HER2-Positive Breast Cancer: A Guide Through an

Evolving Treatment Landscape

This program is supported by educational grants from Genentech,

a member of the Roche Group, and Puma Biotechnology, Inc.
HER2 Testing and Treatment Decisions In Breast
Cancer

Sara M. Tolaney, MD, MPH

Associate Professor of Medicine
Harvard Medical School
Associate Director
Susan F. Smith Center for Women’s Cancer
Director of Clinical Trials, Breast Oncology
Director of Breast Immunotherapy Clinical Research
Senior Physician
Breast Oncology Program
Dana-Farber Cancer Institute
Boston, Massachusetts
Faculty Disclosures
Sara M. Tolaney, MD, MPH, has disclosed that she has received funds for
research support from AstraZeneca, Bristol-Myers Squibb, Cyclacel, Eisai,
Exelixis, Genentech/Roche, Immunomedics/Gilead, Lilly, Merck, Nektar,
Novartis, Odonate, Pfizer, Sanofi, and SeaGen; and consulting fees from
AstraZeneca, Athenex, Bristol-Myers Squibb, Cytomx, Daiichi Sankyo, Eli Lilly,
Eisai, Genentech/Roche, Immunomedics/Gilead, Kyowa Kirin Pharmaceuticals,
Nektar, Novartis, Odonate, OncoPep, Pfizer, Puma, Samsung Bioepsis, Sanofi,
and SeaGen.
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NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®): Breast Cancer. Version 1.2021. January 15, 2021. Available
at: www.NCCN.org. Accessed January 22, 2021.
 HER2 Testing Guidelines Updated: 2018
 2018 update of ASCO/CAP guidelines endorsed 2013 guidelines while
addressing outstanding questions:
‒ Revised definition of IHC 2+
‒ Updated strength of recommendation for retesting following HER2-
negative result on core needle biopsy
‒ Defined appropriate workup to ensure definitive diagnosis for equivocal
HER2 in situ hybridization (ISH) assay results

Wolff. JCO. 2018;36:2105. Slide credit: clinicaloptions.com

 HER2 Testing by IHC for Breast Cancer:
ASCO/CAP Guidelines
HER2 testing (invasive component) by validated ISH assay

Batch controls and on-slide controls show appropriate staining

Circumferential membrane Weak to moderate Incomplete membrane No staining is observed or

staining that is complete, complete membrane staining that is faint/barely membrane staining that is
intense, and in > 10% of staining observed in perceptible and in > 10% of incomplete and is faint/barely
tumor cells > 10% of tumor cells tumor cells perceptible and in ≤ 10% of
tumor cells

IHC 3+ positive IHC 2+ equivocal IHC 1+ negative IHC 0 negative

Must order a reflex test (same specimen using ISH) or order a

new test (new specimen if available, using IHC or ISH)

 2007, 2013/2014, and 2018 guidelines largely ignore both the IHC 0/1+ false negative and the IHC 3+ false positive
Wolff. Arch Pathol Lab Med. 2018;142:1364. Slide credit: clinicaloptions.com
 2018 ASCO/CAP Guidelines Revised the IHC Equivocal
Scoring Criteria
HER2 Testing With IHC

No staining OR
“Unusual staining patterns of HER2
Complete, intense Updated for 2018:
by IHC can be encountered that
Incomplete, faint
circumferential membrane Weak to moderate complete membrane staining incomplete, faint
staining in > 10% of cells membrane staining observed in > 10% of cells membrane staining in
≤ 10% of cells
in 10% of tumor cells
are not covered by these
definitions. In practice, these
patterns are rare and if
encountered should be considered
IHC 2+ equivocal.”
IHC 3+ positive IHC 2+ equivocal IHC 1 + negative IHC 0 negative
Order a reflex test using
ISH, or order a new test,
if a new specimen is
available

 Examples of unusual HER2 staining patterns include:

‒ moderate to intense but incomplete (basolateral or lateral) IHC staining
‒ intense circumferential membrane IHC staining within ≤ 10% of tumor cells
Wolff. JCO. 2018;36:2105. Slide credit: clinicaloptions.com
Patient Case 1
 35-yr-old woman presents with 3.5 cm palpable right-sided breast mass
 US-guided core biopsy performed: Grade 3 invasive ductal carcinoma
‒ ER 0%, PR 0%
 HER2: 35% of tumor cells showed weak to moderate complete
membrane staining  IHC 2+  reflex ISH
Patient Case 1 contd.
 35 yr old woman presents with 3.5 cm palpable right-sided breast mass
 US-guided core biopsy performed: Grade 3 invasive ductal carcinoma
‒ ER 0%, PR 0%
 HER2: 35% of tumor cells show weak to moderate complete membrane
staining  IHC 2+  reflex ISH
 FISH
‒ HER2/CEP17: 2.3
‒ CEP17/cell: 1.5
‒ HER2 signals/cell: 3.5
 HER2 Testing by ISH for Breast Cancer:
ASCO/CAP Guidelines
HER2 testing (invasive component) by validated dual-probe ISH assay

HER2/CEP17 ratio ≥ 2.0 HER2/CEP17 ratio < 2.0

Average
HER2 copy
Group 1 Group 2 Group 3 Group 4 Group 5
number ≥ 4.0 signals/cell < 4.0 signals/cell ≥ 6.0 signals/cell ≥ 4.0 , < 6.0 signals/cell < 4.0 signals/cell

Perform reflex testing on the same specimen using IHC

IHC 0/1+ IHC 2+

HER2 negative Observer blinded to previous results recounts ISH, counting ≥ 20
ISH negative
cells

Recount ISH result = previous ISH result

Recount ISH result does not
equal previous ISH result
Initial ISH: HER2/CEP17 ratio ≥ 2.0; Initial ISH: HER2/CEP17 ratio < 2.0; Initial ISH: HER2/CEP17 ratio < 2.0;
avg HER2 signals/cell < 4.0 avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0

Recount ISH: HER2/CEP17 ratio
≥ 2.0; avg HER2 signals/cell < 4.0
DIAGNOSIS: HER2 NEGATIVE*
Adapted from Wolff. Arch Pathol Lab Med. 2018;142:1364.
Recount ISH: HER2/CEP17 ratio
< 2.0; avg HER2 ≥ 6.0 signals/cell
DIAGNOSIS: HER2 POSITIVE
*with comment
Recount ISH: HER2/CEP17 ratio < 2.0; Adjudicate results per internal
avg HER2 signals/cell ≥ 4.0 , < 6.0
procedures to define final category
DIAGNOSIS: HER2 NEGATIVE*
Slide credit: clinicaloptions.com
 HER2 Testing by ISH for Breast Cancer:
ASCO/CAP Guidelines
HER2 testing (invasive component) by validated dual-probe ISH assay

HER2/CEP17 ratio ≥ 2.0 HER2/CEP17 ratio < 2.0

Average
HER2 copy
Group 1 Group 2 Group 3 Group 4 Group 5
number ≥ 4.0 signals/cell < 4.0 signals/cell ≥ 6.0 signals/cell ≥ 4.0 , < 6.0 signals/cell < 4.0 signals/cell
If sample workup already included IHC,
Perform reflex testing on the same specimen using IHC immediately proceed to blinded recount

IHC 2+ IHC and ISH should be reviewed together by the

IHC 0/1+ same institution to ensure assay quality and guide
HER2 negative Observer blinded to previous results recounts ISH, counting ≥ 20 selection of theISH
area negative
to score by ISH (local practice
cells considerations will dictate the best procedure to
accomplish this concomitant review)
Recount ISH result = previous ISH result
The 20 cells to be recounted include areas
Recount ISH result does not
of invasive cancer with IHC 2+ staining equal previous ISH result
Initial ISH: HER2/CEP17 ratio ≥ 2.0; Initial ISH: HER2/CEP17 ratio < 2.0; Initial ISH: HER2/CEP17 ratio < 2.0;
avg HER2 signals/cell < 4.0 avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0

Recount ISH: HER2/CEP17 ratio
≥ 2.0; avg HER2 signals/cell < 4.0
DIAGNOSIS: HER2 NEGATIVE*
Adapted from Wolff. Arch Pathol Lab Med. 2018;142:1364.
Recount ISH: HER2/CEP17 ratio
< 2.0; avg HER2 ≥ 6.0 signals/cell
DIAGNOSIS: HER2 POSITIVE
*with comment
Recount ISH: HER2/CEP17 ratio < 2.0; Adjudicate results per internal
avg HER2 signals/cell ≥ 4.0 , < 6.0
procedures to define final category
DIAGNOSIS: HER2 NEGATIVE*
Slide credit: clinicaloptions.com
 HER2 Negative With a Comment
 Patient remains in Group 2 after work-up; HER2 negative with a
comment
 ASCO/CAP guidelines recommend the following comment:
“Evidence is limited on the efficacy of HER2‐targeted therapy in the small subset of
cases with a HER2/CEP17 ratio ≥ 2.0 and an average HER2 copy number of < 4.0 per
cell. In the first generation of adjuvant trastuzumab trials, patients in this subgroup
who were randomly assigned to the trastuzumab arm did not seem 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 the 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 the lack of protein overexpression.”

Wolff. JCO. 2018;36:2105. Slide credit: clinicaloptions.com

Patient Case 2
 49-yr-old woman presents with 3.1 cm palpable mammographic
abnormality
 US-guided core biopsy performed: Grade 3 invasive ductal carcinoma
‒ ER 0%, PR 0%
 FISH
‒ HER2/CEP17: 1.9
‒ CEP17/cell: 3.2
‒ HER2 signals/cell: 6.1
 HER2 Testing by ISH for Breast Cancer:
ASCO/CAP Guidelines
HER2 testing (invasive component) by validated dual-probe ISH assay

HER2/CEP17 ratio ≥ 2.0 HER2/CEP17 ratio < 2.0

Average
HER2 copy
Group 1 Group 2 Group 3 Group 4 Group 5
number ≥ 4.0 signals/cell < 4.0 signals/cell ≥ 6.0 signals/cell ≥ 4.0 , < 6.0 signals/cell < 4.0 signals/cell
If sample workup already included IHC,
Perform reflex testing on the same specimen using IHC immediately proceed to blinded recount

IHC 2+ IHC and ISH should be reviewed together by the

IHC 0/1+ same institution to ensure assay quality and guide
HER2 negative Observer blinded to previous results recounts ISH, counting ≥ 20 selection of theISH
area negative
to score by ISH (local practice
cells considerations will dictate the best procedure to
accomplish this concomitant review)
Recount ISH result = previous ISH result
The 20 cells to be recounted include areas
Recount ISH result does not
of invasive cancer with IHC 2+ staining equal previous ISH result
Initial ISH: HER2/CEP17 ratio ≥ 2.0; Initial ISH: HER2/CEP17 ratio < 2.0; Initial ISH: HER2/CEP17 ratio < 2.0;
avg HER2 signals/cell < 4.0 avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0

Recount ISH: HER2/CEP17 ratio Recount ISH: HER2/CEP17 ratio Recount ISH: HER2/CEP17 ratio < 2.0; Adjudicate results per internal
≥ 2.0; avg HER2 signals/cell < 4.0 < 2.0; avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0
procedures to define final category
DIAGNOSIS: HER2 NEGATIVE* DIAGNOSIS: HER2 POSITIVE DIAGNOSIS: HER2 NEGATIVE*

Adapted from Wolff. Arch Pathol Lab Med. 2018;142:1364. *with comment Slide credit: clinicaloptions.com
 If Group 3 Specimen Results In IHC 0/1 After Work-up:
 HER2 negative with a comment
 ASCO/CAP guidelines recommend the following comment:

“There are insufficient data on the efficacy of HER2‐targeted therapy in cases with a
HER2 ratio of < 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 or 1+), it is recommended that the specimen be
considered HER2 negative.”

Wolff. JCO. 2018;36:2105. Slide credit: clinicaloptions.com

Patient Case 3
 45-yr-old woman presents with a 1.8 cm palpable mammographic
abnormality
 US-guided core biopsy performed: Grade 2 invasive ductal carcinoma
‒ ER 80%, PR 70%
‒ HER2: IHC 2+
 FISH
‒ HER2/CEP17: 1.7
‒ CEP17/cell: 2.5
‒ HER2 signals/cell: 4.2
 HER2 Testing by ISH for Breast Cancer:
ASCO/CAP Guidelines
HER2 testing (invasive component) by validated dual-probe ISH assay

HER2/CEP17 ratio ≥ 2.0 HER2/CEP17 ratio < 2.0

Average
HER2 copy
Group 1 Group 2 Group 3 Group 4 Group 5
number ≥ 4.0 signals/cell < 4.0 signals/cell ≥ 6.0 signals/cell ≥ 4.0 , < 6.0 signals/cell < 4.0 signals/cell
If sample workup already included IHC,
Perform reflex testing on the same specimen using IHC immediately proceed to blinded recount
IHC and ISH should be reviewed together by the
IHC 0/1+ IHC 2+ same institution to ensure assay quality and guide
selection of theISH
areanegative
to score by ISH (local practice
HER2 negative Observer blinded to previous results recounts ISH, counting ≥ 20
considerations will dictate the best procedure to
cells
accomplish this concomitant review)
Recount ISH result = previous ISH result The 20 cells to be recounted include areas
Recount ISH result does not
of invasive cancer with IHC 2+ staining
equal previous ISH result
Initial ISH: HER2/CEP17 ratio ≥ 2.0; Initial ISH: HER2/CEP17 ratio < 2.0; Initial ISH: HER2/CEP17 ratio < 2.0;
avg HER2 signals/cell < 4.0 avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0

Recount ISH: HER2/CEP17 ratio Recount ISH: HER2/CEP17 ratio Recount ISH: HER2/CEP17 ratio < 2.0; Adjudicate results per internal
≥ 2.0; avg HER2 signals/cell < 4.0 < 2.0; avg HER2 ≥ 6.0 signals/cell avg HER2 signals/cell ≥ 4.0 , < 6.0
procedures to define final category
DIAGNOSIS: HER2 NEGATIVE* DIAGNOSIS: HER2 POSITIVE DIAGNOSIS: HER2 NEGATIVE*

Adapted from Wolff. Arch Pathol Lab Med. 2018;142:1364. *with comment Slide credit: clinicaloptions.com
 Patient Case 3: Follow-up
 Patient remains in Group 4 work-up; HER2 negative with a comment
 ASCO/CAP guidelines recommend the following comment:
“It is uncertain whether patients with an average of ≥ 4.0 and < 6.0 HER2 signals per
cell and a HER2/CEP17 ratio of < 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.”

Wolff. JCO. 2018;36:2105. Slide credit: clinicaloptions.com

 Should HER2 Testing Always Be Repeated On A Surgical
Specimen If Negative On Core Biopsy?
 “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new
HER2 test may be ordered on the excision specimen” [1] if histopathologic features suggest HER2 test
discordance

 Testing an excision sample no longer mandatory for HER2-negative results from a core biopsy with
features suggestive of histopathologic discordance[1]
 Unchanged from 2013 guidelines:[2]
‒ Retesting an excision specimen may be considered in cases of histopathologic discordance with HER2
negative test result
 Additionally, retesting is recommended when HER2 samples are:
‒ Equivocal; reflexing to the alternate method or ordering a new test on a new specimen, using previous results to
guide which section to score
‒ Indeterminate
‒ Positive with histopathologic discordance

1. Wolff. JCO. 2018;36:2105. 2. Wolff. JCO. 2013;31:3997. Slide credit: clinicaloptions.com

 ASCO/CAP Guidelines: Histopathologic Discordance and
When To Order A New HER2 Test
Do not order a
new HER2 Test if:
Initial result:
HER2- Negative
and
Histologic grade 1 carcinoma of:
 Infiltrating ductal or lobular
carcinoma, 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
Wolff. JCO. 2018;36:2105.
Order a new
HER2 Test if:
Initial result:
HER2- positive
and
One of the following:
Histologic grade 1 carcinoma of:
 Infiltrating ductal or lobular
carcinoma, 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
Order a new
HER2 Test if:
Initial result:
HER2- negative
(core needle biopsy)
and
 Tumor is grade 3
 Amount of invasive tumor in the
core biopsy is small
 Resection specimen contains
high grade carcinoma
morphologically distinct from
that in core
 Questionable specimen handling
of core biopsy or test is
suspected to be negative on
basis of testing error
Slide credit: clinicaloptions.com
 Definition of HER2-Low Breast Cancer
Circumferential membrane
staining that is complete,
intense, and in >10% of
tumor cells → (IHC 3+)
HER2-POSITIVE
Wolff. Arch Pathol Lab Med. 2018;142:1364.
HER testing by
validated IHC assay
Incomplete membrane No staining is observed HER2-
Weak to moderate complete
staining that is faint/barely null or membrane staining
membrane staining in >10%
perceptible and in >10% of that is incomplete and is
of tumor cells → (IHC 2+)
tumor cells → (IHC 1+) faint/barely perceptible and in
<10% tumor cells → (IHC 0+)
Reflex Reflex
ISH test ISH test HER2-LOW HER2-NEGATIVE
POSITIVE NEGATIVE
Slide credit: clinicaloptions.com
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