The slide set describes the staining patterns for the VE1 antibody. The validation and verification process is also described. Additionally external quality assurance and image analysis processes for the VE1 antibody are presented.

1. The production of this presentation has been made possible
through a financial contribution from Health Canada, through the
Canadian Partnership Against Cancer
cIQc

2. B-RAF v600e – GETTING STARTED
• Mouse Monoclonal Antibody
• Very Specific for the B-Raf
v600e mutant protein
• Stains cytoplasm of tumour
cells

3. BRAF V600E
• Mutation is present in numerous cancers
• Colorectal Cancer
• Melanoma
• Papillary Thyroid Caracinoma.
• Brain Cancers
• Lung Cancer
• Hairy Cell Leukemia

4. STEP 1
• Stain cases with a known B-Raf v600e
mutation

5. STAINING SELECTION USING THE VE1
RTU ANTIBODY FROM VENTANA
ANTIBODY SUPPLIED COURTESY OF VENTANA
Tissue Parameter BenchMark XT - 1 BenchMark XT - 2 BenchMark XT - 3
Detection Kit OptiView DAB IHC OptiView DAB IHC OptiView DAB IHC
Procedure Type Paraffin Paraffin Paraffin
Deparaffinization Selected, default
temperature
Selected, default
temperature
Selected, default
temperature
Cell Conditioning 64 min CC1, default
temperature
64 min CC1, default
temperature
32 min CC1, default
temperature
Pre Primary
Peroxidase Inhibitor Selected Selected Selected
Incubation
Temperature 37º C 37º C 37º C
Antibody Incubation
Time 16 minutes 32 minutes 32 minutes
Counterstains/ Time
Hematoxylin II 4 min
Bluing 4 min
Hematoxylin 4 min
Bluing 4 min
Hematoxylin II 4 min
Bluing 4 min

6. S12-2171 Thyroid BRAF V600E
Courtesy of Massachusetts General Hospital
•
• Protocol 1
• 2+ staining of papillary thyroid cancer
• 2+ staining colloid
• 3+ granular staining of histocytes
•
• Protocol 2
• 2+ staining of papillary thyroid cancer
• 1+ staining colloid
• 3+ granular staining of histocytes
•
• Protocol 3
• 1+ staining of papillary thyroid cancer
• 0+ staining colloid
• 2+ granular staining of histocytes
•
• Hematoxylin used in protocols 2 and 3 masked the brown colloid staining

7. S12-24759 Colon Cancer BRAF V600E
Courtesy of Massachusetts General Hospital
•
• Protocol 1
• 2+ staining of colon cancer
• 2+ smooth muscle
• 3+ staining normal nuclei in the crypts
•
• Protocol 2
• 2+ staining of colon cancer
• 1+ smooth muscle
• 3+ staining normal nuclei in the crypts
•
• Protocol 3
• 1+ staining of colon cancer
• Blush of staining in the smooth muscle
• 1+ staining normal nuclei in the crypts

8. S13-8899 LUNG BRAF V600E
Courtesy of Massachusetts General Hospital
• Protocol 1
• 2+ staining of cancer
• 2+ cilia in normal respiratory epithelium
•
• Protocol 2
• 1+ staining of cancer
• 3+ cilia in normal respiratory epithelium
•
• Protocol 3
• Blush of staining of cancer
• 2+ cilia in normal respiratory epithelium

9. STAINING SELECTION USING THE VE1
RTU ANTIBODY FROM VENTANA
ANTIBODY SUPPLIED COURTESY OF VENTANA
Tissue Parameter BenchMark XT - 1 BenchMark XT - 2 BenchMark XT - 3
Detection Kit OptiView DAB IHC OptiView DAB IHC OptiView DAB IHC
Procedure Type Paraffin Paraffin Paraffin
Deparaffinization Selected, default
temperature
Selected, default
temperature
Selected, default
temperature
Cell Conditioning 64 min CC1, default
temperature
64 min CC1, default
temperature
32 min CC1, default
temperature
Pre Primary
Peroxidase Inhibitor Selected Selected Selected
Incubation
Temperature 37º C 37º C 37º C
Antibody Incubation
Time 16 minutes 32 minutes 32 minutes
Counterstains/ Time
Hematoxylin II 4 min
Bluing 4 min
Hematoxylin 4 min
Bluing 4 min
Hematoxylin II 4 min
Bluing 4 min

10. STEP 2
• Stain an array of a variety of different tumours

11. MULTI-TUMOUR ARRAY
1 COLONIC ADENOCARCINOMA 37 BREAST CA
2 COLONIC ADENOCARCINOMA 38 BREAST CA
3 COLONIC ADENOCARCINOMA 39 OVARIAN SEROUS CARCINOMA
4 Colon Adenoma 40 OVARIAN SEROUS CARCINOMA
5 Colon Carcinoma 41 OVARIAN SEROUS CARCINOMA
6 THYROID PAPILLARY CA 42 HEPATOCELLULAR CA
7 THYROID PAPILLARY CA 43 HEPATOCELLULAR CA
8 THYROID PAPILLARY CA 44 HEPATOCELLULAR CA
9 LUNG SQ CELL CARCINOMA 45 RENAL CELL CARCINOMA
10 LUNG SQ CELL CARCINOMA 46 RENAL CELL CARCINOMA
11 LUNG SQ CELL CARCINOMA 47 RENAL CELL CARCINOMA
12 LUNG ADENOCARCINOMA 48 PANCREATIC CARCINOMA
13 LUNG ADENOCARCINOMA 49 PANCREATIC CARCINOMA
14 LUNG ADENOCARCINOMA 50 GASTERIC CARCINOMA
15 LUNG SMALL CELL CARCINOMA 51 GASTERIC CARCINOMA
16 LUNG SMALL CELL CARCINOMA 52 PANCREATIC NEUROENDOCRINE TUMOR
17 LUNG SMALL CELL CARCINOMA 53 PANCREATIC NEUROENDOCRINE TUMOR
18 MELANOMA 54 CARCINOID
19 MELANOMA 55 CARCINOID
20 MELANOMA 56 EMBRYONAL CARCINOMA
21 LEIOMYOSARCOMA 57 SKIN SQ CELL CARCINOMA
22 LEIOMYOSARCOMA 58 SKIN BCC
23 LEIOMYOSARCOMA 59 MESOTHELIAL HYPERPLASIA
24 Malignant fibrous histocytoma 60 Low grade endometrial Carcinoma
25 Malignant fibrous histocytoma 61 High grade endometrial Carcinoma
26 Malignant fibrous histocytoma 62 Normal Colon
27 LARGE CELL LYMPHOMA 63 NORMAL KIDNEY
28 LARGE CELL LYMPHOMA 64 LUNG
29 LARGE CELL LYMPHOMA 65 LIVER
30 EPITHELIOID MESOTHELIOMA 66 FALLOPIAN TUBE
31 EPITHELIOID MESOTHELIOMA 67 Normal Skin
32 EPITHELIOID MESOTHELIOMA 68 SKIN
33 SARCOMATOID MESOTHELIOMA 69 PANCREAS
34 SARCOMATOID MESOTHELIOMA 70 ADRENAL
35 SARCOMATOID MESOTHELIOMA 71 CEREBELLUM
36 BREAST CA 72 NEOCORTEX
73 COLON
74 ANTRUM
75 Heart
76 Normal Squamous Mucosa

12. Colon Adrenal gland
Lung Testis
400x
SPECIFIC, NON-SPECIFIC STAINING

13. LUNG

14. Brush Border Staining

15. Nuclear Staining in Normal Epithelium

16. STEP 3
• Stain a TMA of Colorectal Carcinomas

17. COLON CARCINOMA
• MLH1 results from 18 laboratories
• 32 caseS reviewed in the TMA
• 9 cores showed by IHC to be MLH1 absent.
• 4 cores stained definitively positive for B-raf.
Core 31 had a weak blush of positive staining.
• Core 16 was negative by the four routine
MMR markers but was positive for B-raf

18. COLON CANCER ARRAY STAINED FOR
MLH1 AND B-RAF V600E

19. MORE SPECIFIC NON-SPECIFIC
STAINING - SMOOTH MUSCLE

20. STEP 4
Prepare an EQA run for cIQc
Using MLH1 deleted CRC
Run 37

21. MLH1 AND MUTATIONAL STATUS
Acknowledgements: Roza Bidshahri, UBC
Molecular analysis results using D-PCR
core_id Comment
BRAF Mutation
MT
Frequency
IHC
1 WT 0% Positive
2 V600E 19% Positive
3 MSH2-deficient (control core) WT 0% Negative
4 V600E 17% Positive
5 Not MLH1 deficient! WT 0% Negative
6 V600E 5% Positive
7 V600E 12% Positive
8 WT 0% Negative
9 V600E 9% Positive
10 WT 0% Negative
11 WT 0% Negative
12
Original block is actually S98-
8620D
V600E 8% Positive
13 WT 0% Negative
14 V600E 16% Positive
15 WT 0% Negative
16 V600E 20% Positive
17 V600E 8% Positive
18 V600E 13% Positive
19 V600E 27% Positive
20 Not MLH1 deficient! WT 0% Negative
21 V600E 12% Positive
22 WT 0% Negative
23 V600E 19% Positive
24 V600R 35% Negative
25 V600E 12% Positive
26 WT 0% Negative
27 V600E 25% Positive
28 V600E 47% Positive
29 V600E 14% Positive
30 V600E 14% Positive
31 WT 0% Negative
32 V600E 13% Positive
33 V600E 10% Positive
34 V600E 13% Positive
35 WT 0% Negative
36 V600E 4% Positive
37 From cytology department V600E 8% Positive
38 WT 0% Negative
39 V600E 15% Positive
40 WT 0% Negative
41 WT 0% Negative

22. MLH1 DELETED CRC
stained for B-Raf v600e
24/31 cores positive (mutated)

23. MORE SPECIFIC NON-SPECIFIC
STAINING
• Core 1

24. MLH1 AND MUTATIONAL STATUSMolecular analysis results using D-PCR
core_id Comment
BRAF Mutation
MT
Frequency
IHC
1 WT 0% Positive
2 V600E 19% Positive
3 MSH2-deficient (control core) WT 0% Negative
4 V600E 17% Positive
5 Not MLH1 deficient! WT 0% Negative
6 V600E 5% Positive
7 V600E 12% Positive
8 WT 0% Negative
9 V600E 9% Positive
10 WT 0% Negative
11 WT 0% Negative
12
Original block is actually S98-
8620D
V600E 8% Positive
13 WT 0% Negative
14 V600E 16% Positive
15 WT 0% Negative
16 V600E 20% Positive
17 V600E 8% Positive
18 V600E 13% Positive
19 V600E 27% Positive
20 Not MLH1 deficient! WT 0% Negative
21 V600E 12% Positive
22 WT 0% Negative
23 V600E 19% Positive
24 V600R 35% Negative
25 V600E 12% Positive
26 WT 0% Negative
27 V600E 25% Positive
28 V600E 47% Positive
29 V600E 14% Positive
30 V600E 14% Positive
31 WT 0% Negative
32 V600E 13% Positive
33 V600E 10% Positive
34 V600E 13% Positive
35 WT 0% Negative
36 V600E 4% Positive
37 From cytology department V600E 8% Positive
38 WT 0% Negative
39 V600E 15% Positive
40 WT 0% Negative
41 WT 0% Negative

25. RUN 37 cIQc

26. RUN 37 PROTOCOLS
Optimal and sub-optimal staining

27. RUN 37
Lab 202 Core 10
Sub-Optimal staining
due to low signal to
background ratio
Lab 193 Core 9
Optimal Staining

28. RUN 37 - Core 19
• LAB 175
(Ventana protocol)
LAB 202

29. RUN 37
(A) Intense background
nuclear staining in Core 1 by
Lab 123.
(B) Observed optimal
staining in Core 9 by Lab
193.
(C) Generally weak staining
observed in Core 19 by
several participating
laboratories.
(D) Suboptimal staining in
Core 10 due to low signal to
background ratio (Lab 202).

30. STEP 5
• Collaboration with Horizon Diagnostics and
Visiopharm.
• Develop calibration standards

31. B-Raf v600e CELL LINES
• Cell lines were received from Horizon
Diagnostics fixed in alcohol.
• Prepared a cell blocks using Histogel.
• The cell blocks were placed in
formalin and the processed in the
usual manner into paraffin wax.
• From the wax blocks we prepared two
arrays of 12 cores creating
quadruplicate cores from each of the
cell blocks

32. CALIBRATION CELL LINES

33. Research Use35
Genetically Defined HDx Reference Standard: Engineering the BRAF V600E mutation
and characterization
Single Cell Dilution
Heterogeneous “Wildtype” cell
line
Clonal “Wildtype” cell
line
Generate a pair of isogenic cell lines
that are characterized and validated for
IHC
Clonal
mutant
cell line
Clonal
Wildtype
cell line
Evaluation
 SNP 6.0
 Sanger Sequencing
 Droplet Digital PCR
 RT-PCR
 Western Blot
 IHC
 Quantitative Digital
Pathology
Engineering BRAF V600E into
the WT cell lines
(-
)
(+
)
(+
+)

34. B-Raf v600e Lab A

35. B-Raf v600e Lab B

36. Study
• cIQc Run 37: BRAF V600E (April 2014)
• Blindly reviewed and scored, manually, by 6 assessors
• cIQc selected human samples expanded with cell lines
(Horizon diagnostics)
• cIQc samples analyzed with ONCOtopix and BRAF APP
(Visiopharm)
– Calculated scores where compared to cIQc readings
• Cell lines (Horizon diagnostics) analyzed with
ONCOtopix and BRAF APP (Visiopharm)
– Calculated scores compared to analyzed cIQc samples and
readings

37. TMA layout
1
2
1
1
1
0
9 8 7 6 5 4 3 2 1
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
2
1
2
2
2
3
2
4
3
6
3
5
3
4
3
3
3
2
3
1
3
0
2
9
2
8
2
7
2
6
2
5
3
7
3
8
3
9
4
0
4
1
N
P
1
P
2
P
3
N
P
1
P
2
P
3
N
P
1
P
2
P
3
cIQc Human samples
Cell Lines (Horizon diagnostics)

38. TMA layout
cIQc tissue samples
Cell Lines (Horizon diagnostics)

39. TMA layout - cIQc Tissue Samples
• Core 4 and 11 excluded.
• Relevant areas detected and H-Score calculated
• Predefined profile of positive and negative cores by cIQc
assessors
• Core 19 weak positive

40. cIQc Evaluation
Lab 116 123 175 189 191 202 114 111 193 101
CIQC
Score
Adequate
Adequate
(Bordeline)
Adequate
Adequate
(Bordeline)
Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate
cIQc
assessment

41. Image Analysis - cIQc Tissue Samples
Regions detected automatically

42. Analysis - cIQc Tissue Samples
Separation of cells
based on the
cytoplasm staining
positivity.
0 (negative, blue)
1+ (weak positive,
yellow)
2+ (mid positive,
orange)
3+ (strong positive,
red)
Combined into H-
Score

43. 0
50
100
150
200
250
300
20 22 40 10 41 24 1 3 19 21 33 29 25 17 7 12 16 18 9 14
Lab 193 - Optimal
Analysis - cIQc Tissue Samples
H-Score development from lowest reacting core to highest
(Lab profile)
• Lab 193 was the best performing lab
• Compare to expected output
• Calculate cut-offs for positive,
intermediate and negative
cores
Core 19

44. Analysis - cIQc Tissue Samples
H-Score development from lowest reacting
core to highest (Lab profile)
0
50
100
150
200
250
300
20 22 40 10 41 24 1 3 19 21 33 29 25 17 7 12 16 18 9 14
Lab 111 - Sup. optimal
Core 19
0
50
100
150
200
250
300
20 22 40 10 41 24 1 3 19 21 33 29 25 17 7 12 16 18 9 14
Lab 189 - Adequate
Core 19

45. Lab 116 123 175 189 191 202 114 111 193 101
TISSUE
Percent
agreement
79.5% 61.5% 71.8% 87.2% 54.1% 43.6% 61.5% 52.6% 97.4% 64.1%
Score
Adequate Adequate Adequate Adequate AdequateSub-opt.
Borderlin
e
Sub-opt. Optimal Adequate
CIQC Score
Adequate
Adequate
/Borderline
Adequate
Adequate
/Borderline
AdequateSub-opt.Adequate Sub-opt. Optimal Adequate
Analysis - cIQc Tissue Samples
Calculate comparison metrics (lab vs. expected)
• Percent agreement between lab profile and
expected profile

46. Analysis - cIQc Tissue Samples
Calculate comparison metrics (lab vs. expected concordance)
• Percent agreement between lab profile and expected profile
• Calculate cut-off for lab agreement
Lab 116 123 175 189 191 202 114 111 193 101
TISSUE
Percent
agreement
79.5% 61.5% 71.8% 87.2% 54.1% 43.6% 61.5% 52.6% 97.4% 64.1%
Score
Adequate Adequate Adequate Adequate Adequate Sub-opt.
Borderlin
e
Sub-
opt.
Optimal Adequate
CIQC Score
Adequate
Adequate
/Borderline
Adequate
Adequate
/Borderline
Adequate Sub-opt. Adequate
Sub-
opt.
Optimal Adequate

47. Analysis - cIQc Tissue Samples
Calculate comparison metrics (lab vs. expected concordance)
• Percent agreement between lab profile and expected profile
• Calculate cut-off for lab agreement
• Optimal > 90%
• Adequate > 55%
• Sub-optimal ≤55%
• Convert to lab score
Lab 116 123 175 189 191 202 114 111 193 101
TISSUE
Percent
agreement
79.5% 61.5% 71.8% 87.2% 54.1% 43.6% 61.5% 52.6% 97.4% 64.1%
Score Adequate Adequate Adequate Adequate Sub-opt Sub-opt. Adequate Sub-opt. Optimal Adequate
CIQC Score
Adequate
Adequate
/Borderline
Adequate
Adequate
/Borderline
AdequateSub-opt. Adequate Sub-opt. Optimal Adequate

48. TMA layout - Cell Lines
• Cores designed to express varying
biomarker level
– N = Negative expression
– P1 = Intermediate
– P2 = Intermediate
– P3 = Positive (strong)

49. Analysis - Cell Lines
Separation of cells
based on the
cytoplasm staining
positivity.
0 (negative, blue)
1+ (weak positive,
yellow)
2+ (mid positive,
orange)
3+ (strong positive,
red)
Combined into H-
Score

50. Analysis - Cell Lines
H-Score development from lowest reacting core to highest
(Lab profile)
• Compare to expected output
• Calculate cut-offs for positive,
intermediate and negative
cores
0
50
100
150
200
250
300
N N N E E E E E E P P P
Lab 193 Optimum

51. Analysis - Cell Lines
H-Score development from lowest reacting
core to highest (Lab profile)
0
50
100
150
200
250
300
N N N E E E E E E P P P
Lab 111 - Sub. Optimal
0
50
100
150
200
250
300
N N N E E E E E E P P P
Lab 189 - Adequate

52. Analysis - Cell Lines
Calculate comparison metrics (lab vs. expected)
• Percent agreement between lab profile and
expected profile
Lab 116 123 175 189 191 202 114 111 193 101
CELL
LINES
Percent
agreement
50% 42% 83% 50% 50% 33% 42% 25% 100%
Score
CIQC Score
Adequate
Adequate
/Borderline
Adequate
Adequate
/Borderline
Adequate
Sub-
opt.
AdequateSub-opt. Optimal Adequate

53. Analysis - Cell Lines
Calculate comparison metrics (lab vs. expected)
• Percent agreement between lab profile and expected profile
• Calculate cut-off for lab agreement
Lab 116 123 175 189 191 202 114 111 193 101
CELL
LINES
Percent
agreement
50% 42% 83% 50% 50% 33% 42% 25% 100%
Score
CIQC Score
Adequate
Adequate
(Bordeline)
Adequate
Adequate
(Bordeline)
Adequate
Sub-
opt.
AdequateSub-opt. Optimal Adequate

54. Analysis - Cell Lines
Calculate comparison metrics (lab vs. expected)
• Percent agreement between lab profile and expected profile
• Calculate cut-off for lab agreement
• Optimal > 90%
• Adequate > 40%
• Sub-optimal ≤40%
• Convert to lab score
Lab 116 123 175 189 191 202 114 111 193 101
CELL
LINES
Percent
agreement
50% 42% 83% 50% 50% 33% 42% 25% 100%
Score Adequate Adequate
Adequate
Adequate
Adequate
Sub-opt. Adequate Sub-opt.
Optimal
CIQC Score
Adequate
Adequate
(Bordeline)
Adequate
Adequate
(Bordeline)
Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate

55. CIQC evaluation vs. Cell Lines
Lab 116 123 175 189 191 202 114 111 193 101
TISSUE
Percent
agreement
79.5% 61.5% 71.8% 87.2% 54.1% 43.6% 61.5% 52.6% 97.4% 64.1%
Score Adequate Adequate Adequate Adequate Sub-opt Sub-opt. Adequate Sub-opt. Optimal Adequate
CELLLINES
Percent
agreement
50% 42% 83% 50% 50% 33% 42% 25% 100%
Score Adequate Adequate
Adequate
Adequate
Adequate
Sub-opt. Adequate Sub-opt. Optimal
CIQC
Score
Adequate
Adequate
/Borderline
Adequate
Adequate
/Borderline
Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate
Using Visiopharm image analysis

58. CALIBRATION
Purpose of calibration: The act of evaluating and
adjusting the precision and accuracy of
measurement .Intended to eliminate or reduce bias
in readings over a range for all continuous values.
Precision: Repeated measurements under
unchanged conditions show the same result
Accuracy is the degree of closeness of
measurements of a quantity to its actual true value.

59. Research Use
100% concordance between calibrated BRAF V600E cell lines
and molecular profiled tissue.
Lab A results: Optimal
BRAF V600E staining
Core1.BRAFV600E
NegativeCellline
Core4.BRAFV600E
StrongCellline
Core15.BRAFV600E
(TMA–vebyddPCR)
Lab B results: Suboptimal
BRAF V600E staining
Core2.BRAFV600E
IntermediateCellline
Core3.BRAFV600E
IntermediateCellline
Core16.BRAFV600E
(TMA+vebyddPCR)
Lab A results: Optimal
BRAF V600E staining
Lab B results: Suboptimal
BRAF V600E staining

60. A QC DASHBOARD – A role for
genetically engineered reference
standards and image analysis.
Test Score
B-Raf 98%
ALK 79%
HER2 95%
ER 49%
Failed
Sub-optimal
Adequate
Optimal

61. A QC DASHBOARD – A role for
genetically engineered reference
standards and image analysis.

62. B-Raf v600e Analysis
Deanna Johnson, Lions Gate Hospital
Farah Patell-Socha, Horizon
Martin Kristensson, Visiopharm
Roza Bidshahri, UBC
Katerina Dvorak, Ventana Medical
Systems

64. E Torlakovic MD, B Gilks MD, J Won PhD and J Garratt RT
Department of Pathology, Vancouver General Hospital, Vancouver,
BC, Canada
Department of Pathology and Laboratory Medicine, University of
British Columbia, Canada.
Canadian Immunohistochemistry Quality Control (CIQC) and
Canadian Association of Pathologists, Canada.
Department of Laboratory Medicine and Pathobiology, University of
Toronto, Canada
The Canadian External Quality Assurance Program for
Immunohistochemistry: an initiative of Canadian
Immunohistochemistry Quality Control (cIQc) and Canadian
Association of Pathologists (CAP) National Standards
Committee/Immunohistochemistry