her2 immunohistochemistry, in-situ hybridization and...

HER2

HER2 Immunohistochemistry,

in-situ hybridization and

quality assurance

Søren Nielsen

Scheme Manager & Project coordinator

NordiQC

Aalborg Hospital, Denmark

HER2

I have nothing to declare

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

4

www.nordiqc.org HER2

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Generel Breast


HER-2 ISH

2000 2004 2006 2008 2010 2012 2014

App. 700 laboratories in total

> 70 countries


Semiquantitative IHC/ISH test – number/localization/intensity will define class


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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 !!!!!


60°C 1h. HER-2: 3+ 80°C 16h. HER-2: 1+

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)


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)


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




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….




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




Cold ischemic time – as short as possible < 1 hour

Material: 9 tumours, 3+, low-high.

HercepTest

Oracle

PATHWAY

PathVision


15 H-score: intensity (0-3) x proportion (%)

4°C

20°C/RT


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

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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.




Fixation time in 10% NBF, 6-72 hours

30 min fixation in NBF adequate….


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)


4 h

48 h

24 h

168 h

Breast carcinomas 1+, 3+, HER2 PATHWAY, rmAb 4B5


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


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


Tumour 1 + + + +

Tumour 2 - - - -

Tumour 3 + + + +

Tumour 4 + + + +

Tumour 5 + + + +

Tumour 6+ + + + +

Tumour 7 - - - -

Tumour 8 + + + +

Tumour 9 + + + +

Internal

IHC

validation


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


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


4 h

48 h

24 h

168 h

Breast carcinoma, 1+ Dual SISH CCrb ext, P3. 8 m


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

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CK7

Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0

Optim

al


Poor

> 6.0 2.3 – 2.6 1.3 – 1.7 1.1 – 1.4

10-20% HER2+

found in 2+ cat.

27 27


CK7


Optim

al


Poor

> 6.0 2.3 – 2.6 1.3 – 1.7 1.1 – 1.4


Material processed

according to ASCO/CAP

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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

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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…..

32 32

CK7


Optim

al

Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 2+

Good


ISH: 2.5-2.8

ISH: 2.5-

2.8

ISH: 1.3-1.7

Increased need for

additional ISH

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34


35


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

36


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

37


90% of ins.

results used

approriate

protocol.

38


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


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



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



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 ?

43 43


CK7


Optim

al


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

PATHWAY 1 PATHWAY 2 PATHWAY 3

2+ Unamp

2+ Amp

3+ Amp

CC1_64/32M/OP CC1_64/16M/UV CC1_24/12M/UV



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


Lab 1; scored 1+ Lab 2; scored as 1+

50


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


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)


Digital computer

assisted analysis

to be integrated.


To improve consistency

To reduce cohort of 2+

To serve as internal QC


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

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


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


Thank You for the

attention and…..

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her2 immunohistochemistry, in-situ hybridization and...

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