Chair, Kurt A. Schalper, MD, PhD, Michael F. Press, MD, PhD, Paolo Tarantino, MD, and Zev A. Wainberg, MD, prepared useful Practice Aids pertaining to immuno-oncology biomarkers for this CME/MOC/CC activity titled “Decoding the Latest Evidence and Practical Recommendations on Biomarker Testing for New Therapeutic Options Targeting HER2, HER3, and TROP2 in Solid Tumors.” For the full presentation, downloadable Practice Aids, and complete CME/MOC/CC information, and to apply for credit, please visit us at https://bit.ly/3iQ5A6Y. CME/MOC/CC credit will be available until April 22, 2022.

1. HER2, HER3, and TROP2 as Therapeutic
Targets in Different Cancers
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HER2 Aberrations: Clinical Role and Frequency Across Tumor Types1,2
•
HER2 is an established therapeutic target in breast and gastric cancer
• HER2 alterations, including overexpression, amplifications, and other mutations, are found in a variety of
other solid tumors as well
•
A number of novel HER2-targeted therapies are being evaluated in breast, gastrointestinal, lung, and other
cancers, indicating that the role and impact of these therapies will continue to expand, along with the need
for broader HER2 testing
ERBB2 amplifications/mutations and HER2 overexpression
have been identified in a range of malignancies
Colorectum
5.8 5 2
Pancreas
2 26 1
Ovary
7 27 1
Prostate
5.8-6 10 1
Tumor types
HER2
amplification (%)
HER2
overexpression (%)
HER2
mutation (%)
Breast
20 15-20 2
Salivary gland
12-52 17-44 1
Stomach
11-16 20 3
Bladder
8.6 12.4 9
Cervix
0.5~14 21 3
Uterus
4-69 18-80 2
Lung
2–3 2.5 1-3
Biliary tract
5-15 20 2

2. HER2, HER3, and TROP2 as Therapeutic
Targets in Different Cancers
Full abbreviations, accreditation, and disclosure information available at
PeerView.com/EZK40
1. Oh DY, Bang YJ. Nat Rev Clin Oncol. 2020;17:33-48. 2. Hechtman JF, Ross DS. Cancer Cytopathol. 2019;127:428-431. 3. Mishra R et al. Oncol Rev. 2018;12:355. 4. Guerra E et al. Oncogene. 2013;32:1594-1600.
5. Zeng P et al. Sci Rep. 2016;6:33658.
Mechanism of Action of Agents Targeting HER21
HER3 and TROP2 as Emerging Therapeutic Targets
a.
Single-epitope monoclonal antibodies bind HER2 at a single extracellular domain, inhibiting downstream
signaling, engaging antibody-dependent cytotoxicity, or inhibiting receptor dimerization
b.
ADCs also have antitumor effects through these pathways, but they additionally exhibit cytotoxicity by
releasing a cytotoxic agent close to HER2-positive tumor cells
c.
Bispecific antibodies target more than one extracellular region of HER2
d.
Small-molecule inhibitors bind the intracellular tyrosine-kinase domain
HER33
•
Crucial heterodimeric partner for other EGFR
family members
•
Potential to regulate EGFR/HER2-mediated
resistance
•
Enhanced expression associated with several
cancers, including lung, breast, ovarian, prostate,
gastric, bladder, melanoma, colorectal, and
squamous cell carcinoma
•
Implicated in contributing to treatment failure
through activation of PI3K/AKT, MAPK/ERK, and
JAK/STAT pathways
•
HER3-targeting investigational therapies:
mono and bispecific antibodies targeting
HER3 at multiple subdomains; miscellaneous
HER3-targeting therapies, including antisense
oligonucleotides, HER3-specific peptide
vaccines, ligand traps, molecules targeting HER3
pseudokinase activity, pan-HER approaches,
HER3 ADCs, and HER3 nanobiologic therapeutic
approaches; select examples: patritumab
deruxtecan/U3-1402 (HER3-targeting ADC) and
MCLA-128 (HER2–HER3 bispecific antibody)
TROP24,5
•
Transmembrane glycoprotein overexpressed in
many different tumors, including lung, breast,
pancreatic, cervical, ovarian, colorectal, and
gastric cancers
•
Membrane bound with an extracellular domain
•
Effectively internalized with binding antibody
•
High expression correlates with poor prognosis
•
Rational therapeutic target; TROP2-targeting
therapies: sacituzumab govitecan/IMMU-132,
datopotamab deruxtecan/DS-1062
Pertuzumab
a. Single-Epitope mAbs b. ADCs
d. Small-Molecule
Inhibitors
c. Bispecific Antibodies
Dimerization
domain
Trastuzumab
Margetuximab
Inhibition of receptor
dimerization
Targeted
delivery of
highly
cytotoxic
agents
Direct inhibition of
the downstream
tyrosine-kinase
domain
Promotion of
receptor
internalization
and/or
degradation
Engagement
of ADCC
Tyrosine-kinase
domain
Cell
membrane
Trastuzumab emtansine
Trastuzumab deruxtecan
Dual targeting of
the trastuzumab
and pertuzumab
binding sites
ZW25
Lapatinib
Neratinib
Tucatinib
I
I
I
III
IV
I
I
I
III
IV
Inhibition of PI3-kinase signalling
promoting cell-cycle arrest
I
I
I
III
IV

3. Treatment Recommendations for HER2+ Breast Cancer
Full abbreviations, accreditation, and disclosure information available at PeerView.com/EZK40
a
Alternative taxanes (ie, docetaxel, paclitaxel, albumin-bound paclitaxel) may be substituted for select patients due to medical necessity (ie, hypersensitivity reaction). If substituted for weekly paclitaxel or docetaxel, then the weekly dose of albumin-bound paclitaxel should not
exceed 125 mg/m2
. b
Paclitaxel + trastuzumab may be considered for patients with low-risk T1, N0, M0, HER2+ disease, particularly those not eligible for other standard adjuvant regimens due to comorbidities. c
Trastuzumab given in combination with an anthracycline is associated
with significant cardiac toxicity. Concurrent use of trastuzumab and pertuzumab with an anthracycline should be avoided. d
Consider extended adjuvant neratinib following adjuvant trastuzumab-containing therapy for patients with HR+/HER2+ with a perceived high risk of
recurrence. The benefit or toxicities associated with extended neratinib in patients who have received pertuzumab or ado-trastuzumab emtansine is unknown. e
It is acceptable to change the administration sequence to taxane (with or without HER2-targeted therapy) followed by AC.
1. NCCN Clinical Practice Guidelines in Oncology. Breast Cancer. Version 1.2022. https://www.nccn.org/professionals/physician_gls/pdf/breast.pdf.
Preferred Regimens
• Paclitaxel + trastuzumabb
• TCH (docetaxel/carboplatin/trastuzumab)
• TCHP (docetaxel/carboplatin/trastuzumab/pertuzumab)
• If no residual disease after preoperative therapy or no preoperative therapy: complete up to 1 year of HER2-targeted
therapy with trastuzumabc
(category 1) ± pertuzumab
• If residual disease after preoperative therapy: trastuzumab emtansine (category 1) alone; if trastuzumab emtansine
discontinued for toxicity, then trastuzumab (category 1) ± pertuzumab to complete 1 year of therapyc,d
Useful in Certain Circumstances Other Recommended Regimens
• Docetaxel + cyclophosphamide + trastuzumab
• AC followed by Te
+ trastuzumabc
(doxorubicin/cyclophosphamide followed by paclitaxel +
trastuzumab; various schedules)
• AC followed by Te
+ trastuzumab + pertuzumabc
(doxorubicin/cyclophosphamide followed by paclitaxel +
trastuzumab + pertuzumab; various schedules)
• Neratinibd
(adjuvant setting only)
• Paclitaxel + trastuzumab + pertuzumabc
• Trastuzumab emtansine (adjuvant setting only)
• AC followed by docetaxele
+ trastuzumabc
(doxorubicin/cyclophosphamide followed by docetaxel +
trastuzumab)
• AC followed by docetaxele
+ trastuzumab + pertuzumabc
(doxorubicin/cyclophosphamide followed by docetaxel +
trastuzumab + pertuzumab)
Preoperative/Adjuvant Therapy: Updated Recommendations Based on NCCN Guidelines Version 1.20221,a
Updates in Version 1.2022 of the Guidelines From Version 8.2021
Useful in certain circumstances, options added:
• Neratinib (adjuvant setting only)
• Paclitaxel + trastuzumab + pertuzumab
• Trastuzumab emtansine (adjuvant setting only)
!

4. Treatment Recommendations for HER2+ Breast Cancer
Full abbreviations, accreditation, and disclosure information available at PeerView.com/EZK40
a
Maintenance trastuzumab/pertuzumab after response (with concurrent endocrine therapy if ER+ and HER2+ metastatic breast cancer). b
Regimens may also be used as an option for third line and beyond; the optimal sequence for third-line therapy and beyond is not known.
c
An FDA-approved biosimilar is an appropriate substitute for trastuzumab. d
Trastuzumab deruxtecan may be considered in the first-line setting as an option for select patients (ie, those with rapid progression within 6 months of neoadjuvant or adjuvant therapy [12 months for
pertuzumab-containing regimens]). e
Trastuzumab deruxtecan is contraindicated for patients with pneumonitis or ILD. f
Tucatinib + trastuzumab + capecitabine is preferred in patients with both systemic and CNS progression in the third-line setting and beyond and it may be given
in the second-line setting. g
Multiple lines of concurrent chemotherapy with anti-HER2 therapy (trastuzumab or a TKI) offer clinical benefit for recurrent unresectable HER2+ metastatic breast cancer and have been studied in phase 2 or 3 trials. Clinical experience suggests frequent
clinical benefit for such treatment. However, there are no meaningful data for use of any of these regimens among patients previously treated with pertuzumab-based chemotherapy, trastuzumab emtansine, trastuzumab deruxtecan, or trastuzumab/capecitabine/tucatinib regimens.
Thus, the optimal sequence or true benefit of therapy is not known. h
Trastuzumab given in combination with an AC is associated with significant cardiac toxicity. Concurrent use of trastuzumab and pertuzumab with an AC should be avoided. i
Trastuzumab may be safely combined
with all non−AC-containing preferred and other single agents listed on the NCCN guidelines for systemic therapies for recurrent or metastatic breast cancer (BINV-Q [1 of 8]).
1. NCCN Clinical Practice Guidelines in Oncology. Breast Cancer. Version 1.2022. https://www.nccn.org/professionals/physician_gls/pdf/breast.pdf.
Updates in Version 1.2022 of the Guidelines From Version 8.2021
• Second-line option added: Trastuzumab deruxtecan; this is a category 1 preferred regimen
• Second-line option modified: Trastuzumab emtansine has been changed from a category 1 preferred regimen to a category 2A other recommended regimen
•
Heading modified: Third line and beyond (optimal sequence is not known)
Recurrent Unresectable (Local or Regional) or Stage IV (M1) Disease: Updated Recommendations Based on NCCN
Guidelines Version 1.20221
Setting Regimen NCCN Category of Preference Evidence Level
First linea
Pertuzumab + trastuzumab + docetaxelc Preferred regimen 1
Pertuzumab + trastuzumab + paclitaxelc Preferred regimen 2A
Second lineb
Trastuzumab deruxtecan (T-DXd)b,d,e Preferred regimen 1
Trastuzumab emtansine (T-DM1)b Other recommended regimen 2A
Third line and
beyond
(optimal sequence
unknown)
Tucatinib + capecitabine + trastuzumabc,f Other recommended regimen 1
Trastuzumab + docetaxel or vinorelbinec,g Other recommended regimen 2A
Trastuzumab + paclitaxel ± carboplatinc,g Other recommended regimen 2A
Capecitabine + trastuzumab or lapatinibc,g Other recommended regimen 2A
Trastuzumab + lapatinibc,g (without cytotoxic therapy) Other recommended regimen 2A
Trastuzumab + other agentsc,g,h,i Other recommended regimen
Neratinib + capecitabineg Other recommended regimen 2A
Margetuximab + chemotherapyg (capecitabine, eribulin, gemicitabine, or vinorelbine) Other recommended regimen 2A
2A
•
Footnote b modified: Regimens may also be used as an option for third line and beyond or fourth-line option; the optimal sequence for third-line therapy and beyond is not known
• Footnote d added: Trastuzumab deruxtecan may be considered in the first-line setting as an option for select patients (ie, those with rapid progression within 6 months of
neoadjuvant or adjuvant therapy [12 months for pertuzumab-containing regimens])
• Footnote f modified: Tucatinib + trastuzumab + capecitabine is preferred in patients with both systemic and CNS progression in the third-line setting and beyond
on trastuzumab emtansine. However, tucatinib + trastuzumab + capecitabine and it may be given in the second-line setting
•
Footnote removed: Trastuzumab deruxtecan is preferred in patients with visceral metastases if progression on trastuzumab emtansine
!

5. Testing for HER2 and HER2-Low
Expression in Breast Cancer
Full abbreviations, accreditation, and disclosure information available at
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Must order reflex test
(same specimen using ISH)
or order a new test
(new specimen if available,
using IHC or ISH)
IHC 3+
positive
IHC 1+
negative
IHC 0
negative
No staining is observed
or
Membrane staining that
is faint/barely perceptible
and in ≤10% of tumor cells
IHC 2+
equivocal
Batch controls and on-slide controls show appropriate staining
Incomplete membrane
staining that is faint/barely
perceptible and in
10% of tumor cells
Weak-to-moderate
complete membrane
staining observed in
10% of tumor cells
Circumferential membrane
staining that is complete,
intense, and in
10% of tumor cells
HER2 testing (invasive component) by validated IHC assay
• HER2 IHC 2+ (equivocal) cases with “weak-to-moderate complete membrane staining” observed in 10% of tumor cells
• If the initial HER2 test result in a core needle biopsy is negative, may repeat test in excision specimen based on clinical criteria
• If HER2/CEP17 ratio of ≥2.0, but the average HER2 signals per cell is 4.0, additional workup is needed
• If average of ≥6.0 HER2 signals per cell with an HER2/CEP17 ratio 2.0 (formerly diagnosed as ISH positive for HER2),
additional workup is needed
• If average HER2 signals per tumor cell ≥4.0 and 6.0 and the HER2/CEP17 ratio is 2.0 (formerly diagnosed as ISH equivocal),
additional workup is needed
Updates in the 2018 ASCP/CAP Guidelines1
Recommendations by the ASCO/CAP HER2 Testing Expert Panel are aimed at
improving the analytic validity of HER2 testing and the clinical utility of HER2 as a
predictive biomarker for potential responsiveness to therapies targeting the HER2 protein.
HER2 gene amplification assessed by in situ hybridization (ISH) or protein
overexpression assessed by IHC remains the primary predictor of responsiveness
to HER2-targeted therapies in breast cancer.
Breast Cancer: 2018 ASCO/CAP Guidelines for
Evaluation of HER2 Protein Expression by IHC1

6. Testing for HER2 and HER2-Low
Expression in Breast Cancer
Full abbreviations, accreditation, and disclosure information available at
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ISH
positive
ISH
negative
Evaluation of HER2 Gene Amplification
by ISH Assay Using Dual-Probe ISH¹
Additional
workup required
Additional
workup required
Additional
workup required
HER2 testing (invasive component) by validated dual-probe ISH assay
Evaluation of HER2 Gene Amplification
by ISH Assay Using Single-Probe ISH¹
HER2/CEP17
ratio ≥2.0
HER2/CEP17
ratio 2.0
Group 1
Average HER2
copy number
≥4.0 signals/cell
Group 2
Average HER2
copy number
4.0 signals/cell
Group 3
Average HER2
copy number
≥6.0 signals/cell
Group 4
Average HER2 copy
number ≥4.0 and
6.0 signals/cell
Group 5
Average HER2
copy number
4.0 signals/cell
Batch controls and on-slide controls show appropriate hybridization
ISH
positive
ISH
negative
Average HER2 copy number
≥6.0 signals/cell
Average HER2 copy number
4.0 signals/cell
Concurrent IHC 3+
and/or
Concurrent dual-probe
ISH group 1
Concurrent IHC 0, 1+
and/or
Concurrent dual-probe
ISH group 5
Batch controls and on-slide controls show appropriate hybridization
Average HER2 copy number
≥4.0 and 6.0 signals/cell
Perform dual-probe
ISH for final result
Concurrent
IHC 2+
HER2 testing (invasive component) by validated single-probe ISH assay

7. Testing for HER2 and HER2-Low
Expression in Breast Cancer
Full abbreviations, accreditation, and disclosure information available at
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Summary of the Major Changes in the ASCO/CAP HER2 Testing Guidelines Between 2007 and 2018²
HER2 testing must be performed on
all newly diagnosed and recurrent
breast cancers
Re-emphasized the importance of
testing metastatic breast cancers
At least one HER2 test should
be performed on primary and
metastatic breast cancers
Specimen type Same as 2013
Homogeneous, dark circumferential
membrane staining in 10% of
invasive tumor cells
Homogeneous, dark circumferential
membrane staining in 30% of
invasive tumor cells
IHC positive
HER2 (3+)
Same as 2013
• 0: No staining or incomplete
membranous staining that is
faint/barely perceptible and within
≤10% of invasive tumor cells
• 1+: Incomplete membranous
staining that is faint/barely
perceptible and within 10% of
invasive tumor cells
• 0: No staining
• 1+: Weak incomplete
membrane staining in any
proportion of tumor cells or
weak, complete membrane
staining in 10%
IHC negative
HER2 (0 or 1+) Same as 2013
• Single probe: HER2 copy number
≥4.0 and 6 signals/cell
• Dual probe: HER2/CEP17 ratio
of 2.0 with an average HER2
copy number ≥4.0 and
6 signals/cell
HER2/CEP17 ratio of 1.8-2.2 or
average HER2 gene copy number
4-6 signals/nucleus for test without
an internal control probe
ISH equivocal
Need additional work concomitant
with IHC result to render a
diagnosis whether HER2 is positive
or negative, with an explanatory
comment to be added
Must repeat if:
• Tumor on excision is grade 3
• Invasive tumor in the NCB is small
• Resection specimen contains high-grade
carcinoma that is morphologically distinct
from the prior core
• Core biopsy result is equivocal for HER2
after testing by both ISH and IHC
• There is doubt about the specimen
handling of the core biopsy (long ischemic
time, short time in fixative, different fixative)
or the test is suspected by the pathologist
to be negative on the basis of testing error
No recommendations
NCB negative
Same as 2013 but change
the word “must” to “may”
2013
2007
Item 2018
Circumferential membrane staining
that is incomplete and/or
weak/moderate and with 10% of
invasive tumor cells
Complete and circumferential
membranous staining that is
intense and within ≤10% of
invasive tumor cells
Non-uniform or weak intensity,
circumferential, complete
membranous staining in at
least 10% of invasive tumor cells
Complete and intense membranous
staining of 30% or less of
invasive tumor cells
Complete weak/moderate
membrane staining in 10%
of invasive tumor cells
Complete and circumferential
membranous staining that is
intense and within ≤10% of
invasive tumor cells
• Single probe: HER2 copy number
4.0 signals/cell
• Dual probe: HER2/CEP17 ratio
of 2.0 with an average HER2
copy number 4.0 signals/cell
HER2/CEP17 ratio of 1.8 or
average HER2 gene copy number
4 signals/nucleus for test without
an internal control probe
Same as 2013
IHC equivocal
HER2 (2+)
No specification on the type of
the probe (whether single or dual)
for test without an internal control
probe
• HER2 to CEP17 ratio of 2.2
or average HER2 gene copy
number 6 signals/nucleus
ISH positive
Same as 2013 for group 1
Groups 2 and 3: Need additional
work concomitant with IHC result
to render a diagnosis whether
HER2 is positive or negative,
with an explanatory comment
to be added
Specified criteria for single and
dual probe:
• Group 1: Single-probe average HER2
copy number ≥6.0 signals/cell or dual-probe
HER2/CEP17 ratio of ≥2.0 with an average
HER2 copy number ≥4.0 signals/cell
• Group 2: Dual-probe HER2/CEP17
ratio of ≥2.0 with an average HER2
copy number 4.0 signals/cell
• Group 3: Dual-probe HER2/CEP17
ratio of 2.0 with an average HER2
copy number ≥6.0 signals/cell
ISH negative
6-72 h
6-48 h Same as 2013
Duration of
tissue fixation

8. Testing for HER2 and HER2-Low
Expression in Breast Cancer
Full abbreviations, accreditation, and disclosure information available at
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Going Beyond the Dichotomous
HER2+/- Status?
Proposed Algorithm for Defining
HER2-Low Breast Cancer4
HER2 positive HER2 low HER2 negative
Reflex
ISH test
positive
Reflex
ISH test
negative
Circumferential membrane
staining that is complete,
intense, and in
10% of tumor cells
IHC 3+
Weak-to-moderate
complete membrane
staining in
10% of tumor cells
IHC 2+
Incomplete membrane
staining that is faint/barely
perceptible and in
10% of tumor cells
IHC 1+
No staining is observed:
HER2 null
or
Membrane staining that
is incomplete and is
faint/barely perceptible
and in10% of tumor cells
IHC 0+
HER2 testing by
validated IHC assay
HER2 positive
HER2 negative
Dichotomous
HER2 positive
HER2 negative
HER2 low
New
paradigm?
Challenge: Distinguishing IHC score 0 from score 1+
HER-low BC: ≈55%
• IHC 1+, IHC 2+ with
a negative ISH test
HER2-positive BC: ≈15%
HER2-negative BC: ≈30%
International guidelines currently recommend a binary model (HER2 positive vs negative) to guide
clinicians in treatment decisions. However, a great proportion of patients (≈40%-55%) classified as HER2
negative are, in fact, HER2 low; a population with a high unmet medical need. Despite past drawbacks
with older drugs, a new generation of anti-HER2 agents has recently shown encouraging signs of clinical
activity and safety in HER2-low disease.3

9. Testing for HER2 and HER2-Low
Expression in Breast Cancer
Full abbreviations, accreditation, and disclosure information available at
PeerView.com/EZK40
1. Wolff AC et al. Arch Pathol Lab Med. 2018;142:1364-1382. 2. Zhang H et al. Curr Oncol Rep. 2020;22:51. 3. Eiger D et al. Cancers (Basel). 2021;13:1015. 4. Tarantino P et al. J Clin Oncol. 2020;38:1951-1962.
Novel Agents and Mechanisms Enabling the Targeting of HER2-Low Breast Cancer4
Th1
Vaccines
Bispecific Antibodies
Antibody–Drug Conjugates
Monoclonal
Antibodies
Trastuzumab
Margetuximab
Pertuzumab
RAF/MEK/MAPK
Proliferation
PI3K/AKT/mTOR
Survival
Trastuzumab
Pertuzumab
Margetuximab
TrasGEX
Trastuzumab deruxtecan
Trastuzumab duocarmazine
PF-06804103
A166
RC48-ADC
ARX788
Antitumoral
agent
Linker
Antibody
Proliferation
Survival
MCLA-128
ZW25
Ertumaxomab
MM-111
GBR 1302
Nelipepimut-S
GP2
AE37
CTL
MHC I
TNFα
IFNγ
HER2-
polarized
DC
Novel anti-HER2 drugs enable the targeting of low HER2-expressing cancers through different mechanisms: A. Monoclonal antibodies engineered to enhance
antibody-dependent cellular cytotoxicity, or to more effectively inhibit HER2 heterodimerization, have shown preclinical evidence of activity in HER2-low breast
cancer. B. Novel antibody–drug conjugates enable exploitation of low HER2 expression to direct cytotoxic molecules to tumor cells, showing promising activity
in early clinical trials. C. Bispecific antibodies allow forced connections between cancer and immune cells and/or suppress multiple signaling pathways, with
potential activity in HER2-low breast cancer cells. D. Cancer vaccines enhance antitumor immune response against HER2 and are currently being tested in
the adjuvant setting to reduce relapses in HER2-low breast cancer.
A B C D

10. HER2 Testing in Gastroesophageal Cancer
Guidance From CAP/ASCP/ASCO1
Full abbreviations, accreditation, and disclosure information available at PeerView.com/EZK40
GEA and potential
candidate for
HER2-targeted
therapy
HER2-targeted therapy should not
be initiated until HER2 positivity
is confirmed
Biopsy or resection specimen
from primary or metastatic sites
should be used
Alternative: FNA specimens
(cell blocks) may be used
Request
HER2 test
Equivocal
or negative
result
Inadequate
specimen
tested
No positive
results
Positive
results
Initiate HER2-targeted therapy;
no further HER2 testing is
required
Retest additional available
tissue; if there is no available
tissue, additional tumor tissue
may be obtained for retesting
NGS2
enables assessment
of ERBB2 copy number
and mutations simultaneously,
along with other molecular
events such as TMB
and MSI status; can be performed
on both surgical specimens
or cell blocks and has recently
been shown to work
with cytologic
supernatant

11. HER2 Testing in Gastroesophageal Cancer
Guidance From CAP/ASCP/ASCO1
Full abbreviations, accreditation, and disclosure information available at PeerView.com/EZK40
Perform HER2
test using
IHC
IHC 2+
Equivocal
IHC 1+
Negative
IHC 3+
Positive
Perform ISH
Testing
IHC 0
Negative
Surgical specimen
Strong, complete basolateral or
lateral membranous reactivity in
≥10% of tumor cells
Biopsy specimen
Tumor cell cluster with strong,
complete basolateral or lateral
membranous activity irrespective
of percentage of tumor cells
stained
Surgical specimen
Weak to moderate, complete
basolateral or lateral membranous
reactivity in ≥10% of tumor cells
Biopsy specimen
Tumor cell cluster with weak/
moderate, complete basolateral
or lateral membranous activity
irrespective of percentage of
tumor cells stained
Surgical specimen
Faint/barely perceptible
membranous reactivity in ≥10% of
tumor cells; cells reactive only in
part of their membrane
Biopsy specimen
Tumor cell cluster with faint/barely
membranous activity irrespective
of percentage of tumor cells
stained
Surgical specimen
No reactivity or membranous
reactivity in 10% of tumor cells
Biopsy specimen
No reactivity in any tumor cells
No further ISH testing is required No further ISH testing is required
Tissue sample
from patient
diagnosed
with GEA

12. HER2 Testing in Gastroesophageal Cancer
Guidance From CAP/ASCP/ASCO1
Full abbreviations, accreditation, and disclosure information available at PeerView.com/EZK40
1. Bartley AN et al. Am J Clin Pathol. 2016;146:647-669. 2. Hechtman JF, Ross DS. Cancer Cytopathol. 2019;127:428-431.
Summary of HER2 Testing Recommendations
Strength of Recommendation
Strong recommendation • In patients with GEA who are potential candidates for HER2-targeted therapy, the treating clinician should request HER2 testing on tumor tissue
Strong recommendation
• Laboratories must incorporate GEA HER2 testing methods into their overall laboratory quality improvement program, establishing appropriate quality improvement
monitors as needed to ensure consistent performance in all steps of the testing and reporting process; in particular, laboratories performing GEA HER2 testing must
participate in a formal proficiency testing program, if available, or an alternative proficiency assurance activity
• Laboratories should report HER2 testing results in GEA specimens in accordance with the CAP “Template for Reporting Results of HER2 (ERBB2) Biomarker Testing
of Specimens From Patients With Adenocarcinoma of the Stomach or Esophagogastric Junction”
Strong recommendation
Strong recommendation
• Pathologists should identify areas of invasive adenocarcinoma and also mark areas with the strongest intensity of HER2 expression by IHC in GEA specimens for
subsequent ISH scoring when required
Recommendations
No recommendation • There is insufficient evidence to recommend for or against genomic testing in patients with GEA at this time
Strong recommendation
• When GEA HER2 status is being evaluated, laboratories/pathologists should perform/order IHC testing first, followed by ISH when IHC result is 2+ (equivocal);
positive (3+) or negative (0 or 1+) HER2 IHC results do not require further ISH testing
• Pathologists should select the tissue block with the areas of lowest grade tumor morphology in biopsy and resection specimens; more than 1 tissue block may be
selected if different morphologic patterns are present
Recommendation
• Pathologists should use the Ruschoff/Hofmann method in scoring HER2 IHC and ISH results for GEA
Strong recommendation
Strong recommendation
• Laboratories/pathologists must specify the antibodies and probes used for the test and ensure that assays are appropriately validated for HER2 IHC and ISH on
GEA specimens
Recommendation
• Treating clinicians should offer combination chemotherapy and HER2-targeted therapy as the initial treatment for appropriate patients with HER2-positive tumors
who have metastatic or recurrent GEA
Recommendation
• Treating clinicians or a pathologist should request HER2 testing on tumor tissue in the biopsy or resection specimens (primary or metastasis), preferably before the
initiation of trastuzumab therapy if such specimens are available and adequate; HER2 testing on FNA specimens (cell blocks) is an acceptable alternative

13. HER2, HER3, and TROP2 as Therapeutic
Targets in Lung Cancer
Full abbreviations, accreditation, and disclosure information available at
PeerView.com/EZK40
•
HER2 is overexpressed, amplified, or mutated in a
significant fraction of lung adenocarcinomas, but
prognostic significance is not yet fully defined
–
Frequency of overexpression (IHC 2+ and 3+): 15%-30%
– Frequency of overexpression (IHC 3+): 2%-6%
– Frequency of amplification: 2%-6%
– Frequency of mutations: 1%-5%
•
HER2 dysregulations encompass heterogeneous
and distinct alterations which must be taken into
consideration
–
There is a lack of correlation between overexpression,
amplification, and mutations
–
Cohorts must be defined according to the specific HER2
alteration present
•
The appropriate testing methodology for different HER2
alterations should be used
– Amplification: in situ hybridization (ISH)
– Expression: immunohistochemistry (IHC)
– Mutation: sequencing (NGS or other)
•
Although they have become outdated in the context of
the rapid advances in targeted therapy for lung cancer
and related biomarker testing demands, the latest
published lung cancer molecular testing guidelines state
that HER2 molecular testing is not indicated as a routine
stand-alone assay to guide targeted therapy selection
outside the context of a clinical trial. However, it is
appropriate to include HER2 mutation analysis as part of
a larger testing panel performed either initially or when
routine testing results for other alterations with approved
targeted therapies are negative.
•
There is a rationale for investigating HER2-targeted
therapies in NSCLC
•
Antibody–drug conjugates (ADCs) represent a promising
therapeutic approach; agents under investigation
include:
–
Trastuzumab deruxtecan (T-DXd)—in various settings
of HER2-expressing and HER2-mutated NSCLC
– Ado-trastuzumab emtansine (T-DM1)
• HER3 is highly expressed in NSCLC
•
HER3 overexpression is associated with metastatic
progression and poor outcomes in patients with NSCLC
•
There are currently no established biomarker testing
recommendations for HER3 in NSCLC
•
HER3 alterations are not known to be a mechanism of
resistance to EGFR TKIs in EGFR-mutated NSCLC
•
Targeting HER3 may address multiple EGFR-targeted
therapy resistance mechanisms
•
There is a rationale for investigating HER3-targeted
therapies in NSCLC
•
ADCs represent a promising therapeutic approach: eg,
anti-HER3 ADC patritumab deruxtecan
(HER3-DXd) is under investigation in various
settings of EGFR-mutated advanced NSCLC
HER1 HER2 HER3 HER4
EGFR family
Cytoplasm
Nucleus
Chr17
HER2
• Proliferation
• Motility
• Invasiveness
• Survival
• Angiogenesis
P P
PI3K
Akt
SOS
RAS
RAF
MEK
MAPK
P
P
Ligands
Role of HER2 in NSCLC1-7
Role of HER3 in NSCLC8

14. HER2, HER3, and TROP2 as Therapeutic
Targets in Lung Cancer
Full abbreviations, accreditation, and disclosure information available at
PeerView.com/EZK40
Role of TROP2 in NSCLC9-12
•
TROP2 is highly expressed in NSCLC and has been
associated with poor prognosis
•
There are currently no established biomarker testing
recommendations for TROP2 in NSCLC
•
There is a rationale for investigating TROP2-targeted
therapies in NSCLC
•
ADCs represent a promising therapeutic approach:
eg, the anti-TROP2 ADC datopotamab deruxtecan
(Dato-DXd) is under investigation in various settings
of NSCLC with and without actionable genomic
alterations
1. Li BT et al. J Thorac Oncol. 2016;11:414-419. 2. Li BT et al. International Association for the Study of Lung Cancer 18th World Conference on Lung Cancer (WCLC 2017). Abstract OA14.05. 3. Li BT et al.
J Clin Oncol. 2018;36:2532-2537. 4. Iqbal N et al. Mol Biol Int. 2014;2014:852748. 5. Yan M et al. Cancer Metastasis Rev. 2015;34:157-164. 6. Rolfo C et al. Cancer Discov. 2020;10:643-645. 7. Lindeman NI et al.
Arch Pathol Lab Med. 2018;142:321-346. 8. Scharpenseel H et al. Sci Rep. 2019;9:7406. 9. Mito R et al. Pathol Int. 2020;70:287-294. 10. Inamura K et al. Oncotarget. 2017;8:28725-28735. 11. Jiang A et al.
Oncol Lett. 2013;6:375-380. 12. Lenart S et al. Cancers (Basel). 2020;12:3328.
Fibronectin
Decreased adhesion
Increased cell migration
ErB3 activation
NRG-1 is released
when TROP2 is lost
Recruitment to
tight junctions
NRG-1
TACE
Clauidn 1/7
TROP2
α5 β1
Talin
RACK 1
RACK 1
FAK
P
FAK
Src
Src
P
NRG-1
Endosome