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B-Raf V600E immunohistochemistry

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  • 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

    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 37 C 37 C 37 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

    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 37 C 37 C 37 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

    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

  • 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

    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

  • 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

    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

  • 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)

    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.

    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 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 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

    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

  • 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

  • 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.

    Adequate Sub-opt. Optimal Adequate

  • 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.

    Adequate Sub-opt. Optimal Adequate

  • 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

  • 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 /Borderline

    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