_64/32M/OP CC1_64/16M/UV CC1_24/12M/UV
CONNECT: 0.25
Range 0.20-0.30
CONNECT: 0.40 CONNECT: 0.10
![Page 57: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/57.jpg)
HER2 – NordiQC perspective
Core element for HER2 IHC QC:
Is the method calibrated properly ?
Identified by initial validation on histology samples
Metrix (no of 3+ etc), FISH etc
Is the method successfully performed ?
Identified by interpretation of appropriate control
What is the level of HER2 expression in patient ?
Identified by objective and precise interpretation
![Page 58: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/58.jpg)
HER2 – NordiQC perspective
4 central parametres to adress for accurate HER2 IHC/ISH test
1. Analytical method used – robustness and calibration
2. Control material – precision of information
3. Interpretation – experience, consistency, cut-off value
4. Pre-analytics – mainly time-to and time-in NBF fixation
![Page 59: HER2 Immunohistochemistry, in-situ hybridization and ...cpqa.ca/main/wp-content/uploads/2015/06/Nielsen... · HER2 Immunohistochemistry, in-situ hybridization and quality assurance](https://reader033.fdocuments.us/reader033/viewer/2022042205/5ea716f38bfef76bee1a2788/html5/thumbnails/59.jpg)
HER2 – NordiQC perspective
Thank You for the
attention and…..