Download pdf - B-Raf V600E immunohistochemistry

The production of this presentation has been made possible through a financial contribution from Health Canada, through the

Canadian Partnership Against Cancer

cIQc

B-RAF v600e – GETTING STARTED

• Mouse Monoclonal Antibody

• Very Specific for the B-Raf v600e mutant protein

• Stains cytoplasm of tumour cells

BRAF V600E

• Mutation is present in numerous cancers

• Colorectal Cancer

• Melanoma

• Papillary Thyroid Caracinoma.

• Brain Cancers

• Lung Cancer

• Hairy Cell Leukemia

STEP 1

• Stain cases with a known B-Raf v600e mutation

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


Cell Conditioning 64 min CC1, default temperature

64 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

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

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

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

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



Cell Conditioning 64 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

STEP 2

• Stain an array of a variety of different tumours

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



9 LUNG SQ CELL CARCINOMA 45 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

Colon Adrenal gland

Lung Testis

400x

SPECIFIC, NON-SPECIFIC STAINING

LUNG

Brush Border Staining

Nuclear Staining in Normal Epithelium

STEP 3

• Stain a TMA of Colorectal Carcinomas

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

COLON CANCER ARRAY STAINED FOR MLH1 AND B-RAF V600E

MORE SPECIFIC NON-SPECIFIC STAINING - SMOOTH MUSCLE

STEP 4

Prepare an EQA run for cIQc

Using MLH1 deleted CRC

Run 37

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


5 Not MLH1 deficient! WT 0% Negative

6 V600E 5% 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


15 WT 0% Negative







22 WT 0% Negative


24 V600R 35% Negative


26 WT 0% Negative





31 WT 0% Negative




35 WT 0% Negative


37 From cytology department V600E 8% Positive

38 WT 0% Negative


40 WT 0% Negative

41 WT 0% Negative

MLH1 DELETED CRC stained for B-Raf v600e

24/31 cores positive (mutated)

MORE SPECIFIC NON-SPECIFIC STAINING

• Core 1

MLH1 AND MUTATIONAL STATUS Molecular analysis results using D-PCR

core_id Comment BRAF Mutation

MT Frequency

IHC

1 WT 0% Positive


3 MSH2-deficient (control core) WT 0% Negative



6 V600E 5% 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


15 WT 0% Negative







22 WT 0% Negative


24 V600R 35% Negative


26 WT 0% Negative





31 WT 0% Negative




35 WT 0% Negative


37 From cytology department V600E 8% Positive

38 WT 0% Negative


40 WT 0% Negative

41 WT 0% Negative

RUN 37 cIQc

RUN 37 PROTOCOLS Optimal and sub-optimal staining

RUN 37

Lab 202 Core 10

Sub-Optimal staining

due to low signal to background ratio

Lab 193 Core 9

Optimal Staining

RUN 37 - Core 19

• LAB 175

(Ventana protocol)

LAB 202

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).

STEP 5

• Collaboration with Horizon Diagnostics and Visiopharm.

• Develop calibration standards

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

CALIBRATION CELL LINES

Research Use Only

35

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

(-)

(+)

(++)

B-Raf v600e Lab A

B-Raf v600e Lab B

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

TMA layout

12

11

10

9 8 7 6 5 4 3 2 1

13

14

15

16

17

18

19

20

21

22

23

24

36

35

34

33

32

31

30

29

28

27

26

25

37

38

39

40

41

N

P1

P2

P3

N

P1

P2

P3

N

P1

P2

P3

cIQc Human samples

Cell Lines (Horizon diagnostics)

TMA layout

cIQc tissue samples

Cell Lines (Horizon diagnostics)

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

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

Image Analysis - cIQc Tissue Samples

Regions detected automatically

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

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

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

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.

Borderline

Sub-opt. Optimal Adequate

CIQC Score Adequate

Adequate /Borderline

Adequate Adequate

/Borderline Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate

Analysis - cIQc Tissue Samples

Calculate comparison metrics (lab vs. expected)

• Percent agreement between lab profile and expected profile

Analysis - cIQc Tissue Samples Calculate comparison metrics (lab vs. expected concordance)


• 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.

Borderline

Sub-opt.

Optimal Adequate

CIQC Score Adequate


Adequate Adequate

/Borderline Adequate Sub-opt. Adequate

Sub-opt.

Optimal Adequate

Analysis - cIQc Tissue Samples Calculate comparison metrics (lab vs. expected concordance)



• 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 Adequate

/Borderline Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate

TMA layout - Cell Lines

• Cores designed to express varying biomarker level

– N = Negative expression

– P1 = Intermediate

– P2 = Intermediate

– P3 = Positive (strong)

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

Analysis - Cell Lines


• 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

Analysis - Cell Lines


0

50

100

150

200

250

300


Lab 111 - Sub. Optimal

0

50

100

150

200

250

300


Lab 189 - Adequate

Analysis - Cell Lines Calculate comparison metrics (lab vs. expected)


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.

Adequate Sub-opt. Optimal Adequate




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 Adequate (Bordeline)

Adequate Sub-opt.

Adequate Sub-opt. Optimal Adequate




• 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 Adequate

(Bordeline) Adequate Sub-opt. Adequate Sub-opt. Optimal Adequate

CIQC evaluation vs. Cell Lines

Lab 116 123 175 189 191 202 114 111 193 101

TISS

UE

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

CEL

L LI

NES

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 Sub-opt. Adequate Sub-opt. Optimal Adequate

Using Visiopharm image analysis

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.

Research Use Only

62

100% concordance between calibrated BRAF V600E cell lines and molecular profiled tissue.

Lab A results: Optimal BRAF V600E staining

Co

re 1

. B

RA

F V

60

0E

N

ega

tive

Ce

ll lin

e

Co

re 4. B

RA

F V6

00

E Stro

ng C

ell lin

e

Co

re 15

. BR

AF V

60

0E

( TM

A –ve b

y dd

PC

R)

Lab B results: Suboptimal BRAF V600E staining

Co

re 2

. B

RA

F V

60

0E

In

term

edia

te C

ell

line

Co

re 3

. B

RA

F V

60

0E

In

term

edia

te C

ell

line

Co

re 16

. BR

AF V

60

0E

( TMA

+ve b

y dd

PC

R)

Lab A results: Optimal BRAF V600E staining

Lab B results: Suboptimal BRAF V600E staining

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

A QC DASHBOARD – A role for genetically engineered reference

standards and image analysis.

B-Raf v600e Analysis

Deanna Johnson, Lions Gate Hospital

Farah Patell-Socha, Horizon

Martin Kristensson, Visiopharm

Roza Bidshahri, UBC

Katerina Dvorak, Ventana Medical Systems

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