Welcome to CAP’s “Hot Topics in Pathology” Webinar Series ... · 453 (61.5%) cases had an TTF less than 1 hour and 283 (38.5%) had TTF greater than an hour . 2013 HER2 Testing

This webinar on “HER2 Testing Revision” is presented

by David G. Hicks, MD, FCAP and Stephen J.

Sarewitz, MD, FCAP

Your host is Jill Kaufman, PhD. For comments about

this webinar or suggestions for upcoming

webinars, please contact Jill Kaufman at

[email protected]

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Welcome to CAP’s “Hot Topics in Pathology”

Webinar Series sponsored by the

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HER2 Testing Revision David G Hicks, MD, FCAP, and Stephen J. Sarewitz, MD, FCAP

December 3, 2013

Hot Topics in

Pathology

• Professor of Pathology and Laboratory

Medicine, University of Rochester

School of Medicine

• Director of Surgical Pathology at the

University of Rochester Medical Center

• Current research interests focus on the

molecular genetic profiling of clinical

samples from patients with cancer

• Authored or co-authored over 140

peer reviewed articles

• Member of the CAP’s Breast Pathology

Certificate Program Work Group and

Breast Predictive Factors Testing

Program Group

David G. Hicks, MD, FCAP


• Current CAP Board of

Governors

• Vice-chair for CAP’s Council

on Accreditation

• Member of CAP’s Risk

Management Committee,

Council on Education and

Constitution and Bylaws

Committee

Stephen J. Sarewitz, MD, FCAP


Disclaimer

The College does not permit reproduction of any substantial portion of the material in this Webinar without its written

authorization. The College hereby authorizes attendees of the

CAP Webinar to use the pdf presentation solely for educational

purposes within their own institutions. The College prohibits use of the material in the Webinar – and any unauthorized use of

the College’s name or logo – in connection with promotional

efforts by marketers of laboratory equipment, reagents,

materials, or services.

Opinions expressed by the speaker are the speaker’s own and

do not necessarily reflect an endorsement by CAP of any

organizations, equipment, reagents, materials or services used

by participating laboratories.


• Dr. Hicks – member of a speaker’s bureau

sponsored by Genentech BioOncology that support

educational programs on breast cancer testing

• Dr. Sarewitz – no disclosures

Disclosure


Why is HER2 Testing Different?

• HER2 testing is more like doing a frozen section than looking at a special stain:

– A single observation leads to a critical treatment decision

• The test is assumed to be accurate and precise every time by both clinician and patient

• ASCO/CAP HER2 testing guidelines have provided standards for improving the accuracy and reliability for testing

To address ongoing testing needs and challenges in light of new published literature, the 2007 ASCO/CAP HER2 guideline have been updated in 2013

2013 HER2 Testing Guidelines Update, What Changed?

Updates From 2007

8

Wolff AC et al. J Clin Oncol. 2013; Oct 7. [Epub ahead of print]

2013 Updates

2013 Recommendations

• All new tumors should be tested (including

metastatic tumors)

• Highlights the need for enhanced communication

between pathologists and oncologists

• Guidance for communicating with patients

• Optimal tissue specimen handling procedures

• Maximum time in fixative: 72 hours

• New algorithms for test interpretation and reporting

• Language on repeat testing (reflex and new tests)

2013 HER2 Testing Guidelines Update: What Remains the Same From 2007

9

1. Wolff AC et al. J Clin Oncol. 2007;25:118-145; 2. Wolff AC et al. J Clin Oncol. 2013; Oct 7. [Epub ahead of print]

No Change

From 20071

Recommendations1,2

• Optimal tissue specimen handling procedures

• Tissue acquisition (ie, minimize cold

ischemic time): <1 hour

• Fixative: 10% neutral buffered formalin

(NBF)

• Minimum duration of fixation: 6 hours

• Must document fixation time points in

accession or report

• Laboratory quality assurance processes,

including proficiency testing and lab

accreditation

2013 HER2 Testing in BC Guidelines Update: Tumor Specimens to be Tested

10


2007 Guidelines1

• All primary breast cancer

specimens and metastases should

have at least one HER2 test

performed

• Perform HER2 testing on every

• Primary invasive tumor

• Metastatic sites

2013 Guideline Update2

2007 Guidelines

• Resection specimens preferred

sample for HER2 testing

• More representative sample of

the patient’s tumor, more

tumor tissue for evaluation

2013 Guideline update

• Increasing use of core for testing

• Core biopsies can be used for initial

test (likely better pre-analytics)

• However, repeat testing on the

excision may be necessary if a HER2

result is negative on the core in

certain circumstances

2013 HER2 Testing in BC Guidelines Update: Recommended Tumor Specimens to be Tested

2013 HER2 Testing in BC Guidelines Update: Tumor Specimen Selection

Core samples may not be optimal in some situations

Crushing and surface artifacts in cores may hamper interpretation

Tumor on resection may show morphologic heterogeneity

Tumor on resection may show intratumoral heterogeneity

Tissue is not fixed for adequate length of time

12

Heterogeneity HER2 IHC stain obtained

by core needle biopsy

Edge Artifact Crush Intratumoral heterogeneity

If core results are questionable, test the excision specimen

• Breast tissue undergoes ischemic changes from the minute it is

removed from a patient

• Enzymatic activity in the tissue is not stopped until fixation begins

(stabilizing tissue)

• Unabated enzymatic degradation of tissue may lead to false

results

– Critical antigens may be altered or lost.

– Test results may be indeterminate because of artifacts caused

by improper handling.

– Breast tissue should be incised and placed into an appropriate

fixative (10% NBF) within <1 hr from removal from the patient

13

Specimen Handling is Critical!

Tumor stained as ‘2+’ for HER2 at 0.5 h of delayed fixation (a), but

demonstrated reduction in staining at 3 h (b) and was completely negative

at 24 h (c) and 48 h (d). Yildiz-Aktas IZ, et al. Mod Pathol. 2012 Aug;25(8):1098-105.

Time to Fixation: HER2 Testing IHC and FISH

15 a, 30 min IHC; b, 30 min FISH; c, 4 h immunohistochemistry; d, 4 h FISH

HER2/CEP17 = 0.98

HER2/CEP17 = 0.29

Khoury T, et al., Mod Pathol. 2009 Nov;22(11):1457-67

30

min

ute

s 4

ho

urs

736 breast cancer specimens, the mean TTF was 53.8 min (SD=37.8min). 453 (61.5%) cases had an TTF less than 1 hour and 283 (38.5%) had TTF greater than an hour

2013 HER2 Testing Guidelines Updates: Duration of Fixation

17



• Time in fixative

• 6 – 72 hours

2007 Guidelines1

• Time in fixative

• 6 – 48 hours

Both the ASCO/CAP HER21 and ER/PgR3 Testing Guidelines now share the same recommendation for the duration of fixation

IHC 2+ with appropriate fixation time

IHC 0 after extended fixation time

Core require same fixation time as excisions

2013 HER2 Testing Guidelines Update: Summary of Changes to the Testing Algorithm

18 Wolff AC et al. J Clin Oncol. 2013; Oct 7. [Epub ahead of print]

HER2 Result

IHC ISH

Positive

•> 10% of invasive tumor cells display strong circumferential membrane staining

•Amplified ratio of HER2/CEP17 of ≥ 2.0 or average HER2 signals ≥6

signals/cell (regardless of ratio) in population of >10% of tumor cells

Negative

•IHC 0: No staining observed or membrane staining

that is incomplete, faint/barely perceptible and within ≤10% of the invasive tumor cells

•IHC 1+: Incomplete membrane staining that is faint/barely perceptible and within >10% of the invasive tumor cells

•HER2/CEP17 ratio < 2 and

HER2 signals/cell < 4, regardless of ratio

Equivocal Must reflex test

•2+ based on circumferential membrane staining, incomplete, weak, or moderate within >10% of the

invasive tumor cells; or complete & circumferential

membrane intense staining within ≤10% of the invasive tumor cells

•Dual Probe HER2/CEP17

ratio <2.0 with an average

HER2 copy number ≥4.0 and <6.0 signals/cell

Indeterminate

• Technical issues prevent assay from being conclusive (e.g., issues with controls, specimen handling, artifacts, or analytical failure)

• Assay must be repeated before diagnosis can be rendered

2013 HER2 Testing in BC Guidelines Update: IHC HER2 Positive Interpretation Criteria Redefined

19

HER2 (3+) in

10% of tumor

(HER2 Positive)

If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed.


*Readily appreciated at low power.

2007 Guidelines1

• Positive for HER2 is 3+ (defined as

uniform intense membrane staining

of > 30% of invasive tumor cells)

• Positive for HER2 is 3+ (defined as

uniform intense membrane staining

of >10% of invasive tumor cells*


HER2 (3+) in

>90% of tumor

(HER2 Positive)

2013 HER2 Testing in BC Guidelines Update: IHC HER2 Negative Interpretation Criteria

20

2007 Guidelines1

Negative result for HER2 IHC is 0 or 1+ • IHC 0: no staining

• IHC 1+: weak, incomplete

membrane staining in any

proportion of tumor cells or weak, complete staining in <10% of cells

Negative result for HER2 IHC is 0 or 1+

• IHC 0: No staining* or incomplete

membrane staining (faint/barely

perceptible) and within ≤ 10% of

tumor cells

• IHC 1+: Incomplete membrane

staining (faint/barely perceptible)

and within > 10% of tumor cells*



2013 HER2 Testing in BC Guidelines Update: HER2 Testing of the Invasive Component by IHC

21


*Readily appreciated at low power.

If the pathologist or oncologist observes an apparent histopathologic discordance after

HER2 testing, the need for additional HER2 testing should be discussed.

2007 Guidelines

Positive for HER2 is FISH amplified

(ratio of HER2 to CEP17 of > 2.2

or average HER2 gene copy

number > six signals/nucleus for

those test systems without an

internal control probe)


Positive for HER2 is amplified ratio of

HER2/CEP17 of ≥ 2.0 (with

average HER2 signals >4*) or if

average HER2 signals are ≥6

signals/cell (regardless of ratio) in

population of >10% of tumor cells.

*Patient’s with ratio of >2 where eligible

for the adjuvant trial, even if HER2< 4.

no good data on benefit

2013 HER2 Testing in BC Guidelines Update: ISH HER2 Positive Interpretation Criteria, Redefined

If the pathologist or

oncologist observes an

apparent histopathologic

discordance after HER2

testing, the need for

additional HER2 testing

should be discussed.

2013 HER2 Testing in BC Guidelines Update: ISH HER2 Negative Interpretation Criteria, Redefined

2007 Guidelines

Negative for HER2 ISH is FISH

HER2/CEP17 ratio of < 1.8 or

average HER2 gene copy

number of < 4 signals/nucleus

for test systems without an

internal control probe


Negative for HER2 ISH is

HER2/CEP17 ratio < 2 and

HER2 signals/cell < 4*,

regardless of ratio

*Patient’s with ratio of >2 where eligible for the

adjuvant trial, even if HER2< 4, however no

good data on benefit


2013 HER2 Testing in BC Guidelines Update: HER2 Testing of the Invasive Component by Dual Probe ISH

24


Look at ratio,

then HER2#


2013 HER2 Testing in BC Guidelines Update: HER2 Equivocal Results Require a Reflex or Repeat Test

2007 Guidelines

Equivocal for HER2 IHC is 2+

ISH: FISH HER2/CEP17 ratio of 1.8-

2.2 or average HER2 gene

copy number 4-6 HER2

signals/nucleus for test systems

without an internal control

probe


Must report HER2 test result as

Equivocal (HER2 tumor status

Unknown) and order reflex test

using the alternative test if:

IHC: (2+) circumferential membrane

staining, incomplete and/or weak/

moderate in >10% of the invasive

tumor cells; or complete and

circumferential membrane intense

staining within ≤10% of the invasive

tumor cells

ISH: Dual Probe HER2/CEP17 ratio <2.0

with an average HER2 copy

number ≥4.0 and <6.0 signals/cell

If a reflex test on a HER2 Equivocal result

does not render a (+) or (-) HER2 result, must

review clinical and pathologic features of case

and should confer with the oncologist about

additional testing

• New ISH algorithm and interpretive guideline address

unusual cases with:

o Intratumoral heterogeneity

o Chromosomal abnormalities involving CEP17

(aneusomy)

2013 HER2 Testing Guidelines Update: Changes to the Testing Algorithm Help Address:

Some illustrative cases

•New language on repeat HER2 testing

Case#1 History (Heterogeneity)

• 71 year old female

• Palpable mass lesion

• Left breast @ 11:00

• Core needle biopsy

performed

• ER: Negative

• PR: Negative

• HER2 IHC: Shown on

next slide

H&E Stain

HER2 Immunohistochemistry

~10-20% on invasive tumor IHC (3+)

Low Power Scan, HER2 FISH

Low power scan for HER2 IHC versus FISH

*observed in a homogeneous and

contiguous population of at least 10%

HER2 IHC versus FISH

HER2/CEP17 = 1.7

HER2/CEP17 = 7.5

HER2/CEP17 combined = 2.9

33

Reporting Consideration with Heterogeneity

• This case should be reported as HER2

positive, and the percentage of the invasive

tumor involved should be provided on the

report.

• Do not combine the ratios!

• Could result in an equivocal or even negative

result depending on which cells counted

• HER2 signals (and ratio) should be reported for both

the minority amplified (>10%) and the majority non-

amplified portions of the tumor.

Patterns of Intratumor

Heterogeneity for HER2

Mixed Histology

Homogeneous Histology

clusters of HER2+ cells vs scattered + cell

Intratumor Heterogeneity for HER2: IHC vs FISH

Mean score/ratio depends on the

number of amplified cells counted

and gene copy number after dilution

with the non-amplified cells

Fields selected for evaluation will

determine whether or not the tumor is

reported as amplified

Heterogeneity easier to detect by IHC

IHC can be used to target “hot-spots” for FISH analysis

Heterogeneity has implications for core versus excisional biopsies

H&E

Slides courtesy of Dr. Ken Bloom

Core needle biopsy 53 year old female

An Illustrative Case

HER2 IHC

Slides courtesy of Dr. Ken Bloom

An Illustrative Case (cont)

Clinical Significance of Heterogeneity???

• Perez EA, et al, Predictability of adjuvant trastuzumab benefit in N9831

patients using the ASCO/CAP HER2-positivity criteria. JNCI 2012 Jan

18;104(2):159-62

– 107 [3.7%] of 2904 patients on trial HER2 (3+) in 10-30% of tumor

10% threshold (FDA)

30% threshold (ASCO/CAP)

Conclusion:

Following the 2007

ASCO/CAP 30% criteria

for HER2 positivity would

potentially negate the

option of possible life-

saving trastuzumab

therapy for a small but

meaningful group of

patients.

http://jnci.oxfordjournals.org/content/104/2/159/F1.large.jpg

• Intratumoral heterogeneity for HER2 can be seen in breast cancer by IHC and FISH – Can lead to discordant results for HER2 analysis

• Between IHC and FISH, cores vs excision, between blocks

– Easier to detect with IHC (can be used to target FISH)

• Clinical significance of heterogeneity remains unclear however: – Patients with HER2 IHC 3+ (10-30%) and FISH ratio (2-2.2)

appear to benefit from treatment with HER2-targeted therapy

• ASCO/CAP updated HER2 guideline definition for HER2+ – HER2 IHC - >10% of tumor cells with strong circumferential

membrane staining (readily appreciated at low power) – HER2/CEP17 ratio of >2, observed in a homogenous

contiguous population of at least 10% of tumor cells

Pearls of Pathology: Heterogeneity

Case #2: Repeat Testing

• 50 year-old woman, core biopsy for mass, Jan 2011

• Diagnosis: Grade 3, triple negative invasive ductal carcinoma,

positive lymphovascular invasion, negative for DCIS

Case provided by

Stephen Naber, MD, PhD

Core Biopsy IHC Markers

Estrogen Receptor HER2 (Score 1+)

• Progesterone receptor negative

• Cold ischemic and fixation times within CAP/ASCO guidelines • HER2 FISH not performed at this time

Mastectomy and Lymphadenectomy

• Primary tumor: 0.8 cm, Grade 3, triple negative (September 2011)

• 8 of 13 lymph nodes positive for metastasis

Estrogen Receptor HER2 (Score 0)

• Cold ischemic and fixation times within CAP/ASCO guidelines

• HER2 FISH not performed at this time

Contralateral Axillary Node Biopsy

Estrogen Receptor HER2 (Score 1+)

• Core needle biopsy of contralateral axillary node (Jan 2012)

• Cold ischemic and fixation times within CAP/ASCO guidelines

•HER2 FISH assay ordered: • HER2:CEP17 = 2.6 at reference lab

• HER2:CEP17 = 2.7 at hospital lab

Follow-up HER2 FISH

• HER2 FISH then performed in-house on tumor from

mastectomy: HER2:CEP17 = 2.3

• Dual ISH for HER2 performed in-house on tumor from core biopsy:

Numerous HER2 amplified

cells present

Black signals = HER2

Red signals = CEP17

HER2-Negative (1+) Breast Cancer with Unfavorable

Prognostic Features: to FISH or not to FISH? (Viale 2012)

Concordance study IHC vs FISH for

HER2

IHC Score % of case amplified

by FISH

0 3.2%

1+ 6.7%

2+ 23.9%

3+ 89.3%

Overall Concordance, IHC vs FISH

82%

• Given possible association between HER2 positive disease and unfavorable prognostic tumor characteristics

• Conducted a prospective study to assess

the incidence of HER2 gene amplification in selected tumors with adverse prognostic features scored (1+) IHC

• 492 invasive breast cancer IHC (1+)

• 84 cases selected for FISH with one or more of the following histopathologic features

– High grade, high Ki67 (>14%), LVI, LN+

– 13% these cases amplified by FISH (confidence interval 7% to 22%)

– 2 fold higher than what was observed in previous study (6.7%)

• Authors recommended considering FISH for IHC (1+) cancers with adverse prognostic features

• A new HER2 test should be considered following a HER2 negative result, if the following suspicious pathologic findings are observed

– Tumor with a high nuclear grade or Nottingham score

• A new HER2 test should not be ordered if the following histopathologic findings occur and the initial HER2 test was negative:

– Histologic grade 1 carcinoma of the following types:

• Infiltrating ductal or lobular carcinoma, grade 1, ER and PgR positive

• Tubular (at least 90% pure)

• Mucinous (at least 90% pure)

• Cribriform (at least 90% pure)

• Adenoid cystic carcinoma (90% pure) and often triple negative

2013 HER2 Testing Guidelines Update:

Histopathologic Discordance and Repeat Testing


Pearls of Pathology: Repeat Testing

• Under what circumstances should you consider reflex testing an invasive breast cancer that is IHC HER2 (0) or (1+) by FISH?

– Cases with high risk, unfavorable histopathologic features

(increased likelihood of HER2+ disease)

• 50 years of age or less

• ER negative

• ER positive & PR low/negative

• High proliferative index (Ki-67 > 20%)

• Grade II or III

• Peritumoral lymphatic invasion

The yield will be low, however the magnitude of the benefit of Trastuzumab justifies dual

testing for those patients who are at increase risk of HER2+ disease. Patient’s who have a

primary (-) FISH test with these characteristic should be consider for reflex IHC testing

Clinical History Case 3: Aneusomy

• 68 year old male presents

with palpable right sided breast mass

• calcifications seen on

mammographic imaging

• US guided core needle biopsy performed

• Invasive ductal carcinoma

– Size: 2.5 cm

– Nottingham grade: 3 of 3

– LVI: Present

– Margins: Negative

– Axillary nodes: (7/31)

– ER: (+), 70% of cells, strong

– PR: (+), 5% of cells, moderate

– Ki67: 40% of cell positive

– HER2 IHC: (image on next slide)

• pT2 pN2, cMx

• Standardized tissue handling, fixation and processing was

followed

Right Breast, Mastectomy:

HER2 Immunohistochemistry

How would you interpret the HE2 IHC results? Are any further steps needed?

HER2 FISH Assay Results

HER2/CEP17 ratio 1.7

Total # of tumor cells

counted

40 + 40

Average # HER2

signals/nucleus

7.5

Average # CEP17

signals/nucleus

4.5

By ratio, this tumor would be interpreted as non-amplified. What is the

most appropriate interpretation of the HER2 status for this patient?

HER2 FISH

52

• A result like this will be confusing to clinicians (ratio <2, HER2#

>6 = amplified)

• Include a comment to help guide communication between

the clinician and patient.

– For example “Although the ratio is < 2, there are more than

6 HER2 gene signals per cell. The data indicates that this

case is HER2 amplified based on the HER2 copy number.”

Reporting Considerations – Case #3

Aneusomy of Chromosome 17

53

HER2 amplification defined by

Ratio criterion (≥2)

HER2 copy number criterion (≥6)

Both ratio and copy number

criteria

Co-amplification of CEP17 may

lead to HER2/CEP17 <2.0,

suggesting lack of HER2

amplification

If the HER2 copy number is ≥6, the

test is positive regardless of the

HER2/CEP17 ratio HER2 copy number 10.1

CEP17 copy number 7.4

HER2/CEP17 ratio 1.34

Some labs may choose to repeat HER2 testing in the same specimen and using an alternative chromosome 17 reference probe (another gene on chromosome 17 not expected to co-amplified with HER2)

Patient Communication Issues

54

• Clinicians or pathologists should understand and be able to

explain:

– Importance of tumor biologic characteristics

– Importance of HER2 testing

– Types of tissue used for HER2 testing

– Types of tests used for HER2 testing

– Various test results and their significance and requirements

for further action

– Importance of evaluating the HER2 status on each new

tumor (primary or metastasis)

– Value of guidelines being followed to assure accuracy

55

2007 Guidelines

• Initial test validation requires

25-100 samples tested by alternative validated method in the same laboratory or by validated method in another laboratory


• Initial test validation*: 20 negative and 20 positive for FDA-approved assays and 40 negative and 40 positive for LDTs

Note: Requirement does not apply to assays that were previously validated in conformance with the 2007 HER2 testing guideline, and routinely participating in external proficiency testing for HER2 tests, such as the program offered by CAP.

*Conforms to the published 2010 ASCO/CAP Validation Recommendations for

IHC Testing of ER and PgR Guideline (Fitzgibbons, Arch Pathol and Lab Med, 2010)

2013 HER2 Testing in BC Guidelines Update:

Initial Test Validation

2013 HER2 Testing in BC Guidelines Update: Guideline Update Addresses QA

• Specimen handling

• Lab validation

• Test Interpretation

and diagnosis

• Test reporting

requirements

• Proficiency testing

• Laboratory accreditation

56

QA elements will be specified and monitored.

Pearls of Pathology: Guidelines

• Guidelines are living document which change

– From user feedback

– From new publications and data

• Iteration of guidelines leads to greater clarity

• Algorithm changes in the guideline update provide better safety

for patients

– Positive patients will be found and treated

– Equivocal patients will have further work done to better define

their HER2 status

– Negative patients will be spared unnecessary treatment

– Close scrutiny of cases by physicians (oncologists &

pathologists) will find patients with unusual situations

57

Suggested Action Items for Labs Performing HER2 Testing

(Check-List)

Review your lab’s algorithms for HER2 testing and adjust them to

fit the new guideline.

Change length of fixation policies to 72 hours maximum.

Educate other pathologists at your location about the new

interpretation criteria, look at sample cases together.

Review the issues of polysomy, monosomy and tumor heterogeneity with other pathologists and oncologists

interpreting or acting on HER2 tests at your location.

Plan how ISH slides will be optimally handled and interpreted by pathologists

58

• ASCO Guideline Disclaimer:

o “Use of the information is voluntary.”

• CAP Guideline Disclaimer:

o “…adherence to any practice guideline…is

voluntary…”

• A CAP accreditation checklist requirement is NOT

voluntary

o Checklist requirements reflect essence of

guideline but may not exactly = guideline

Guidelines vs. CAP Checklist Requirements


CAP Checklist, in contrast to Guideline

• Requirements must be

o Practical for labs to implement

o Readily evaluable by inspector

o Consistent with FDA for cleared/approved tests

• A guideline element based purely on expert

opinion may not be appropriate as a checklist

requirement

• Some details in a guideline may not be appropriate

for checklist


o Some guideline elements may be gradually

introduced as checklist requirements

− Phase I, then phase II

− Progressive changes in wording (“consider”,

“should”, “must”)

CAP Checklist, in contrast with Guideline, cont.


• ANP.22969 (II) Information on report

o Fixation, antibody clone or probe, scoring system

• ANP.22970 (II) Annual result comparison with

published benchmarks, & interobserver variability

(95% concordance)

• ANP.22973 (II) Proficiency testing

o “Interpretation-only” PT: OK for IHC…NOT for

(F)ISH!

Checklist elements applicable to HER2


• ANP.22978 (II) Validation / CYG.48399 / MOL.39323

o REVISED

• ANP.22983 (II) Fixation / CYG.48932 / MOL.39358

o REVISED

• ANP.22985 (I) Decalcified tissue

Checklist elements applicable to HER2, cont.


• ANP.22999 (II) IHC scoring

o REVISED

• ANP.23002 (II) / CYG.49465 / MOL.39393 ISH/FISH

scoring

o REVISED

• NEW: Testing on primary and metastatic lesions

o Additional testing for histologic discordance /

limited samples

Checklist Elements Applicable to HER2, cont.


• ANP.22978 (II) / CYG.48399 / MOL.39323

o 2013 checklist edition: minimum 25 cases;

recommended 25-100.

o CHANGED in 2014 to:

− 20 + / 20 neg samples for FDA

cleared/approved tests

− 40 + / 40 neg samples for lab-developed tests

o No required concordance level of new test vs

comparative test

Analytic validation


• Data should show concordance between methods

for each possible result

o IHC: 0, 1+, 2+, 3+

o (F)ISH: +, neg, indeterminate (if applicable),

equivocal [new]

• Compare new test to validated alternative method

in same lab, or same validated method in another

lab, using same set of cases

• If fixative other than 10% NBF used, must validate

with NBF-fixed samples

Analytic validation detail – 2013 checklist edition,

unchanged for 2014, except as noted below


• For previous validations not meeting the current

requirements: data from previous PT samples may

be used; or unstained slides from recent cases may

be sent to reference laboratory

o Lab should document the additional data (PT or

reference laboratory studies)

Validation – 2013 checklist edition – no change


• ANP.22983 / MOL.39358 / CYG.48932 / 2013 edition

o Current: 6 – 48 hours

o CHANGED to 6 – 72 hours for 2014 (now = ER /

PgR fixation requirement)

o Lab should communicate fixation guidelines to

clinical personnel

− Record time of tissue removal / time of

immersion in formalin

o Lab should consider monitoring compliance &

contacting clients if these guidelines not met

Fixation


• ANP.22999

o Lab uses ASCO/CAP criteria or mfg’s instructions

o NEW: Laboratories using FDA approved/cleared

tests should follow interpretation criteria in mfg

instructions, unless the lab has performed a

validation study to allow use of the ASCO/CAP

criteria

o NEW: If using ASCO/CAP criteria, report indicates

year of guideline publication

IHC Scoring – additional wording for 2014 edition

as noted below


• Specific IHC scoring not in checklist, but guideline is

referenced:

o Current positive: > 30% invasive tumor cells

stained

o CHANGED to: positive is > 10% of invasive tumor

cells stained

o Intense, complete staining in >10% and <= 30% is

rare (as few as 0.15% of patients)

IHC scoring


• Equivocal…= 2+: complete weak or nonuniform

circumferential membrane staining in >= 10% of

cells; OR intense, complete staining in <= 30% of

cells Order F(ISH)

• Negative…= 0 and 1+, 0 = no staining; 1+ = weak,

incomplete membrane staining in any % of cells

IHC Scoring, 1st edition of Guideline


• Positive HER2 = 3+

o Circumferential, intense, complete membrane

staining in > 10% of invasive tumor cells

• Equivocal HER2 = 2+

o Incomplete and/or weak/moderate

circumferential staining in >10 % of cells OR

o Complete, intense, circumferential staining in

<= 10% of cells

o Order (F)ISH

New Guideline: IHC Scoring (Referenced in

Checklist)


• Negative HER2: = 1+ or 0

o 1+: faint, incomplete membrane staining in >10%

of cells

o 0: no staining, or faint, incomplete membrane

staining in <= 10% of cells

New Guideline, IHC Scoring, cont.


• ANP.23002 / CYG.49465 / MOL.39393

o Current, with internal control probe:

− + HER2/CEP17 > 2.2, neg HER2/CEP17 < 1.8

− Equivocal: ratio 1.8 – 2.2

o Current, no internal control probe

− +: > 6 signals/nucleus; neg: < 4 signals/nucleus

− Equivocal: 4-6 signals/nucleus

ISH scoring…current 2013 checklist


• Positive: HER2/CEP17 >= 2.0 or average HER2 copy

number >= 6.0 signals / cell

• Negative:

o Dual probe: HER2/CEP17 < 2.0 and average HER2

copy number < 4.0 signals / cell

o Single probe: avg. <4.0 signals/cell

• Equivocal:

o Dual probe: HER2/CEP17 < 2.0 with avg. HER2

copy no. >= 4.0 and < 6.0 signals / cell

o Single probe: avg. copy no. >=4.0 and < 6.0

REVISED ISH scoring for 2014 (in checklists ANP,

CYG, MOL):


• ADDITION: Variable positivity (tumor heterogeneity)

in (F)ISH testing

o Scan ISH slides at low power

o If there is subpopulation of >=10% of tumor cells

with amplified HER2: report case as HER2-positive

o Count at least 20 cells in the subpopulation

• No change: For FDA cleared/approved tests

without an equivocal category, follow mfg.

instructions

Revised ISH Scoring (in ANP, CYG, MOL), cont.:


• At least one tumor sample from all patients (early-

stage, recurrent or metastatic disease) is tested for

either HER2 protein expression (IHC) or HER2 gene

expression ([F]ISH)) using a validated HER2 test if

tissue is available.

NEW checklist element for 2014 – Phase I


• Repeat HER2 test for:

o Histologic discordance (ex., + test, Gr I ER+/Pgr+

tumor)

o Core biopsy—repeat test on excisional spec. if:

− Negative test with limited amount of tumor in

core

− Equivocal test by IHC and ISH

Note to NEW checklist element for 2014 – Phase I,

cont.


• “Practical Issues in Surgical Pathology that Enhance

Ancillary Molecular Testing”

o January 23 at 11 am Central

o Presented by John Pfeifer, MD, PhD, FCAP

• “Molecular and Diagnosis of Respiratory Viruses”

o February 27 at 10 am Central

o Presented by Frederick L Kiechle MD, PhD, FCAP

View all past webinars by going to www.cap.org/webinars

Upcoming Free Webinars


http://www.cap.org/webinars

• Pathology SPECs are:

o short PowerPoint presentations, created for pathologists, focused on

selected diseases where molecular tests play a key role in patient

management.

o valuable resource for your discussions with Tumor Boards or other

physician colleagues.

• Now Available:

― Emerging Concepts in the Diagnosis of Respiratory Viruses (NEW)

― Emerging Concepts in Molecular Testing in Breast Cancer (NEW)

― Emerging Concepts in the Workup of Colorectal Cancer

― Emerging Concepts in Therapeutic Guidance for Metastatic Melanoma

― Emerging Concepts in the Diagnosis and Workup of Thyroid Cancer

― Emerging Concepts in Colorectal Cancer Hereditary Non-Polyposis

Cancer (Lynch Syndrome)

― Emerging Concepts in the Workup of Polycythemia and

Thrombocythemia: JAK2

To register, go to the CAP Member tab on cap.org 80

Short Presentations on Emerging Concepts

(SPECS)

https://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=membership


The CAP has created the Pathology Resource Guides, a tool

(updated Oct 2013) to assist pathologists in understanding key

emerging technologies. These Resource Guides are a CAP member

benefit available at no charge.

Molecular Pathology (single gene, small panel)

Genomic Analysis (large panels, exome, genome)

Digital Pathology

In Vivo Microscopy

Register through the CAP member tab. Once registered, you will be

notified when a new issue is released.

Questions? Contact [email protected].

CAP’s Pathology Resource Guides

81 © 2013 College of American Pathologists. All rights reserved.



CAP Learning – HER2 Revision by ASCO and CAP

82

Course Learning Objectives

ASCO/CAP HER2 Guidelines

CE/CME/SAM – 0.0

Recommendations in the 2007 ASCO/CAP Guideline for HER2 Testing have

been reconciled with the April 2010 ASCO/CAP ER PgR Guideline so that

cancer specimens will be handled in a uniform manner for ER, PgR and HER2 in

breast cancer specimens.

Genomic Aberrations in Salivary

Duct Carcinoma Arising in

Warthin Tumor of Parotid Gland:

DNA Microarray and HER2

Fluorescence In Situ Hybridization

CE/CME/SAM – 0.0

Carcinoma arising from Warthin tumor is extremely rare. A 79-year-old man was

admitted for a firm, well-defined, 5-cm left infra-auricular mass. Aspiration

cytology showed many lymphohistiocytes and oncocytes in a proteinaceous

background, compatible with Warthin tumor. A left superficial parotidectomy

showed a solid mass around the cyst wall. The tumor cells of the solid area were

arranged as infiltrative ducts with a few foci of malignant transformation. Virtual

karyotyping disclosed a complex pattern of genetic aberrations with a focal

amplification in 12q14–q21.2. This chromosomal region contains the MDM2

(murine double minute) gene, which regulates p53 inactivation. HER2

fluorescence in situ hybridization showed a focal amplification. Subsequently,

the patient underwent total parotidectomy and ipsilateral neck dissection for a

recurrence. To our knowledge, this is the first case of salivary duct carcinoma

arising from Warthin tumor. The essential molecular pathway has not been

reported, we presume an important role of MDM2 amplification–P53

inactivation.

© 2013 College of American Pathologists. All rights reserved.

CAP Learning – HER2 Revision by ASCO and CAP

83

Course Learning Objectives

Herceptin HER2+ Breast Cancer

Treatment Information

CE/CME/SAM – 0.0

Herceptin is approved for the treatment of early-stage breast cancer that is

Human Epidermal growth factor Receptor 2-positive (HER2+) and has spread

into the lymph nodes, or is HER2+ and has not spread into the lymph nodes. If it

has not spread into the lymph nodes, the cancer needs to be estrogen

receptor/progesterone receptor (ER/PR)-negative or have one high risk feature.

Standardized Assessment of the

HER2 Status in Breast Cancer by

Immunohistochemistry

CE/CME/SAM – 0.0

Immunohistochemistry (IHC) is widely used in surgical pathology, but it has been

plagued by problems with reproducibility and lack of standardization resulting

in poor concordance between laboratories. In particular, inaccuracy of routine

human epidermal growth factor receptor 2 (HER2) testing in breast cancer

patients has been a major issue. In 2006 this led the American Society of

Clinical Oncologists (ASCO) and College of American Pathologists (CAP) to

charge an expert panel with developing recommendations for HER2 testing.

After subsequent publication and adoption of these guideline

recommendations through dissemination of best practices, variation in clinical

practice is expected to diminish and result in improved accuracy.

In this article, we review the role of genomic HER2 alterations in the

development and treatment of breast cancer, highlight the importance of

accurate and reproducible HER2 testing, and discuss practical approaches to

standardize HER2 testing by IHC. Pre-analytic and analytic variables are

addressed, and a practical algorithm for test interpretation is introduced.


CAP Learning Portal

84

CAP Learning Portal

• The CAP Learning Portal includes content and tools designed to support the learning needs of pathologists . A user must login to cap.org in order to access the portal. In the portal, you will find: o Learning Options search/catalog

o Competency Model for Pathologists

o Personal Progress Check

o My Learning Plan

o Help Center (Guides, Video, FAQs)

• Benefits Increase effectiveness to plan and manage learning

Increase efficiency to target learning needs and identify premium learning solutions Increase satisfaction with learning solutions that meet specific learner needs Increase capability to maintain professional certifications


To learn more…

85

• For more details and to register for/access educational offerings:

1. Log in to the cap.org website

2. Click on the “Learning Portal” tab.

3. Click on the “Browse Our Learning Catalog” tab

4. Type your desired topic in the “Search” box or make a selection from the list provided.

A list of available learning options displays



THANK YOU!

Thank you for attending our webinar

““HER2 Testing Revision” by David G. Hicks, MD, FCAP and

Stephen J. Sarewitz, MD, FCAP

For comments about this webinar

or suggestions for upcoming

webinars, please contact

Jill Kaufman, PhD,

Director of Personalized Health Care at [email protected]

NOTE: There is no CME/CE credit available for

today’s free webinar.

88


Documents

Welcome to CAP’s “Hot Topics in Pathology” Webinar Series ... · 453 (61.5%) cases had an TTF less than 1 hour and 283 (38.5%) had TTF greater than an hour . 2013 HER2 Testing