Template for Reporting Results of Biomarker Testing of Specimens

Template for Reporting Results of Biomarker Testing of

Specimens from Patients with Carcinoma of the Breast

Version: 1.5.0.1

Protocol Posting Date: March 2023

This biomarker template is not required for accreditation purposes but may be used to facilitate

compliance with CAP Accreditation Program Requirements

Authors

Patrick L. Fitzgibbons, MD, FCAP*; James L. Connolly, MD*.

With guidance from the CAP Cancer and CAP Pathology Electronic Reporting Committees.

* Denotes primary author.

Accreditation Requirements

Completion of the template is the responsibility of the laboratory performing the biomarker testing and/or

providing the interpretation. When both testing and interpretation are performed elsewhere (eg, a

reference laboratory), synoptic reporting of the results by the laboratory submitting the tissue for testing is

also encouraged to ensure that all information is included in the patient’s medical record and thus readily

available to the treating clinical team. This template is not required for accreditation purposes.

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Summary of Changes

v 1.5.0.1

• Added a display item to clarify HER2 Low score answers

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Reporting Template

Protocol Posting Date: March 2023

Select a single response unless otherwise indicated.

CASE SUMMARY: (Breast Biomarker Reporting Template)

Includes interpretative content from the CAP/ASCO HER2 Guidelines (2018)

Completion of the template is the responsibility of the laboratory performing the biomarker testing and / or providing the

interpretation. When both testing and interpretation are performed elsewhere (e.g., a reference laboratory), synoptic reporting of the

results by the laboratory submitting the tissue for testing is also encouraged to ensure that all information is included in the patient's

medical record and thus readily available to the treating clinical team.

Core data elements in this template comply with the CAP Accreditation requirements for HER2 and hormone receptor testing. Core

data elements should be reported only for tests performed. If some studies were performed on different specimen(s), the specimen

number(s) should be provided.

TEST(S) PERFORMED

Test(s) Performed (Note A

) (select all that apply)

___ Estrogen Receptor (ER) Status (Note B)

Estrogen Receptor (ER) Status

___ Positive (greater than 10% of cells demonstrate nuclear positivity)#

Percentage of Cells with Nuclear Positivity#

___ Specify %: _________________ %

--OR--

Select range below:

___ 11-20%

___ 21-30%

___ 31-40%

___ 41-50%

___ 51-60%

___ 61-70%

___ 71-80%

___ 81-90%

___ 91-100%

Average Intensity of Staining

___ Weak

___ Moderate

___ Strong

___ Low Positive (1-10% of cells with nuclear positivity)##

+Specify Percentage of Cells with Nuclear Positivity: _________________ %

Average Intensity of Staining

___ Weak

___ Moderate

___ Strong

Status of Internal Controls

___ Internal control cells present and stain as expected

___ Internal control cells absent###

___ Other (specify): _________________

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___ Negative (less than 1%)

___ Internal control cells present and stain as expected

___ Internal control cells absent###

___ Other (specify): _________________

___ Cannot be determined (indeterminate)####

___ Internal control cells present; no immunoreactivity of either tumor cells or internal controls

___ Other (specify): _________________

# Percentage of cells with nuclear positivity for ER may be reported as a specific number or a range if more than

10%.

## Invasive carcinoma cases with 1 to 10% of cells staining for ER (not PgR) are reported as “Low Positive” and

the following report comment is recommended: “The cancer in this sample has a low level (1-10%) of ER

expression by IHC. There are limited data on the overall benefit of endocrine therapies for patients with low level

(1-10%) ER expression but they currently suggest possible benefit, so patients are considered eligible for

endocrine treatment. There are data that suggest invasive cancers with these results are heterogeneous in both

behavior and biology and often have gene expression profiles more similar to ER negative cancers.” The Low

Positive designation applies only to invasive carcinoma, and is not used for Progesterone receptor or DCIS.

### For cases in which no internal controls are present and the ER result is either negative or Low Positive, the

following report comment is recommended: “No internal controls are present, but external controls are

appropriately positive. If needed, testing another specimen that contains internal controls may be warranted for

confirmation of ER status.” When a tumor is negative but no internal control cells are present, the pathologist must

exercise judgment as to whether the assay can be interpreted as a true negative. This should include

consideration of histologic type and grade, cold ischemia and fixation times, and the status of external controls. If

the pathologist decides that hormone receptor status cannot be determined, the test should be reported as such

and repeated on another block or specimen.

#### Technical issues prevent the test from being reported as positive, negative, or equivocal. This may occur if

specimen handling was inadequate, if artifacts (crush or edge artifacts) make interpretation difficult, or if the

analytic testing failed.

Test Type

___ Food and Drug Administration (FDA) cleared (specify test / vendor): _________________

___ Laboratory-developed test

+___ Non-U.S.-based health systems

+___ Health Canada Approved (specify test / vendor): _________________

+___ Other (specify): _________________

Primary Antibody

___ SP1

___ 6F11

___ 1D5

___ Other (specify): _________________

+Scoring System

___ No separate scoring system used

___ Allred

+Proportion Score: _________________

+Intensity Score: _________________

+Total Allred Score: _________________

___ Other scoring system

+Specify System: _________________

+Specify Score Result: _________________

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___ Progesterone Receptor (PgR) Status (Note B

)

Progesterone Receptor (PgR) Status

___ Positive#

Percentage of Cells with Nuclear Positivity

___ Specify %: _________________ %

--OR--

Select range below:

___ 1-10% (specify)#: _________________ %

___ 11-20%

___ 21-30%

___ 31-40%

___ 41-50%

___ 51-60%

___ 61-70%

___ 71-80%

___ 81-90%

___ 91-100%

Average Intensity of Staining

___ Weak

___ Moderate

___ Strong

___ Negative (less than 1%)

___ Internal control cells present and stain as expected

___ Internal control cells absent##

___ Other (specify): _________________

___ Cannot be determined (indeterminate)###

___ Internal control cells present; no immunoreactivity of either tumor cells or internal controls

___ Other (specify): _________________

# Percentage of cells with nuclear positivity may be reported as a specific number or a range if more than 10%.

## When a tumor is negative but no internal control cells are present, the pathologist must exercise judgment as to

whether the assay can be interpreted as a true negative. This should include consideration of histologic type and

grade, cold ischemia and fixation times, and the status of external controls. If the pathologist decides that hormone

receptor status cannot be determined, the test should be reported as such and repeated on another block or

specimen.

### Technical issues prevent the test from being reported as positive, negative, or equivocal. This may occur if

specimen handling was inadequate, if artifacts (crush or edge artifacts) make interpretation difficult, or if the

analytic testing failed.

Test Type

___ Food and Drug Administration (FDA) cleared (specify test / vendor): _________________

___ Laboratory-developed test

+___ Non-U.S.-based health systems

+___ Health Canada Approved (specify test / vendor): _________________

+___ Other (specify): _________________

Primary Antibody

___ 1E2

___ 636

___ 16

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___ SP2

___ 1A6

___ 1294

___ 312

___ Other (specify): _________________

+Scoring System

___ No separate scoring system used

___ Allred

+Proportion Score: _________________

+Intensity Score: _________________

+Total Allred Score: _________________

___ Other scoring system

+Specify System: _________________

+Specify Score Result: _________________

___ HER2 by Immunohistochemistry (Note C

)

HER2 by Immunohistochemistry

___ Negative (Score 0)

# Breast cancers with HER2 IHC score 1+ or HER2 IHC score 2+ and a negative ISH result are eligible for clinically appropriate

HER2-targeted therapy and may be reported as “HER2 Low”.

___ Negative (Score 1+)#

___ Equivocal (Score 2+)#

Percentage of Cells with Uniform Intense Complete Membrane Staining

___ Specify percentage: _________________ %

___ Other (specify): _________________

___ Cannot be determined

___ Positive (Score 3+)

Percentage of Cells with Uniform Intense Complete Membrane Staining

___ Specify percentage: _________________ %

___ Other (specify): _________________

___ Cannot be determined

___ Cannot be determined (indeterminate) (explain): _________________

Test Type

___ Food and Drug Administration (FDA) cleared (specify test / vendor): _________________

___ Laboratory-developed test

+___ Non-U.S.-based health systems

+___ Health Canada Approved (specify test / vendor): _________________

+___ Other (specify): _________________

Primary Antibody

___ 4B5

___ HercepTest

___ A0485

___ SP3

___ CB11

___ Other (specify): _________________

___ HER2 by in situ Hybridization (Note C

)

HER2 by in situ Hybridization

"Number of Observers" and "Number of Invasive Tumor Cells Counted" are required only when Negative or Positive is selected.

___ Negative (not amplified)

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___ Positive (amplified)

___ Cannot be determined (indeterminate) (explain): _________________

___ Not performed

___ Pending

Number of Observers (required only if applicable): _________________

Number of Invasive Tumor Cells Counted (required only if applicable): _________________ cells

Method (required only if applicable) (select all that apply)

___ Not applicable (not performed)

___ Dual probe assay

Average Number of HER2 Signals per Cell: _________________

Average Number of CEP17 Signals per Cell: _________________

HER2 / CEP17 Ratio: _________________

___ Single probe assay

Average Number of HER2 Signals per Cell: _________________

+Aneusomy (as defined by vendor kit used)

___ Not identified

___ Present (explain): _________________

+Heterogeneous Signals

___ Not identified

___ Present

+Percentage of Cells with Amplified HER2 Signals

___ Specify percentage: _________________ %

___ Other (specify): _________________

___ Cannot be determined

Test Type

___ Food and Drug Administration (FDA) cleared (specify test / vendor): _________________

___ Laboratory-developed test

+___ Non-U.S.-based health systems

+___ Health Canada Approved (specify test / vendor): _________________

+___ Other (specify): _________________

___ Ki-67 (Note D

)

Ki-67 Percentage of Positive Nuclei: _________________ %

+Primary Antibody

___ MIB1

___ SP6

___ MM1

___ 30-9

___ IR / IS626

___ Other (specify): _________________

Cold Ischemia and Fixation Times

___ Meet requirements specified in latest version of the ASCO / CAP Guidelines

___ Do not meet requirements specified in latest version of the ASCO / CAP Guidelines

___ Cannot be determined (explain): _________________

+Cold Ischemia Time (minutes): _________________ min

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+Fixation Time (hours): _________________ hours

+Testing Performed on Block Number(s) (specify): _________________

METHODS

+Fixative

___ Formalin

___ Other (specify): _________________

+Image Analysis

___ Not performed

___ Performed

+Specify Method: _________________

+Biomarkers Scored by Image Analysis (select all that apply)

___ ER

___ PgR

___ HER2 by IHC

___ HER2 by ISH

___ Ki-67

___ Other (specify): _________________

COMMENTS

Comment(s): _________________

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Explanatory Notes

A. Results

It is recommended that hormone receptor and HER2 testing be done on all primary invasive breast

carcinomas and on recurrent or metastatic tumors.

1,2,3,4

If hormone receptors and HER2 are both negative

on a core biopsy, repeat testing on a subsequent specimen should be considered, particularly when the

results are discordant with the histopathologic findings. When multiple invasive foci are present, the

largest invasive focus should be tested. Testing smaller invasive carcinomas is also recommended if they

are of different histologic type or higher grade. Other biomarker tests (e.g., Ki-67 or multigene expression

assays) are optional and are not currently recommended for all carcinomas. Fresh tissue should not be

used for special studies (e.g., RNA expression profiling or investigational studies) unless the invasive

carcinoma is of sufficient size that histologic evaluation and ER, PgR, and HER2 assessment will not be

compromised.

Guidelines published by the American Society of Clinical Oncology (ASCO) and the College of American

Pathologists (CAP) require recording specific pre-analytic and analytic variables that can affect test

results.

5,6

Such variables include:

• Cold ischemia time (time between tissue removal and initiation of fixation) and time of fixation.

Alternatively, laboratories may record the time the specimen was removed from the patient and

the time the specimen was placed in formalin. Both the time the tissue is removed from the

patient and the time it is placed in fixative must be communicated to the processing laboratory.

These times are used to determine if the specimen meets requirements specified in latest version

of the ASCO/CAP guidelines for cold ischemia time and fixation time. Reporting these times in

the pathology report is optional.

• Type of fixative, if other than buffered formalin

• Treatment of the tissue that could potentially alter immunoreactivity (e.g., decalcification)

• Status of controls:

o Internal – normal epithelial cells positive or negative for ER and PgR

o External – type and expected level of expression

• Adequacy of sample for evaluation

• Primary antibody clone

• Regulatory status (FDA cleared versus laboratory-developed test)

Information regarding assay validation or verification should be available in the laboratory. Any

deviation(s) from the laboratory’s validated methods should be recorded. Appropriate positive and

negative controls should be used and evaluated.

References

1. National Comprehensive Cancer Network (NCCN) Clinical Practice Guideline in Oncology,

Version 3.2017. www.nccn.org/professionals/physician_gls/PDF/breast.pdf

. Accessed December

18, 2017.

2. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of

recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007;25(33):1-26.

3. Pusztai L, Viale G, Kelly CM, Hudis CA. Estrogen and HER-2 receptor discordance between

primary breast cancer and metastasis. Oncologist. 2010;15(11):1164-1168.

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4. Arslan C, Sari E, Aksoy S, Altundag K. Variation in hormone receptor and HER-2 status between

primary and metastatic breast cancer: review of the literature. Expert Opin Ther Targets.

2011;15(1):21-30.

5. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and progesterone receptor testing in

breast cancer: ASCO/CAP guideline update. Arch Pathol Lab Med doi: 10.5858/arpa.2019-0904-

SA.

6. Wolff AC, Hammond MEH, Allison KH, et al. HER2 testing in breast cancer: American Society of

Clinical Oncology/College of American Pathologists clinical practice guideline focused update.

Arch Pathol Lab Med. 2018;142(11):1364-1382.

7. Arber JM, Arber DA, Jenkins KA, Battifora H. Effect of decalcification and fixation in paraffin-

section immunohistochemistry. Appl Immunohistochem. 1996;4:241-248.

B. Estrogen Receptor and Progesterone Receptor Testing

Scientific rationale: Normal breast epithelial cells have receptors for estrogen and progesterone and

proliferate under their influence. Most breast carcinomas also express these receptors and may be

stimulated to grow by these hormones. Removal of endogenous hormones by oophorectomy or blocking

hormonal action pharmaceutically (e.g., with tamoxifen or aromatase inhibitors) can slow or prevent tumor

growth and prolong survival.

Clinical rationale: Hormone receptor status is determined primarily to identify patients who may benefit

from hormonal therapy.

About 75% to 80% of invasive breast cancers are positive for ER and PgR,

including almost all well-differentiated cancers and most moderately differentiated cancers, and studies

have shown a substantial survival benefit from endocrine therapy among patients with ER-positive

tumors.

True ER-negative, PgR-positive carcinomas are extremely rare, but patients with such tumors

are also considered eligible for hormonal therapy. Receptor status is only a weak prognostic factor.

Method: Hormone receptor status is most often determined in formalin-fixed, paraffin-embedded tissue

sections by immunohistochemistry (IHC). Only nuclear staining is considered positive. Use of single-gene

expression assays are not recommended for routine use.

Quality assurance: There are many tissue and technical variables that can affect test results,

2,3,4,5

and the

assays must be validated to ensure their accuracy.

External proficiency testing surveys for ER and PgR

are invaluable tools to help ensure that assays perform as expected, and they are available from the CAP

and other organizations.

False-negative results: Failure to detect ER or PgR is the greatest problem with this assay because

patients may not receive effective therapy. This may occur if specimen handling was inadequate, if

artifacts (crush or edge artifacts) make interpretation difficult, or if the analytic testing failed. To avoid

false-negative results, appropriate internal and external controls should be positive. When a tumor is

negative (non-immunoreactive), it is essential that the internal control cells be assessed to ensure that

they show positive staining (as expected). If the internal controls are also negative, the test should not be

reported as negative but should be considered indeterminate (“Cannot be determined”).

The test should

be repeated on another block or specimen.

When a tumor is negative but no internal control cells are present in the test section, the pathologist must

exercise judgment as to whether the assay can be interpreted as a true negative. This should include

consideration of histologic type and grade, cold ischemia and fixation times, and the status of external

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controls. If the pathologist decides that hormone receptor status cannot be determined, the test should be

reported as such and repeated or performed on another block or specimen.

Reasons for false-negative results include the following:

• Exposure of tumor cells to heat (eg, carcinomas transected by using cautery during surgery)

• Prolonged cold ischemic time, which may result in antigenic degradation. One hour or less is

preferable

7,8

• Under or overfixation; fixation for at least 6 hours in buffered formalin is recommended,

and

prolonged fixation can also diminish immunoreactivity

5,9

• Type of fixative: ER is degraded in acidic fixatives such as Bouin’s and B-5; formalin should be

buffered to ensure pH range between 7.0 and 7.4

• Decalcification, which may result in loss of immunoreactivity

• Nonoptimized antigen retrieval

• Type of antibody

• Dark hematoxylin counterstain obscuring faintly positive diaminobenzidine (DAB) staining

False-positive results: False-positive results occur less frequently.

Rare reasons would be the use of an

impure antibody that cross-reacts with another antigen or misinterpretation of entrapped normal cells or

an in situ component as invasive carcinoma. False-positive tests can also be generated by image

analysis devices that mistakenly count overstained nuclei. It has been suggested that highly sensitive

assays may detect very low levels of ER in cancers that will not respond to hormonal therapy, but that

has not been proven by a clinical trial.

False-negative and false-positive results can be reduced by paying attention to the following:

• Staining of normal breast epithelial cells. Normal epithelial cells serve as a positive internal

control and should always be assessed. If the normal cells are negative, repeat studies on the

same specimen or on a different specimen should be considered. If normal cells are not present

(eg, core biopsy) and the test results are negative, testing should be repeated on another block or

subsequent specimen.

• External controls (must stain as expected). These controls help ensure that the reagents have

been appropriately dispensed onto the slide with the clinical sample.

• Correlation with histologic type and grade of the cancer. The study should be repeated if the

results are discordant (e.g., ER-negative low-grade carcinoma).

Reporting guidelines: ASCO and the CAP have issued recommendations for reporting the results of

immunohistochemical assays for ER and PgR (Table 1).

Studies using both IHC and the ligand binding

assay suggest that patients with higher hormone receptor levels have a higher probability of response to

hormonal therapy, but expression as low as 1% positive staining has been associated with clinical

response. As a result, the guidelines recommend classifying all cases with at least 1% positive cells as

receptor positive.

For patients with low ER expression (1% to 10% weakly positive cells), the decision on

endocrine therapy should be based on an analysis of its risks and potential benefits.

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Table 1. Reporting Results of Estrogen Receptor (ER) and Progesterone Receptor (PgR) Testing

Result

Criteria

Comments

Positive

Immunoreactive

tumor cells present

(≥1%)

Invasive carcinomas with 1 to 10% of cells staining for ER (not PgR) are reported

as “Low Positive” and the following report comment is recommended:

“The cancer in this sample has a low level (1-10%) of ER expression by

IHC. There are limited data on the overall benefit of endocrine therapies

for patients with low level (1-

10%) ER expression but they currently

suggest possible benefit, so patients are considered eligible for endocrine

treatment. There are data that suggest invasive cancers with these

results are heterogeneous in both behavior and biology and often have

gene expression profiles more similar to ER negative cancers.”

The Low Positive designation applies only to invasive carcinoma, and is not used

for Progesterone receptor or DCIS.

Negative

<1%

immunoreactive

tumor cells present

Definition of a negative result: The ASCO/CAP guidelines recommend that carcinomas with <1% positive

cells be considered negative for ER and PgR.

In the Allred system (see Table 2), the survival of patients

whose carcinomas had a score of 2 (corresponding to <1% weakly positive cells) was similar to that of

patients whose carcinomas were completely negative for ER.

Therefore, a score of 2 was considered to

be a negative result. Carcinomas with <1% positive cells and intensity scores of 2 or 3 would have a total

score of 3 or 4 and be considered positive. These are rare carcinomas, and their response to hormonal

therapy has not been specifically studied.

Quantification of ER and PgR: There is a wide range of receptor levels in cancers as shown by the

biochemical ligand binding assay and as observed with IHC. Patients whose carcinomas have higher

levels have improved survival when treated with hormonal therapy.

9,11

Quantification systems may use

only the proportion of positive cells or may include the intensity of immunoreactivity:

• Number of positive cells: The number of positive cells can be reported as a percentage or within

discrete categories (figure 1 below).

• Intensity: Refers to degree of nuclear positivity (i.e., pale to dark). The intensity can be affected

by the amount of protein present, as well as the antibody used and the antigen retrieval system.

In most cancers, there is heterogeneous immunoreactivity with pale to darkly positive cells

present.

Figure 1. Quantification of Immunohistochemical Findings. The percentage of positive cells can be visually

estimated.

Two methods of quantifying ER by using both intensity and percentage of positive cells are the Allred

score

(Table 2) and the H score

(Table 3). The 2 systems classify carcinomas into similar, but not

identical, groups.

If high-affinity antibodies are used with sensitive detection systems, most carcinomas

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will fall into clearly positive (score 7 or 8) or clearly negative (score 0) categories by Allred score.

14,15

small group of carcinomas (<1% of total) show intermediate levels of immunoreactivity.

Quantitation can also be performed by using the proportion of positive cells. In one study, carcinomas

were scored as 0 (<1% positive), 1 (1% to 25% positive), 2 (>25% to 75% positive), and 3 (>75%

positive).

The same results were obtained when scored by visual analysis or by image analysis. The

proportion of positive cells correlated with the results of the biochemical assay and with prognosis. In

another study, carcinomas with small numbers of positive cells (between 1% and 10%) had a prognosis

between cancers with no or rare positive cells (<1%) and cancers with >10% positive cells.

Table 2. Allred Score* for Estrogen and Progesterone Receptor Evaluation

Proportion Score

Positive Cells, %

Intensity

Intensity Score

None

Weak

1 to 10

Intermediate

11 to 33

Strong

34 to 66

≥67

* The Allred score combines the percentage of positive cells and the intensity of the reaction product in most of the

carcinoma.

The 2 scores are added together for a final score with 8 possible values. Scores of 0 and 2 are

considered negative. Scores of 3 to 8 are considered positive.

Table 3. H Score* for Estrogen and Progesterone Receptor Evaluation

Calculation of H Score

Cell Signal

Percentage of Cells

Value Multiplied

Cells with no signal

% x 0 = 0

Cells with weak signal

% x 1 =

Cells with moderate signal

% x 2 =

Cells with strong signal

% x 3 =

Total score =

* The H score is determined by multiplying the percentage of cells demonstrating each intensity (scored from 0 to 3)

and adding the results.

There are 300 possible values. In this system, <1% positive cells is considered to be a

negative result.

References

1. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of

recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007;25(33):1-26.

2. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and progesterone receptor testing in

breast cancer: ASCO/CAP guideline update. Arch Pathol Lab Med doi: 10.5858/arpa.2019-0904-

SA.

3. Yaziji H, Taylor CR, Goldstein NS, et al. Consensus recommendations on estrogen receptor

testing in breast cancer by immunohistochemistry. Appl Immunohistochem Mol Morphol.

2008;16(6):513-520.

4. Allred DC. Problems and solutions in the evaluation of hormone receptors in breast cancer. J Clin

Oncol. 2008;26(15):2433-2435.

5. Arber DA. Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast

markers. Appl Immunohistochem Mol Morphol. 2002;10(2):183-186.

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6. Fitzgibbons PL, Murphy DA, Hammond EH, Allred DC, Valenstein PN. Recommendations for

validating estrogen and progesterone receptor Immunohistochemistry assays. Arch Pathol Lab

Med. 2010;134(6):930-935.

7. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R. The effect of cold ischemic time on the

immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2

expression in invasive breast carcinoma. Mod Pathol. 2012;25(8):1098-1105.

8. Neumeister VM, Anagnostou V, Siddiqui S, et al. Quantitative assessment of effect of preanalytic

cold ischemic time on protein expression in breast cancer tissues. J Natl Cancer Inst.

2012;104(23)1815-1824.

9. Harvey JM, Clark GM, Osborne CK, et al. Estrogen receptor status by immunohistochemistry is

superior to the ligand binding assay for predicting response to adjuvant endocrine therapy in

breast cancer. J Clin Oncol. 1999;17(5):1474-1481.

10. Arber JM, Arber DA, Jenkins KA, Battifora H. Effect of decalcification and fixation in paraffin-

section immunohistochemistry. Appl Immunohistochem. 1996;4:241-248.

11. Viale G, Regan MM, Maiorano E, et al. Prognostic and predictive value of centrally reviewed

expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and

tamoxifen adjuvant therapy for postmenopausal early breast cancer: BIG 1-98. J Clin Oncol.

2007:25(25):3846-3852.

12. McCarty KS Jr, Miller LS, Cox EB, et al. Estrogen receptor analyses: correlation of biochemical

and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab

Med. 1985;109(8):716-721.

13. Shousha S. Oestrogen receptor status of breast carcinoma: Allred/H score conversion table.

Histopathology. 2008;53(3):346-347.

14. Collins LC, Botero ML, Schnitt SJ. Bimodal frequency distribution of estrogen receptor

immunohistochemical staining results in breast cancer: an analysis of 825 cases. Am J Clin

Pathol. 2005:123(1):16-20.

15. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, Morales AR. Immunohistochemistry of estrogen

and progesterone receptors reconsidered: experience with 5,993 breast cancers. Am J Clin

Pathol. 2005:123(1);21-27.

16. Turbin DA, Leung S, Cheang MC, et al. Automated quantitative analysis of estrogen receptor

expression in breast carcinoma does not differ from expert pathologist scoring: a tissue

microarray study of 3,484 cases. Breast Cancer Res Treat. 2008;110(3):417-426.

C. HER2 (ERBB2) Testing

Scientific rationale: A subset of breast carcinomas (approximately 15% to 20%) overexpress human

epidermal growth factor receptor 2 (HER2; HUGO nomenclature ERBB2). Protein overexpression is

usually due to gene amplification. Assays for gene copy number, mRNA quantity, and protein generally

give similar results; gene amplification correlates with protein overexpression in about 95% of cases. In a

small subset of carcinomas (probably <5%), protein overexpression may occur by different mechanisms.

Overexpression is both a prognostic and predictive factor.

Clinical rationale: HER2 status is primarily evaluated to determine patient eligibility for anti-HER2 therapy.

It may identify patients who have a greater benefit from anthracycline-based adjuvant therapy.

Methods: HER2 status can be determined in formalin-fixed paraffin-embedded tissue by assessing

protein expression on the membrane of tumor cells using IHC or by assessing the number of HER2 gene

copies using in situ hybridization (ISH). When both IHC and ISH are performed on the same tumor, the

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results should be correlated. The most likely reason for a discrepancy is that 1 of the assays is incorrect,

but in a small number of cases there may be protein overexpression without amplification, amplification

without protein overexpression, or marked intratumoral heterogeneity.

HER2 (ERBB2) Testing by Immunohistochemistry

Factors altering the detection of HER2 (ERBB2) by IHC have not been studied as well as for ER and

PgR. It is recommended that tissue be fixed in buffered 10% formalin for at least 6 hours unless another

fixative has been validated. External proficiency testing surveys for HER2 are available from the CAP and

other organizations. These surveys are invaluable tools to ensure that the laboratory assays are working

as expected.

False-positive IHC results for HER2 may be due to:

• Edge artifact. This is usually seen in core biopsies, where cells near the edges of the tissue stain

stronger than in the center, possibly because antibody pools at the sides. Specimens with

stronger staining at the edge of the tissue should be interpreted with caution.

• Cytoplasmic positivity, which can obscure membrane staining and make interpretation difficult.

• Overstaining (strong membrane staining of normal cells). May be due to improper antibody

titration (concentration too high).

• Misinterpretation of ductal carcinoma in situ (DCIS). High-grade DCIS is often HER2 positive. In

cases with extensive DCIS relative to invasive carcinoma (particularly microinvasive carcinoma),

HER2 scoring may mistakenly be done on the DCIS component. Care must be taken to score

only the invasive component.

False-negative IHC results for HER2 may be due to:

• Prolonged cold ischemia time.

• Tumor heterogeneity. When a negative result is found, but only a small biopsy sample was

tested, repeat testing on a subsequent specimen with a larger area of carcinoma should be

considered, particularly if the tumor has characteristics associated with HER2 positivity (ie, tumor

grade 2 or 3, weak or negative PgR expression, increased proliferation index).

• Improper antibody titration (concentration too low)

False-negative and false-positive results can be reduced by paying attention to the following:

• Tissue controls. External controls must stain as expected. There are no normal internal controls

for HER2 protein assessment by IHC.

• Correlation with histologic and other biomarker results. If the HER2 test is negative by IHC, but

the tumor has characteristics associated with HER2 positivity (see above), repeating the test by

ISH should be considered.

Reporting guidelines: ASCO and CAP have issued recommendations for reporting the results of HER2

testing by IHC (Table 4).

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Table 4. Reporting Results of HER2 Testing by Immunohistochemistry (IHC)

Result

Criteria

Negative (Score 0)

No staining observed

Membrane stating that is incomplete and is faint/barely perceptible and within ≤10% of

tumor cells

Negative (Score 1+)

Incomplete membrane staining that is faint/barely perceptible and within >10% of tumor

cells*

Equivocal (Score 2+)†

Weak to moderate complete membrane staining in >10% of tumor cells

Complete membrane staining that is intense but within ≤10% of tumor cells*

Positive (Score 3+)

Complete membrane staining that is intense and >10% of tumor cells*

* Readily appreciated using a low-power objective and observed within a homogeneous and contiguous population of

invasive tumor cells.

† Must order reflex test (same specimen using ISH) or order a new test (new specimen if available, using IHC or

ISH).

HER2 Testing by In Situ Hybridization

Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and silver-enhanced

in situ hybridization (SISH) studies for HER2 determine the presence or absence of gene amplification.

Some assays use a single probe to determine the number of HER2 gene copies present, but most assays

include a chromosome enumeration probe (CEP17) to determine the ratio of HER2 signals to copies of

chromosome 17. Although 10% to 50% of breast carcinomas have more than 2 CEP17 copies, only 1%

to 2% of carcinomas show true polysomy (ie, duplication of the entire chromosome).

Failure to obtain results with ISH may be due to the following:

• Prolonged fixation in formalin (>1 week)

• Fixation in non-formalin fixatives

• Procedures or fixation involving acid (e.g., decalcification) may degrade DNA

• Insufficient protease treatment of tissue

External proficiency testing surveys for HER2 by ISH are available from CAP and other organizations.

These surveys are invaluable tools to ensure that the laboratory assays are working as expected.

Reporting guidelines: ASCO and CAP have issued recommendations for reporting the results of HER2

testing by ISH (Tables 5 and 6).

Dual Probe ISH Group Definitions:

Group 1 = HER2/CEP17 ratio ≥2.0; ≥4.0 HER2 signals/cell

Group 2 = HER2/CEP17 ratio ≥2.0; <4.0 HER2 signals/cell

Group 3 = HER2/CEP17 ratio <2.0; ≥6.0 HER2 signals/cell

Group 4 = HER2/CEP17 ratio <2.0; ≥4.0 and <6.0 HER2 signals/cell

Group 5 = HER2/CEP17 ratio <2.0; <4.0 HER2 signals/cell

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Table 5. Reporting Results of HER2 Testing by In Situ Hybridization (single-probe assay)

Result

Criteria (single-probe assay)

Negative

· Average HER2 copy number <4.0 signals/cell

· Average HER2 copy number ≥4.0 and <6.0 signals/cell and concurrent IHC 0, 1+ or 2+

· Average HER2 copy number ≥4.0 and <6.0 signals/cell and concurrent dual probe ISH Group 5

Positive

· Average HER2 copy number ≥6.0 signals/cell

· Average HER2 copy number ≥4.0 and <6.0 signals/cell and concurrent IHC 3+

· Average HER2 copy number ≥4.0 and <6.0 signals/cell and concurrent dual probe ISH Group 1

Table 6. Reporting Results of HER2 Testing by In Situ Hybridization (dual-probe assay)

Result

Criteria (dual-probe assay)

Negative · Group 5

Negative*

(see comment)

· Group 2 and concurrent IHC 0-1+ or 2+

· Group 3 and concurrent IHC 0-1+

· Group 4 and concurrent IHC 0-1+ or 2+

Positive*

· Group 2 and concurrent IHC 3+

· Group 3 and concurrent IHC 2+ or 3+

· Group 4 and concurrent IHC 3+

Positive · Group 1

*For Groups 2-4 final ISH results are based on concurrent review of IHC, with recounting of the ISH test by a second

reviewer if IHC is 2+ (per 2018 CAP/ASCO Update recommendations).

Comment for Group 2 Negative result: Evidence is limited on the efficacy of HER2-targeted therapy in the

small subset of cases with HER2/CEP17 ratio ≥2.0 and an average HER2 copy number <4.0/cell. In the

first generation of adjuvant trastuzumab trials, patients in this subgroup who were randomized to the

trastuzumab arm did not appear to derive an improvement in disease free or overall survival, but there

were too few such cases to draw definitive conclusions. IHC expression for HER2 should be used to

complement ISH and define HER2 status. If IHC result is not 3+ positive, it is recommended that the

specimen be considered HER2 negative because of the low HER2 copy number by ISH and lack of

protein overexpression.

Comment for Group 3 Negative result: There are insufficient data on the efficacy of HER2-targeted

therapy in cases with HER2 ratio <2.0 in the absence of protein overexpression because such patients

were not eligible for the first generation of adjuvant trastuzumab clinical trials. When concurrent IHC

results are negative (0-1+), it is recommended that the specimen be considered HER2 negative.

Comment for Group 4 Negative result: It is uncertain whether patients with ≥4.0 and <6.0 average HER2

signals/cell and HER2/CEP17 ratio <2.0 benefit from HER2 targeted therapy in the absence of protein

overexpression (IHC 3+). If the specimen test result is close to the ISH ratio threshold for positive, there is

a high likelihood that repeat testing will result in different results by chance alone. Therefore, when IHC

results are not 3+ positive, it is recommended that the sample be considered HER2 negative without

additional testing on the same specimen.

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Important issues in interpreting ISH are the following:

• Identification of invasive carcinoma: A pathologist should identify on the hematoxylin and eosin

(H&E) or HER2 IHC slide the area of invasive carcinoma to be evaluated by ISH.

• Identification of associated DCIS: In some cases, DCIS will show gene amplification, whereas the

associated invasive carcinoma will not. ISH analysis must be performed on the invasive

carcinoma.

Some cancers have a low level of HER2 expression as determined by equivocal results by both IHC and

ISH analysis. Repeat testing may be helpful to exclude possible technical problems with the assays but

often does not result in definitive positive or negative results.

Either the number of HER2 genes or the ratio of HER2 to CEP17 can be used to determine the presence

of amplification. In the majority of carcinomas, both methods give the same result. In unusual cases, the 2

methods give different results, usually due to variation in the number of CEP17 signals. Some studies

have shown that chromosome 17 abnormalities can lead to alterations of the HER2/CEP17 ratio,

potentially leading to equivocal or incorrect ISH results.

In such cases, gene copy number may be a

more accurate reflection of HER2 status. If there is a second contiguous population of cells with

increased HER2 signals/cell, and this cell population consists of more than 10% of tumor cells on the

slide (defined by image analysis or by visual estimation of the ISH or IHC slide), a separate counting of at

least 20 non-overlapping cells must also be done within this cell population and also reported. An overall

random count is not appropriate in this situation.

References

1. Wolff AC, Hammond MEH, Allison KH, et al. HER2 testing in breast cancer: American Society of

Clinical Oncology/College of American Pathologists clinical practice guideline focused update.

Arch Pathol Lab Med. 2018;142(11):1364-1382.

2. Selvarajan S, Bay B-H, Choo A, et al. Effect of fixation period on HER2/neu gene amplification

detected by fluorescence in situ hybridization in invasive breast carcinoma. J Histochem

Cytochem. 2002;50(12):1693-1696.

3. Willmore-Payne C, Metzger K, Layfield LJ. Effects of fixative and fixation protocols on

assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization.

Appl Immunohistochem Mol Morphol. 2007;15(1):84-87.

4. Brown RS, Edwards J, Bartlett JW, Jones C, Dogan A. Routine acid decalcification of bone

marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer. J

Histochem Cytochem. 2002;50(1):113-115.

5. Gunn S, Yeh IT, Lytvak I, et al. Clinical array-based karyotyping of breast cancer with equivocal

HER2 status resolves gene copy number and reveals chromosome 17 complexity. BMC Cancer.

2010;10:396.

D. Ki-67 Testing

Ki-67 is a nuclear protein found in all phases of the cell cycle and is a marker of cell proliferation. The

monoclonal antibody MIB-1 is the most commonly used antibody for assessing Ki-67 in formalin-fixed

paraffin-embedded tissue sections. The percentage of Ki-67 positive tumor cells determined by IHC is

often used to stratify patients into good and poor prognostic groups, but there is a lack of consensus on

scoring, definition of low versus high expression, an appropriate cut point for positivity, or which part of

the tumor should be scored (e.g., leading edge, hot spots, overall average).

There is also a paucity of

data on the effects of pre-analytic variables (e.g., ischemic time, length of fixation, antigen retrieval) on Ki-

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67 staining. For these reasons, routine testing of breast cancers for Ki-67 expression is not currently

recommended by either ASCO or the National Comprehensive Cancer Network (NCCN).

References

1. Dowsett M, Nielsen TO, A'Hern R, et al. Assessment of Ki67 in breast cancer: recommendations

from the International Ki67 in breast cancer working group. J Natl Cancer Inst.

2011;103(22):1656-1664.