QuantifyHER: Quantitative Immunofluorescence and/or RT-qPCR for Measuring HER2 in HER2-low Metastatic Breast Cancer
Conditions: HER2-positive Metastatic Breast Cancer
Sponsor: Abramson Cancer Center at Penn Medicine

 
 
TBCRC 066 Protocol 
 
Version 1.19.24 
 
 
QuantifyHER 
Quantitative Immunofluorescence and/or RT-qPCR for Measuring HER2 in HER2-low 
Metastatic Breast Cancer  
 
Protocol Number 
 
TBCRC 066   
 
Protocol Chair 
 
Angela DeMichele MD, MSCE 
 
 
 
 
Rowan Breast Center of the Abramson Cancer Center 
University of Pennsylvania  
Perelman Center, 3rd Floor  
3400 Civic Center Blvd, Philadelphia PA 19104 
Phone:  215-615-6820 
Fax:  215-615-3349 
Email: angela.demichele@pennmedicine.upenn.edu  
 
Co-Chair  
David Rimm MD, PhD  
 
Yale University School of Medicine  
 
david.rimm@yale.edu  
 
Co-Chair  
 
Co-Investigators 
Eleanor Taranto MD  
 
Department of Hematology & Oncology 
Hospital of the University of Pennsylvania  
Eleanor.taranto@pennmedicine.upenn.edu 
 
 
Statistician 
E. Paul Wyleto, PhD  
 
 
Perelman School of Medicine at the University of Pennsylvania   
 
Supporters 
Johns Hopkins University on behalf of the Translational Breast 
Cancer Research Consortium (TBCRC) 
 
Danaher/Cepheid, Inc  
 
American Society of Clinical Oncology, via Gianni Bonadonna 
Breast Cancer Research Fellowship  
 
Coordinating Center  
University of Pennsylvania  
 
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QuantifyHER 
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TBCRC 066  
 
 
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QuantifyHER 
Quantitative Immunofluorescence and/or RT-qPCR for Measuring HER2 in HER2-low 
Metastatic Breast Cancer  
 
- Protocol Revision Record - 
 
 
Original Protocol: 
 
1/19/2024 
 
 
 
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Table of Contents 
1 .      SUMMARY / SCHEMA ................................................................................................................................... 5 
2. 
OBJECTIVES .................................................................................................................................................... 7 
2.1 
PRIMARY OBJECTIVE ................................................................................................................................... 7 
2.2 
SECONDARY OBJECTIVES ............................................................................................................................ 7 
3. 
BACKGROUND ................................................................................................................................................ 8 
3.1 
HER2-LOW METASTATIC BREAST CANCER ................................................................................................ 8 
3.2 
HER2 ASSESSMENT METHODS ..................................................................................................................... 8 
3.3 
QUANTITATIVE HER2 ASSESSMENT METHODS: IMMUNOFLUORESCENCE .................................................. 9 
3.4 
QUANTITATIVE HER2 ASSESSMENT METHODS: MRNA ............................................................................. 9 
3.5 
RATIONALE ............................................................................................................................................... 10 
4. 
PARTICIPANT SELECTION ........................................................................................................................ 11 
4.1 
INCLUSION CRITERIA ................................................................................................................................. 11 
4.2 
EXCLUSION CRITERIA ............................................................................................................................... 11 
4.3 
INCLUSION OF UNDERREPRESENTED POPULATIONS .................................................................................. 11 
5. 
REGISTRATION PROCEDURES ................................................................................................................ 12 
5.1 
GUIDELINES FOR REGISTRATION ............................................................................................................... 12 
6. 
STUDY CALENDAR ...................................................................................................................................... 13 
7. 
STUDY PLAN .................................................................................................................................................. 14 
7.1 
RECRUITMENT ........................................................................................................................................... 14 
7.2 
INTERVENTION PLAN ................................................................................................................................. 14 
7.3 
SAFETY ASSESSMENTS .............................................................................................................................. 14 
7.4 
DURATION OF FOLLOW-UP ....................................................................................................................... 14 
7.5 
WITHDRAWAL FROM STUDY ..................................................................................................................... 15 
7.6 
RETURN OF RESULTS ................................................................................................................................. 15 
7.7 
ADDITIONAL INFORMATION ...................................................................................................................... 15 
8. 
SAFETY ASSESSMENTS .............................................................................................................................. 16 
9. 
CORRELATIVE STUDIES ............................................................................................................................ 17 
9.1 
TISSUES SAMPLES ..................................................................................................................................... 17 
9.2 
COLLECTION ............................................................................................................................................. 17 
9.3 
PROCESSING AND STORAGE ....................................................................................................................... 17 
9.4 
LABORATORY ANALYSIS METHODS .......................................................................................................... 17 
9.5 
SPECIMEN SUBMISSION REQUIREMENTS ................................................................................................... 18 
9.6 
ADDITIONAL INFORMATION ...................................................................................................................... 18 
10. 
SPECIMEN BANKING .............................................................................................................................. 18 
11. 
MEASUREMENT OF EFFECT ................................................................................................................ 19 
11.1 
EVALUABLE FOR OBJECTIVE RESPONSE .................................................................................................... 20 
11.2 
RESPONSE CRITERIA.................................................................................................................................. 20 
11.3 
RESPONSE REVIEW .................................................................................................................................... 21 
12. 
DATA AND SAFETY MONITORING ..................................................................................................... 21 
12.1 
DATA MANAGEMENT AND REPORTING ..................................................................................................... 21 
12.2 
SOURCE DOCUMENTS ................................................................................................................................ 21 
12.3 
CASE REPORT FORMS ................................................................................................................................ 22 
12.4 
MEETINGS ................................................................................................................................................. 22 
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12.5 
MONITORING ............................................................................................................................................. 22 
12.6 
CONFIDENTIALITY ..................................................................................................................................... 22 
13. 
REGULATORY CONSIDERATIONS ..................................................................................................... 22 
13.1 
PROTOCOL REVIEW AND AMENDMENTS .................................................................................................... 23 
13.2 
INFORMED CONSENT ................................................................................................................................. 23 
13.3 
ETHICS AND GOOD CLINICAL PRACTICE ................................................................................................... 23 
13.4 
COMPLIANCE WITH TRIAL REGISTRATION AND RESULTS POSTING REQUIREMENTS ................................. 24 
14. 
MULTI-CENTER GUIDELINES ............................................................................................................. 24 
14.1 
STUDY DOCUMENTATION .......................................................................................................................... 24 
14.2 
RECORDS RETENTION ................................................................................................................................ 24 
14.3 
PUBLICATION ............................................................................................................................................ 24 
15. 
STATISTICAL CONSIDERATIONS ....................................................................................................... 25 
15.1 
STUDY DESIGN .......................................................................................................................................... 25 
15.2 
STUDY ENDPOINTS .................................................................................................................................... 25 
15.3 
SAMPLE SIZE/ ACCRUAL RATE .................................................................................................................. 26 
15.4 
ANALYSIS OF PRIMARY ENDPOINT ............................................................................................................ 26 
15.5 
ANALYSIS OF SECONDARY ENDPOINTS ..................................................................................................... 27 
15.6 
REPORTING AND EXCLUSIONS ................................................................................................................... 27 
REFERENCES .......................................................................................................................................................... 28 
APPENDICES ............................................................................................................................................................ 30 
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1 .      SUMMARY / SCHEMA 
 
Metastatic breast cancer remains incurable despite significant advances in both understanding of 
the disease and treatment innovations. Balancing treatment effectiveness with quality of life is 
therefore integral to the care of the patient. Trastuzumab-deruxtecan (T-DXd), an antibody-drug 
conjugate, was recently approved for patients with metastatic breast cancer expressing low levels 
of HER2 by immunohistochemistry (IHC 1+ or 2+, FISH negative). However, there is 
significant inter-reader variability in calling HER2 IHC 1+ vs IHC 0, raising concern for 
inappropriate patient selection for T-DXd.  
 
Rimm et al. have developed a quantitative immunofluorescence (QIF) HER2 assay that, in 
serially collected samples, has identified up to 20% of IHC 1+ samples below the level of 
quantification using QIF.  HER2 messenger RNA (mRNA) assessment is also being explored as 
an alternative quantitative method.  
 
We now propose to validate these quantitative assays by conducting a multi-site, longitudinal 
cohort study (“QuantifyHER”) within the Translational Breast Cancer Research Consortium 
(TBCRC). This study will assess whether a quantitative, immunofluorescence-based HER2 assay 
and/or a HER2 mRNA RT-qPCR assay can accurately and reliably discriminate between 
responders and non-responders among patients with HER2 IHC1+ metastatic breast cancer who 
are receiving T-DXd.  
 
Patients with biopsy-proven metastatic breast cancer that is HER2 1+ by IHC and who are 
initiating T-DXd will be enrolled prospectively and followed.  Key eligibility criteria include 
available tissue, measurable disease, and ability to provide informed consent. Patients with prior 
HER2 positive disease are excluded. Patient data (demographics, clinical features, and follow 
up) will be recorded in case report forms and stored in a Penn-managed REDCap database. For 
each patient, 9 unstained FFPE sections and 1 H&E section will be sent to Dr. David Rimm at 
Yale for quantitative immunofluorescence analysis (a.k.a. the Troplex assay) yielding a HER2-
expression value (QIF) and ERBB2 mRNA level assessment via Cepheid Xpert Breast Cancer 
STRAT4 (RUO) assay.  Slides will be collected for these two purposes, but remaining slides will 
be stored and may be used for testing beyond these two planned assays (the informed consent 
will include the possibility of additional research to assess resistance factors or related 
biomarkers). Clinical endpoints (response, death) will be assessed by local investigators using 
real-world response categories. The primary objective is to determine if there is a relationship 
between QIF of HER2 protein expression and/or quantitative mRNA levels of ERBB2 and real-
world objective response (rwORR). For the planned 200 patients and a one-sided type-one error 
of 2.5%, we will have 80% power to detect a difference in proportions of 9 to 10 percentage 
points for each standard deviation increase in QIF or mRNA across the range of interest, such as 
29% versus 20%, or 34.5% versus 25%.  This corresponds to an odds ratio of approximately 1.6.  
A non-binding interim review at n=70 will assess preliminary biomarker distribution and allow 
for necessary sample size adjustment. Secondary endpoints will include real-world progression-
free survival, overall survival, threshold QIF value for response, relationship between 
QIF/mRNA level and estrogen-receptor status, and relationship between HER2 mRNA level and 
response. We will conduct an exploratory analysis of rwORR using QIF and mRNA jointly to 
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determine whether a combination is better than either alone at discriminating responders from 
non-responders.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
QuantifyHER Study Schema
Eligibility:
• Metastatic breast 
cancer, 
histologically 
confirmed
• HER2 IHC 1+ (per 
local pathology lab)
• Measurable 
disease
• Available tissue for 
biomarker 
assessment
• Intention to initiate 
therapy with T-DxD
10 unstained FFPE slides 
submitted to Rimm Lab 
(Yale) after enrollment
N=200 evaluable 
participants
Patient consent
Off study at T-DXd 
discontinuation
Treatment with T-DxD 
per standard of care 
5.4 mg/kg given as an 
intravenous infusion once every 
3 weeks (21-day cycle)
Site to submit slides from 
(1) most recent metastatic 
biopsy and (2) any prior 
biopsy that has been read 
as Her2 1+ by IHC, not to 
exhaust tumor sample
Analysis of HER2 
expression via QIF and 
mRNA assays
Endpoints*:
Primary: Real-World ORR
Secondary: Real-World PFS
*per standard real world 
published methodologies 
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2.    OBJECTIVES 
2.1   Primary Objective 
2.1.1 To determine whether there is a relationship between low levels of tumor HER2 
measured by quantitative immunofluorescence (QIF) and/or mRNA by RT-qPCR and 
real-world response (rwORR) to T-DXd among patients with metastatic breast cancer 
whose tumors are deemed HER2 1+ by immunohistochemistry.   
Primary Endpoint:  Association between quantitative HER2 expression (as a 
continuous variable) and real-world objective response rate (rwORR) 
2.2   Secondary Objectives 
2.2.1 To determine whether there is a relationship between low levels of tumor HER2 
measured by QIF and/or mRNA by RT-qPCR and real-world progression-free 
survival (rwPFS) to T-DXd among patients with tumors deemed HER2 1+ by 
standard immunohistochemistry. 
Endpoint: Association between quantitative HER2 expression (as a continuous 
variable) and rwPFS. 
2.2.2 To determine the relationship between HER2 expression by QIF and mRNA and both 
rwPFS and rwORR, stratified by estrogen receptor (ER) expression status. 
Endpoint: Association between HER2 expression by quantitative 
immunofluorescence and mRNA in HER2 IHC 1+ tumors and both rwORR and 
rwPFS, stratified by mRNA ER expression.  
2.2.3 To determine whether a cut-off value exists for HER2 QIF, mRNA or the 
combination below which patients will not respond to T-DXd.  
Endpoint: ROC thresholds for HER2 QIF and/or mRNA levels that discriminate 
responders from non-responders by identifying the lowest levels that perform 
better than a random classifier.   
2.2.4 To determine, based on rwORR, whether a linear combination of HER2 QIF and 
mRNA is better than either alone at discriminating T-DXd responders from non-
responders. 
Endpoint: Association between combined mRNA + QIF and rwORR  
 
 
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3.    BACKGROUND 
3.1  HER2-low Metastatic Breast Cancer  
Metastatic breast cancer (MBC) remains incurable despite significant advances in both 
our understanding of the disease as well as treatment innovations. Therefore, balancing 
treatment effectiveness with quality of life is integral to the overall care of the patient given that 
therapies are palliative in nature. Although HER2-targeted therapies have historically been 
restricted to MBC that is defined as HER2+ by standard ASCO/CAP guidelines, recent 
advances in antibody-drug conjugate (ADC) design have led to striking results, as exhibited by 
one such ADC, trastuzumab deruxtecan (T-DXd), in patients with HER2-negative MBC that 
expressed low levels of HER2 (1+ or 2+ by immunohistochemistry [IHC]). T-DXd has 
demonstrated objective response rates (ORR) of up to 50% in patients with estrogen-receptor 
(ER)-positive and ER-negative MBC expressing HER2 IHC 1+/2+ and has doubled 
progression-free survival (PFS) and improved overall survival (OS) compared to standard 
chemotherapies. This has led to classification of HER2 IHC 1+ or 2+/FISH non-amplified 
tumors as “HER2-low.” 
 
3.2  HER2 assessment methods  
The standard immunohistochemistry (IHC) assays using antibodies against HER2 were 
initially designed to reliably identify patient tumors with very high levels of HER2, without 
focus on the low end of the expression spectrum. HER2 grading by IHC has also been found to 
be subjective and pathologist dependent. A recent study surveying 1400 laboratories around the 
world suggested < 70% interrater agreement between HER2 IHC 0 and 1+ among 19% of the 
laboratories surveyed, and a real world sub-study of 170 tissue sections showed only a 26% 
concordance rate among pathologists in calling HER2 IHC 0 (see Figure 1).1  Another study 
investigating HER2 mRNA levels showed similar levels between HER2 0 and HER2 IHC 1+, 
and significantly different compared to HER2 2+ FISH non-amplified2, suggesting 
misclassification by IHC. Given the toxicity profile for T-DXd, including rates up to 12% for 
interstitial lung disease (ILD)/pneumonitis3, a reliable quantitative assay with high precision for 
the presence of low levels of HER2 tumor expression is urgently needed to ensure optimal 
patient selection. 
Figure 1: Percent HER2 Status By Pathologist 
 
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3.3  Quantitative HER2 Assessment Methods: Immunofluorescence  
The Rimm Lab at Yale University School of Medicine has developed a quantitative 
immunofluorescence (QIF) assay, known as the Troplex Assay, using automated quantitative 
analysis (AQUA).4   This technique has been used to look at HER2 expression in a serial 
collection of breast cancer cases, with work that has now defined the limits of detection (LOD), 
quantification (LOQ), and linearity 
(LOL) for the HER2 QIF assay 
using standard analytic methods.4  
The assay has now been validated 
on over 400 serially collected 
samples from 2011-2014 and over 
250 breast core needle biopsies 
collected prospectively. Rimm and 
colleagues have found that many 
cases considered HER2 negative 
by conventional assay have 
quantifiable levels of HER2 
protein expression (above the 
LOQ), and that a significant proportion of cases called IHC 1+ in fact show HER2 protein levels 
below the limit of quantification (see Table 1, Rimm et. al, Manuscript in process).    
 
3.4  Quantitative HER2 Assessment Methods: mRNA 
 
Cepheid’s Xpert® Breast 
Cancer STRAT4 test can assess 
mRNA for ESR1, PGR, ERBB2, 
and MKi67 in <3 hours.  The 
STRAT4 test, currently available 
for research use only (RUO) in 
the United States but 
commercially available in several 
other countries, produces 
reproducible results, 
demonstrating high concordance 
with central IHC measures.5 In a 
retrospective validation study of 
500 breast cancer cases, overall 
percent agreement between 
STRAT4 and centrally tested 
IHC/FISH was 97.8% for ESR1, 
90.4% for PGR, 93.3% for 
ERBB2, and 78.6% for MKi67 
(see Figure 2).6 Another 
independent validation study of 
                               
                                      
                                                            
        
        
        
        
                                                      
                                                                                                                      
Table 1: Comparison of HER2 by IHC Versus QIF 
Figure 2: Xpert Breast Cancer STRAT4 Performance 
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200 samples found similar rates of concordance between STRAT4 and IHC7, and an unpublished 
pooled analysis representing ~1000 unique breast cancers has further confirmed the correlation.  
In the bottom panel of Figure 2, the cases outlined by the (red) dashed lines represent the lower 
end of the mRNA spectrum, highlighting significant overlap of ERBB2 mRNA expression, 
quantified as delta cycle threshold (delta CT, or dCt) levels across multiple IHC levels (0, 1+, 
2+).  In-house experiments have characterized the limits of quantification (LoQ) and detection 
(LoD) for ERBB2, but these proposed dCt detection cutoffs have not yet been tested or 
correlated with outcomes in patients with “HER2-low” breast cancer treated with HER2-ADCs. 
3.5 Rationale  
 
Given the challenges with interpreting semi-quantitative immunohistochemistry (IHC) 
results—especially when it comes to low levels of HER2 expression—this prospective 
observational study will assess two new quantitative approaches to measure HER2 protein 
expression, and their individual and collective abilities to predict response to treatment among 
those with metastatic breast cancer who are starting treatment with Trastuzumab Deruxtecan (T-
DXd).  We will identify a cohort of patients newly diagnosed with HER2 low (1+) metastatic 
breast cancer who are starting treatment with T-DXd. We will assess their biopsy samples at the 
time of diagnosis with metastatic disease (as well as prior HER2 low samples) for HER2 
expression via the QIF Troplex and mRNA STRAT4 assays.  We will then follow them 
prospectively throughout the course of their treatment with T-DXd.  
 
We propose to prospectively validate the QIF assay as a way to more reliably measure 
low levels of HER2 surface protein, and to assess whether QIF or mRNA levels can discriminate 
responders from non-responders to T-DXd in the HER2 1+ population. We also plan to examine 
the relationship between QIF scores and other key clinical endpoints (ORR, PFS, OS) using “real 
world” response assessment, and to stratify results by estrogen receptor status. If cut-off points 
can be identified that discriminate responders from non-responders to T-DXd, this assay could 
not only improve patient selection for T-DXd but could be scaled and adapted for next 
generation ADCs for any measurable target.  
 
Another key objective will be to investigate the utility of measuring HER2 messenger 
RNA (mRNA) levels as a separate predictor for response to T-DXd, and to assess whether HER2 
QIF and mRNA are jointly superior to either alone for predictive modeling.  By collecting real-
world response data in patients with metastatic breast cancer treated with T-DXd, this approach 
will not only seek to validate the ability of the assays to discriminate between responders and 
non-responders to T-DXd but will also produce results that are immediately generalizable to a 
broader population and to clinical practice.  
 
 
 
 
 
 
 
 
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4.     PARTICIPANT SELECTION 
4.1   Inclusion Criteria 
4.1.1 Women and men age > 18 years  
4.1.2 Metastatic breast cancer, histologically- confirmed. Any estrogen receptor (ER) status 
is allowed. ER status will be determined by local laboratory assessment utilizing 
ASCO/CAP guidelines.  
4.1.3 Primary and/or metastatic tumor with 1+ level of expression of HER2 by 
immunohistochemistry as determined by local laboratory assessment utilizing 
ASCO/CAP guidelines.   
4.1.4 Measurable disease by cross-sectional imaging at the start of treatment. Patients with 
measurable bone-only disease or active brain metastases are eligible.   
4.1.5 Archival tissue available for biomarker assessment. One specimen should be the most 
recent metastatic biopsy. If HER2 1+ status was determined on a different specimen 
(either primary or metastatic tissue), that specimen is also required.  Samples obtained 
from bone metastases that were processed via decalcification methods are not 
eligible.  
4.1.6 Intention to initiate therapy with T-DXd (Enhertu) at FDA-approved dose and 
schedule as next line of therapy. If T-DXd was already initiated, patients must be 
registered within 30 days of initiation.  
4.1.7 Ability to provide informed consent.  
4.2   Exclusion Criteria 
4.2.1 Concurrent Her2-overexpressing metastatic breast cancer (as confirmed by a 
metastatic biopsy with IHC 3+ or IHC 2+ with FISH amplified as per standard 
ASCO/CAP guidelines)  
4.3   Inclusion of Underrepresented Populations 
Individuals of all races and ethnic groups are eligible for this trial. There is no bias towards 
age or race in the clinical trial outlined. This trial is open to the accrual of both men and 
women. 
 
 
 
 
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5.    REGISTRATION PROCEDURES 
 
The University of Pennsylvania (Penn) will serve as the lead institution.  Penn will serve 
as the central site for protocol development and amendments, data management, and study 
monitoring. All subjects must be registered through the study specific REDCap data portal. 
All subjects who sign an informed consent document will be entered into and tracked in 
REDCap, including for collection of demographics and outcome of eligibility. Subject 
eligibility must be reviewed and confirmed by the lead institution prior to formal 
registration.  Confirmation of registration will be communicated by the lead site to the local 
site via email. A subject is considered registered when an “On Study” date is entered into 
the Registration case report form (CRF) in REDCap. This process will be managed 
centrally by the lead institution. 
 
5.1 Guidelines for Registration 
 
Key steps for site personnel to register participants and generate the study ID are 
summarized below. Additional details can be found in the accompanying Study 
Manual. 
 
• Documentation of informed consent  
• Entry into the study specific REDCap data portal which generates a unique 
participant study ID 
• Completion of the eligibility checklist with accompanying source 
documentation uploaded 
• Confirmation of subject eligibility by the lead site and formal registration 
• Following successful registration, the site will gain access to all necessary 
forms (including biospecimen submission forms, clinical, imaging, and 
pathology CRFs) 
 
Participants must be registered with the lead institution prior to archival tissue 
submission. 
 
Participants must be registered within 30 days of the initiation of T-DXd. Participants 
should begin T-DXd treatment as per their treating physician. Issues that cause 
treatment delays should be documented in CRFs. If a participant does not receive T-DXd 
or if samples are not received by the Rimm Lab, the participant’s registration on the study 
may be canceled.  The lead site should be notified of events leading to cancellation as soon 
as possible. 
 
Any requests for eligibility exceptions and/or deviations must be approved by the 
Protocol Chair or designee prior to execution.    
 
Please contact the Penn project manager or designee via study specific email address with 
any questions regarding this process. 
 
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Registration will only be conducted during the business hours of 8 am – 6 pm (EST) 
Monday through Friday.  
 
 
6.    STUDY CALENDAR 
 
Procedure 
Baseline1 
Every  
6 weeks (2 cycles) 
 
Every Scan Visit 
(approximately every 3 
months)  
 
End of SOC treatment 
with T-DXd 
CLINICAL ASSESSMENT: 
 
 
 
 
Informed consent 
X 
 
 
 
Medical History  
X 
 
 
 
Performance Status  
X 
 
 
 
DEMOGRAPHICS 
X 
 
 
 
IMAGING: 
 
 
 
 
CT/MRI/Bone scan/PET 
X 
 
X 
X 
SAMPLES: 
 
 
 
 
Archival tumor sample2 
X 
 
 
 
OTHER ASSESSMENTS: 
 
 
 
 
Follow-up/Survival3,4 
 
X 
X 
X 
 
Note:  Additional tests may be performed at the discretion of the treating physician as 
clinically indicated.  The schedules outlined above are based on an ideal subject, and all 
visits will occur as per the treating physician and the standard of care.  The schedule may be 
modified due to problems such as scheduling delays or conflicts (e.g., clinic closure, poor 
weather conditions, vacations, etc.). 
 
1. Information will be collected about the breast cancer diagnosis, pathology, 
imaging/radiology, treatment(s) received. This should occur within 4 weeks prior to 
starting treatment, unless otherwise noted. 
2. Samples collection is outlined in Section 9.  
3. Subjects will be followed approximately every 6-12 weeks per local clinical practice for 
disease status and survival until discontinuation of T-DxD, the study has ended or is 
cancelled, or the subject asks to discontinue participation. The exact frequency of these 
visits will be determined by the treating physician.  
4. At each follow-up visit (i.e. each infusion visit, each scan review visit, and any visits that 
lead to treatment modification or discontinuation), the plan for treatment continuation or 
discontinuation will be recorded in CRFs, with input from the treating physician as needed. 
The reason for discontinuation will be recorded, including whether for disease progression, 
drug toxicity, or other reasons.     
 
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7.    STUDY PLAN 
7.1   Recruitment  
Patients will be recruited through the breast cancer clinics at each of the participating 
centers. Patients will be automatically assigned a sequential participant Study ID when 
their enrollment information is initially submitted to REDCap.  The instructions for 
subject registration are noted above, with further details supplied in the study manual. 
Patients must enroll at a participating site, but may receive treatment with T-DXd locally, 
so long as biopsy samples and source data—including records of visits with clinical 
status, imaging reports, treatment administration, and treatment discontinuation (with 
reason for discontinuation)—are available to the study team. Patients must be registered 
within 30 days of T-DXd initiation.  
7.2   Intervention Plan  
 
This is a prospective, observational study of patients receiving T-DXd per standard of care. 
The patient and treating physician will make clinical decisions involving treatment without 
knowledge of the findings of any investigational laboratory studies conducted using 
collected specimens or generated research data. The protocol does not dictate T-DXd dose 
reductions; any other drug-related decisions will be made per normal clinical practice and 
the standard of care. Information regarding decisions to discontinue therapy, whether due 
to toxicity, progression, or other reasons, will be recorded in visit-based case report forms 
(CRFs) that will be submitted to the central coordinating center via electronic database 
(REDCAP).  
7.3   Safety Assessments   
 
This is a prospective observational study of a standard-of-care medical intervention. It 
involves only the collection of data at the time of standard clinical assessments.  Because 
there is no investigational therapy, collection and documentation of individual participant 
adverse events per CTCAE are not warranted. The study will collect information 
regarding drug toxicity only as it pertains to T-DXd dose reductions and discontinuation 
of therapy.  This information will be recorded in visit-based case report forms (CRFs) 
that will be submitted to the central coordinating center via electronic database. 
7.4   Duration of Follow-Up  
Participants will be followed until T-DxD discontinuation, death, or written 
withdrawal from study participation, whichever occurs first.  
 
In the event that a subject does not continue care at the treating institution, every attempt 
will be made to collect this information by direct contact or through communication with 
the outside physician(s).  
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7.5   Withdrawal from Study 
 
Participants may choose to no longer participate in this project at any time. Specifically, 
withdrawal of consent to participate in the study must be requested in writing to the lead site. 
Participants may also ask that the previously collected samples be destroyed; however, data 
on any research done until the date of the request will be retained. The decision to withdraw 
will not affect any research already conducted and/or results generated. All reasons for study 
discontinuation should be documented clearly in the medical record and in the REDCAP 
survey.  
 
The reasons for withdrawal from the study include: 
 
• 
Subject withdraws consent for follow-up. 
• 
Subject is lost to follow-up. 
• 
The study is terminated for any reason. 
The decision to no longer take part in this project will not affect a participant’s care at the 
enrolling institution.  
7.6   Return of Results  
 
During the consent process, patients will be able to opt in to return-of-results.  The results 
they will receive will be the QIF assay results, which are performed in a CLIA 
Laboratory. Results will be provided to patients who consent for return of results at the 
completion of their participation in the trial. Patients will be given the option to have their 
results shared with them and their treating physician, along with a patient education 
packet about the results. This report will go initially to the participating site where the 
patient was enrolled, and then the site will contact patients to disclose and share results.  
Patients will not be given the results of the mRNA/RT-qPCR testing as this test is not yet 
clinically validated.  
7.7   Additional Information  
 
Patients will not be paid for their participation in this observational study. Remuneration 
for participants in appreciation for their time (e.g., parking stickers) is not currently 
provided by this trial, however may be given per institutional standards.  
 
At the conclusion of the study and upon publication of any manuscript(s) or posting of data 
on public websites (e.g., Clinicaltrials.gov per federal guidelines), consideration will be 
made to providing participants with a notification of available results. Any such 
notification will be approved by the respective IRB at each site prior to dissemination to 
participants, as needed.  
 
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8.    SAFETY ASSESSMENTS 
 
This is a prospective, observational study of standard treatment in the clinical setting.  
There is no risk to patient safety specifically from participating in the study. This study 
involves the collection of data likely to occur at the time of planned clinical assessments 
and sample collection for routine care. Therefore, collection of toxicity or other events 
related to the standard treatment and procedures being received will be collected only as 
it pertains to discontinuation of T-DXd.   
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9.    CORRELATIVE STUDIES 
 
Patients enrolled on this study are required to have tissue samples available at the time of 
enrollment.  
 
NOTE: A separate laboratory worksheet/manual will be provided to outline the specific specimen 
identification/collection parameters and shipping instructions for all research samples.  
9.1   Tissues Samples 
Tumor tissue samples from a primary tumor and/or metastatic biopsy which 
demonstrate HER2-low (1+) disease by immunohistochemistry at the local site, must 
be available. Samples will be shipped directly to Dr. David Rimm’s lab at Yale 
University as per the laboratory manual. If the HER2-low (1+) disease was 
established from a prior biopsy, samples from both the biopsy at the time of 
metastatic disease and the biopsy demonstrating HER2 1+ status must be shipped to 
the Rimm Lab.  
9.2   Collection  
Participants will be asked permission in the consent form to access tissue collected as 
part of routine care at the time of initial diagnosis/diagnostic core biopsy and again at 
surgical resection for correlative studies. Either sections/slides or blocks may be 
provided at the discretion of the pathologist.  
 
The specifics of slide requirements: Formalin Fixed Paraffin Embedded (FFPE) 
unstained sections on charged glass slides, including an H&E stained section, must be 
collected from the metastatic biopsy that demonstrated HER2 1+ status by 
immunohistochemistry. 9 unstained FFPE sections (5-micron thick) and 1 H&E 
section will be collected. Site collection should avoid exhausting available tumor for 
a given patient.    
9.3   Processing and storage   
All tissue samples will be sent to and stored at Yale University until analysis. A 
separate laboratory worksheet/manual will be provided to outline the specific 
collection parameters and shipping instructions for all research samples. Slides will 
be stored at -80 until processing for HS-HER2 or RT-qPCR.  Remaining slides will 
be stored at -80 until further usage. 
The study specimens will be recorded, de-identified, handled, processed, and shipped 
according to the protocol standard operating procedures detailed in the REDCap 
study manual and the study laboratory manual. 
9.4   Laboratory Analysis Methods  
Correlative samples will be analyzed with collaboration from the University of 
Pennsylvania, Yale University, and Cepheid, Inc.  
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9.4.1  HER2 Quantitative Immunofluorescence (QIF) Assessment  
Measurement of HER2 QIF via Troplex Assay is based on quantitative 
immunofluorescence (QIF) as described in Moutafi et al.4 This approach uses a 
series of cell lines with known levels of HER2 protein as previously determined by 
mass spectrometry. These lines are used in a cell line microarray (CMA) used as a 
standard in every assay. For every stainer batch (up to 9 cases), one CMA slide is 
included and used to create a stainer run-specific standard curve to calculate the 
HER2 level (in attomole/mm2) for each case. One section is stained with H&E stain 
and is used to define the region of the tissue section that is measured.  A pathologist 
views each case and, using a pseudo-IHC image generated by the Cytefinder II scan, 
software conversion and the scanned H&E if necessary, chooses the area of the slide 
to be measured that represents the diagnostic metastatic tumor. The measurement is 
provided in amol/mm2 as an average across the region of interest selected by the 
pathologist.  
 
9.4.2 HER2, ER/PR mRNA Assessment 
Messenger RNA levels will be measured quantitatively using the Cepheid STRAT4 
assay. This assay, run on the Cepheid GeneXpert platform, measures the cycle 
threshold in a sensitive closed system quantitative RT-qPCR approach normalized 
by an internal “housekeeping” gene mRNA (CYFIP1) as described previously6,8. In 
this study, two unstained slides will be placed on top of a tumor-annotated H&E and 
the tumor region will be scraped and processed per STRAT4 protocol. A cycle 
threshold is determined for each case and normalized by the CYFIP1 gene to return a 
deltaCT (dCT) value.  
9.5   Specimen Submission Requirements 
The above specimens are to be submitted to the indicated lab. Refer to the separate 
laboratory manual for additional processing and shipping instructions. Sample FFPE 
lysis kits and STRAT4 carts will be provided to the testing lab by Cepheid.  
9.6   Additional Information 
The laboratory technicians will document information with the collection conditions, 
processing and storage information for correlative samples.  
 
The laboratory and radiology investigators will be blinded to subject identifiers and 
clinical data while generating the research data. Additionally, the reported results will 
not disclose any unique patient identifiers.  
 
A separate laboratory worksheet/manual will be provided to outline the specific 
collection parameters and shipping instructions for all research samples.  
 
10.     SPECIMEN BANKING 
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Any leftover study tissue samples may be stored for future research studies. The subjects will 
consent to the future use of samples in the consent form for the study. Any samples will only be 
released for use in future studies after approval by the Protocol Chair and other regulatory 
bodies, as appropriate.  
 
The study Protocol Chair and collaborators have approval by the TBCRC to use all research 
biospecimens collected during the conduct of this trial to address the research questions 
described in the protocol document. All future use of leftover/residual or repository specimens 
collected in this trial for purposes not prospectively defined will require review and approval by 
the TBCRC according to its established policies, whether the specimens are stored in a central 
site or at a local institution in a virtual repository as described in TBCRC Protocol ADM-002.  
Secondary use of biospecimens for new endpoints must be submitted to the TBCRC Central 
Office for possible review by the TBCRC Correlative Science Review Committee.  
 
Once the trial has closed to accrual and primary study objectives have been reported, all 
biospecimens and data will “roll over” (be virtually stored) under TBCRC Protocol ADM-002: 
Translational Breast Cancer Research Consortium Biospecimen and Outcomes Repository, to 
allow TBCRC Investigators accessibility for future research. Research participants that have opt-
ed in under this protocol will not be approached for additional consent at the time of roll over.  
 
 
11.   MEASUREMENT OF EFFECT  
 
For the purposes of this study, patients should be re-evaluated for response as per the standard of 
care, with a case report form (CRF) completed in REDCap at all visits, including routine treatment, 
imaging, and follow-up visits and as otherwise dictated by the treating physician, roughly as per 
the study calendar.  This CRF will document treatment response and disease status, any changes 
to treatment, and the basis for these changes—whether progression, toxicity, or otherwise.  
 
Since this is a real-world study without the use of RECIST for measuring tumor response, we will 
employ real-world response methodology9-11 to derive real-world (rw) measures of efficacy, 
including response and progression, (rwORR, rwPFS) based on interpretation of treating 
clinicians’ assessments (in office notes), radiology reports, pathology reports, and laboratory 
studies.  
 
Real-world response categories, as defined by Bartlett et al.10 are listed in Appendix A. These real-
world methodologies have been validated against matched clinical trial cohorts and have been 
shown to be feasible, reliable and highly correlated with a Spearman’s  = 0.99.11 Clinical 
endpoints will be correlated with HER2 QIF and ERBB2 mRNA dCT by STRAT4.   
 
Study personnel at each site will be trained to extract this data from electronic medical records and 
complete electronic CRFs for data collection. It will be expected that the site research personnel 
will complete these CRFs using the accompanying study manual along with input from the treating 
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physician after each infusion visit, each scan review visit, and any visits that lead to treatment 
modification or discontinuation.  
 
After site startup, data monitoring by the lead site will occur for the first several patients at each 
site to confirm that real world response assessment is consistent across the different sites and 
matches real-world response categories as defined by Bartlett et al.11  
11.1   Evaluable for Objective Response  
 
All patients who initiate treatment with T-DXd will be evaluable for objective response 
based on real-world response categories. These patients will have their responses 
classified according to the definitions in Appendix A. (Note:  Patients who exhibit 
objective disease progression prior to the end of cycle 1 will also be considered 
evaluable.) 
11.2    Response Criteria  
11.2.1 Real world Response Rate (rwORR) 
At every visit, with timing determined as per the treating physician/standard of care, 
clinical and imaging reports will be used to determine the extent of disease response 
to therapy, and whether there will be a change in treatment.  
The dates of therapy initiation, confirmed disease progression or death, and treatment 
changes (including the reason for treatment changes) will be collected, as well as 
response based on imaging and clinical status at each visit.  After each scan review 
visit, information will be collected regarding overall response assessment and the 
number and size of changing lesions to document best response.  For every visit the 
patient has as per the treating physician, information about treatment continuation and 
discontinuation—and the reasons for discontinuation, including imaging progression, 
biochemical progression, symptomatic deterioration, drug toxicity, or other—will be 
recorded. This information will be documented in CRFs by the site coordinator after 
each visit, with input as needed from the treating physician about overall response.  
Real world objective response rate (rwORR) will be calculated based on the number 
of those with partial or complete response as determined by the criteria in Appendix 
A over the absolute number of patients.   
11.2.2 Progression due to Symptomatic Deterioration 
Subjects with global deterioration of health status requiring discontinuation of 
treatment without objective imaging evidence of disease progression at that time, and 
not either related to study treatment or other medical conditions, should be reported as 
PD due to “symptomatic deterioration.” Every effort should be made to document 
objective progression even after discontinuation of treatment due to symptomatic 
deterioration.  
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11.2.3 Real-world Progression-Free Survival (rwPFS) 
Real-world Progression-free survival (rwPFS) is defined as the time from the date of 
initiation of the study treatment until the date of objective disease progression, death, 
or discontinuation of T-DXd for any reason in the absence of progression. 
11.3    Response Review 
There is no independent or central review of the radiology assessments planned for this 
trial. Response determination will be performed by the local site radiologist in 
conjunction with the treating physician, as per the standard of care. The Protocol Chair 
(or designee) may choose to review select cases including imaging reports, pathology 
reports, and reports about clinical history from the electronic medical record.  
 
 
12.    DATA AND SAFETY MONITORING 
12.1 
   Data Management and Reporting  
All information will be collected on study-specific case report forms (CRFs) in REDCAp 
by the study staff at each institution. The necessary forms will be provided to each site by 
the Coordinating Center. The following procedures will apply to the REDCap electronic 
database:  
 
• 
The database will be password protected; only authorized staff may enter and view 
study data.  
• 
Passwords and system IDs will not be shared.  
• 
Physical security of the workstations/files will be maintained.  
• 
Staff is trained on the data entry system and importance of security procedures.  
• 
Workstations with the database open will not be left unattended.  
 
All data will be forwarded via secure REDCap to the Coordinating Center for central 
review and inclusion in the overall study dataset, with relevant source documentation as 
outlined in the case report forms.  
 
Study data will be reviewed for completeness and accuracy by the Protocol Chair or 
designees as needed, with initial monitoring of data collection by clinical trial monitors at 
the lead site to ensure accuracy of data collection at each participating site. The Principal 
Investigator (or her/his designee) at each respective institution is responsible for review, 
and ensuring the completeness and accuracy, of the data generated by her/his institution. 
12.2 
     Source Documents  
Source data is all information, original records of clinical findings, observations, or 
other activities in a clinical trial necessary for the reconstruction and evaluation of the 
trial. Source data are contained in source documents. Examples of these original 
documents, and data records include: hospital records, clinical and office charts, 
laboratory notes, memoranda, evaluation checklists, pharmacy dispensing records, 
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recorded data from automated instruments, copies or transcriptions certified after 
verification as being accurate and complete, x-rays, and patient files. 
12.3 
    Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the 
study. All data requested on the CRF must be recorded. All entries will be entered 
into an electronic data capture system (EDC) via REDCap. 
12.4 
     Meetings  
The Coordinating Center will schedule regular teleconferences to take place which will 
include the Protocol Chair and site investigators and/or study personnel. The following 
study team members involved with the conduct of the trial will be included as 
appropriate: study site coordinators, data managers, research nurses, sub-investigators, 
patient advocates, collaborators (if applicable), and statistician.  
 
During these meetings matters related to the following will be discussed: enrollment rate 
relative to expectation, characteristics of participants, retention of participants, adherence 
to protocol (potential or real protocol violations), validity and integrity of the data, safety 
data, analysis of samples, and progress of data for objectives. 
12.5 
    Monitoring   
This study does not include an investigational drug, agent, or device. No Data Safety 
Monitoring Board applies to this study; the Protocol Chair/Coordinating Center will 
monitor data and study conduct as above.  
 
12.6 
     Confidentiality  
Information about study patients will be kept confidential and managed according to 
the requirements of the Health Insurance Portability and Accountability Act 
(HIPAA). Those regulations require a signed patient authorization informing the 
patient of the following: 
 
• What protected health information (PHI) will be collected from patients in this 
study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research patient to revoke their authorization for use of their PHI. 
 
In the event that a patient revokes authorization to collect or use PHI, the investigator, 
by regulation, retains the ability to use all information collected prior to the 
revocation of patient authorization. For patients that have revoked authorization to 
collect or use PHI, attempts should be made to obtain permission to collect at least 
vital status (i.e. that the patient is alive) at the end of their scheduled study period. 
 
13.    REGULATORY CONSIDERATIONS 
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13.1 
  Protocol Review and Amendments 
 
Unless otherwise specified, each participating institution must obtain its own IRB approval. 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations.  The IRB should approve the consent form and 
protocol. 
 
Information regarding study conduct and process will be reported to the Institutional 
Review Board (IRB) per the current institutional standards of each participating center. 
Any changes to the protocol will be made in the form of an amendment and must be 
approved by the IRB of each institution prior to implementation.  
 
The Protocol Chair (or her designee) is responsible for the coordination and development 
of protocol amendments, and will disseminate this information to the participating centers 
for local submission.  
13.2 
  Informed Consent 
The investigator (or her/his designee) will explain to each subject the nature of the study, 
its purpose, the procedures involved, the expected duration, the potential risks and benefits 
involved and any discomfort it may entail.  Each subject will be informed that participation 
in the study is voluntary, that s/he may withdraw from the study at any time, and that 
withdrawal of consent will not affect her subsequent medical treatment or relationship with 
the treating physician(s) or institution.  The informed consent will be given by means of a 
standard written statement, written in non-technical language, which will be IRB approved.  
The subject should read and consider the statement before signing and dating it, and will 
be given a copy of the document.  No subject will enter the study before his/her informed 
consent has been obtained. 
 
In accordance with the Health Information Portability and Accountability Act (HIPAA), 
the written informed consent document (or a separate document to be given in conjunction 
with the consent document) will include a subject authorization to release medical 
information to the study sponsor and supporting agencies and/or allow these bodies, a 
regulatory authority, or Institutional Review Board access to subjects’ medical information 
that includes all hospital records relevant to the study, including subjects’ medical history. 
 
13.3 
  Ethics and Good Clinical Practice  
This study will be carried out in compliance with the protocol and Good Clinical Practice, 
as described in: 
 
1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 
and 56 concerning informed consent and IRB regulations). 
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3. Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
 
The investigator agrees to adhere to the instructions and procedures described in it and 
thereby to adhere to the principles of Good Clinical Practice that it conforms to. 
 
13.4 
  Compliance with Trial Registration and Results Posting Requirements 
Under the terms of the Food and Drug Administration Modernization Act (FDAMA) and 
the Food and Drug Administration Amendments Act (FDAAA), the sponsor-investigator 
of the trial is solely responsible for determining whether the trial and its results are subject 
to 
the 
requirements 
for 
submission 
to 
the 
Clinical 
Trials 
Data 
Bank, 
http://www.clinicaltrials.gov. Information posted will allow subjects to identify potentially 
appropriate trials for their disease conditions and pursue participation by calling a central 
contact number for further information on appropriate trial locations and study site contact 
information. 
 
14. MULTI-CENTER GUIDELINES 
14.1 
   Study Documentation 
Each participating site is responsible for submitting copies of all relevant regulatory 
documentation to the Coordinating Center/Lead Site. The required documents include, but 
are not limited to the following: local IRB approvals (i.e., protocol, consent form, 
amendments, patient brochures and recruitment material, etc.), IRB membership rosters, 
summary of unanticipated problems or protocol deviations, and documentation of expertise 
of the investigators. The Coordinating Center will provide each participating site with a 
comprehensive list of the necessary documents. It is the responsibility of the participating 
sites to maintain copies of all documentation submitted to the Coordinating Center. 
14.2 
   Records Retention 
Following closure of the study, each participating center will maintain a copy of all site 
study records in a safe and secure location. The Coordinating Center will inform the 
investigator at each site at such time that the records may be destroyed. 
14.3 
   Publication 
It is understood that any manuscript or releases resulting from the collaborative research 
must be approved by the Protocol Chair and will be circulated to applicable participating 
sites/investigators prior to submission for publication or presentation.   
 
Additionally, any publication of study data and results must conform to the publications 
policy as stated the Translational Breast Cancer Research Consortium’s (TBCRC) 
“Policies and Procedures”. 
 
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15.     STATISTICAL CONSIDERATIONS  
15.1   Study Design 
15.1.1 Study Overview 
 
QuantifyHER is a prospective, longitudinal cohort study of patients with metastatic 
breast cancer whose tumors express HER2 identified as IHC 1+ by pathologists’ local 
assessment as per ASCO/CAP guidelines, and who are initiating therapy with T-DXd. 
See further eligibility criteria in Section 4. If a subject is included for the screen and 
then ultimately excluded, this subject will not be included in the N=200 sample size 
below.  
15.1.2 Clinical Data Collection 
A REDCap database will be constructed to maintain participant information. Clinical 
data will be abstracted from patient charts at participating sites by designated site 
personnel into electronic case report forms (CRFs), and will include baseline 
demographics, clinical and pathologic characteristics, treatment history, imaging 
report review, toxicity assessment as it relates to drug discontinuation, and real-world 
data for endpoint assessment.   
15.1.3 Measurement of Effect  
Since this is a real-world study without the use of RECIST for measuring tumor 
response, we will employ real-world response methodology9-11 to derive real-world 
(rw) measures of efficacy, including response and progression, (rwORR, rwPFS) 
based on interpretation of treating clinicians’ assessments (in office notes), radiology 
reports, pathology reports, and laboratory studies. Real-world response categories, as 
defined by Bartlett et al.10 are listed in Appendix A.  
These real-world methodologies have been validated against matched clinical trial 
cohorts and have been shown to be feasible, reliable and highly correlated with 
a Spearman’s  = 0.99.11 Clinical endpoints will be correlated with HER2 QIF and 
mRNA.  Study personnel at each site will be trained to extract this data from 
electronic medical records and complete electronic CRFs for data collection. Please 
see sections 9 and 11 for further information.  
 
15.2   Study Endpoints  
15.2.1 Primary Endpoint 
Association between quantitative HER2 and/or ERBB2 expression (as a continuous 
variable) and real-world objective response rate (rwORR) 
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15.2.2 Secondary Endpoint(s) 
• Association between quantitative HER2 and/or ERBB2 expression (as a 
continuous variable) and rwPFS. 
• Association between HER2 expression by quantitative immunofluorescence 
in HER2 IHC 1+ tumors and both rwORR and rwPFS, stratified by ER 
expression. 
• ROC-based cut-points for HER2 QIF and/or mRNA levels that discriminate 
responders from non-responders by identifying the lowest levels that perform 
better than a random classifier. 
• Association between combined mRNA + QIF and rwORR  
 
15.3   Sample Size/ Accrual Rate 
15.3.1 Sample Size: 200 evaluable patients.   
With our full sample of 200 patients and a one-sided type-one error of 2.5%, we will 
have 80% power to detect a difference in proportions of 9 to 10 percentage points for 
each standard deviation increase in QIF or mRNA across the range of interest, such as 
29% versus 20%, or 34.5% versus 25%.  This corresponds to an odds ratio of 
approximately 1.6.  Because there are no prior studies evaluating quantitative HER2 
expression and response rates, we will structure this study as a two-stage trial, with a 
full sample size of N=200, and a non-binding interim review at N=70. The interim 
review will give us a preliminary look at biomarker distribution through descriptive 
statistics. We will also calculate conditional power to achieve our target odds ratio 
(OR=1.6). Power considerations are identical for the mRNA measure.   
15.3.2 Length of Enrollment 
We estimate that it will take approximately 2 years to enroll 200 evaluable patients.  
15.3.3 Accrual rate 
Planned 1-2 / month / institution. 
 
15.4   Analysis of Primary Endpoint 
For our primary objective (real world response rate, rwORR), we will test the 
hypothesis (H1) that there is a relationship between QIF measures of HER2 protein 
expression and/or ERBB2 mRNA levels as measured by dCt values in STRAT4 and 
rwORR, against the null hypothesis (H0) of no-relationship. We will analyze the data 
using logistic regression, with the primary outcome variable (rwORR) being binary, 
and with two separate continuous predictor variables (HER2 QIF value, and/or 
ERBB2 dCt). The hypothesis will be tested using the z-score corresponding to the 
odds-ratio, using a one-sided type-one error of 2.5%.  Proportions such as rwORR 
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will be analyzed using logistic regression and tested using the z-value corresponding 
to the odds ratio.   
 
15.5  Analysis of Secondary Endpoints  
 
Time to event measures, to assess real-world PFS (rwPFS), will be analyzed using 
Kaplan-Meier methods and summarized as survival curves and time percentiles with 
95% CI.    
 
For assessment of the interplay between ER status and HER protein or ERBB2 
mRNA quantitative assessment, we will summarize any association between HER2 
QIF or ERBB2 dCt and ER expression as a Pearson Correlation with 95% CI.  
 
If we find a relationship between QIF or ERBB2 by STRAT4 and rwORR, we will 
use ROC curve methodology to determine whether there is predictive potential in the 
model comparing AUC to a value of 50% (random prediction), and in turn identify 
the lowest level (threshold) QIF and ERBB2 dCT values that do better than random 
prediction in differentiating responders from non-responders. We will ultimately 
identify (estimate) the QIF values corresponding to multiple rwORR values (10%, 
20%, 30% response) using the logistic model, with standard errors calculated using 
the delta method.   
 
Because the logistic regression analysis presumes a linear response of the logit to our 
continuous biomarkers, we will explore any relationship between QIF or ERBB2 by 
STRAT4 and rwORR for evidence of non-linearity by examining binned Pearson 
residuals, and the Box-Tidwell transformation.     
 
Lastly, we will conduct an analysis of rwORR using QIF and mRNA jointly, to 
determine whether a linear combination of the two values is better than either alone at 
discriminating responders from non-responders. We will use ROC in conjunction 
with cross-validation methods to assess the predictive performance of the model. 
 
15.6   Reporting and Exclusions 
15.6.1 Interim Review 
Because this is the first study comparing HER2 QIF or ERBB2 mRNA levels against 
standard IHC for response to T-DXd, we will perform a non-binding interim review 
after enrolling the first 70 patients to assess biomarker distribution and test 
performance, allowing us to modify the sample size and inclusion criteria as needed. 
This interim review will give us a preliminary look at biomarker distribution through 
descriptive statistics. We will also calculate conditional power to achieve our target 
odds ratio (OR = 1.6), which would be the basis for any sample size re-estimation.  
 
  
 
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REFERENCES 
 
1. 
Fernandez AI, Liu M, Bellizzi A, et al. Examination of Low ERBB2 Protein 
Expression in Breast Cancer Tissue. JAMA Oncol. 2022;8(4):607-610. 
doi:10.1001/JAMAONCOL.2021.7239 
2. 
Shu L, Tong Y, Li Z, Chen X, Shen K. Can HER2 1+ Breast Cancer Be 
Considered as HER2-Low Tumor? A Comparison of Clinicopathological 
Features, Quantitative HER2 mRNA Levels, and Prognosis among HER2-
Negative Breast Cancer. Cancers (Basel). 2022;14(17). 
doi:10.3390/CANCERS14174250 
3. 
Modi S, Jacot W, Yamashita T, et al. Trastuzumab Deruxtecan in Previously 
Treated HER2-Low Advanced Breast Cancer. N Engl J Med. 2022;387(1):9-20. 
doi:10.1056/NEJMOA2203690/SUPPL_FILE/NEJMOA2203690_DATA-
SHARING.PDF 
4. 
Moutafi M, Robbins CJ, Yaghoobi V, et al. Quantitative measurement of HER2 
expression to subclassify ERBB2 unamplified breast cancer. Lab Invest. 
2022;102(10):1101-1108. doi:10.1038/S41374-022-00804-9 
5. 
Erber R, Hartmann A, Fasching PA, Ruebner M, Stöhr R, Beckmann MW, 
Zentgraf M, Popp V, Weidler J, Simon I, Becker S, Huebner H, Fischer J, Guerini 
Rocco E, Viale G, Cayre A, Penault-Llorca F, Caniego Casas T, Pérez-Miés B, 
Palacios J, Jank P, Denkert C, Khoury L, Mairinger T, Ferrazzi F. Reproducibility 
of mRNA-Based Testing of ESR1, PGR, ERBB2, and MKI67 Expression in 
Invasive Breast Cancer: A Europe-Wide External Quality Assessment. Cancers 
(Basel). 2021 Sep 21;13(18):4718. doi: 10.3390/cancers13184718. PMID: 
34572945 
6. 
Wu NC, Wong W, Ho KE, et al. Comparison of central laboratory assessments of 
ER, PR, HER2, and Ki67 by IHC/FISH and the corresponding mRNAs (ESR1, 
PGR, ERBB2, and MKi67) by RT-qPCR on an automated, broadly deployed 
diagnostic platform. Breast Cancer Res Treat. 2018;172(2):327-338. 
doi:10.1007/S10549-018-4889-5 
7. 
Wu NC, Wong W, Acca B, et al. A multicenter clinical study of Xpert® breast 
cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, 
and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues. Presented at 
San Antonio Breast Cancer Symposium; Poster P2-03-03. Abstract available at 
https://www.sabcs.org/Portals/SABCS2016/Documents/SABCS-2017-
Abstracts.pdf  
8. 
Gupta S, Mani NR, Carvajal-Hausdorf DE, et al. Macrodissection prior to closed 
system RT-qPCR is not necessary for estrogen receptor and HER2 concordance 
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with IHC/FISH in breast cancer. Lab Invest. 2018;98(8):1076-1083. 
doi:10.1038/S41374-018-0064-1 
9. 
DeMichele A, Cristofanilli M, Brufsky A, et al. Comparative effectiveness of 
first-line palbociclib plus letrozole versus letrozole alone for HR+/HER2− 
metastatic breast cancer in US real-world clinical practice. Breast Cancer Res. 
2021;23(1):1-10. doi:10.1186/S13058-021-01409-8/TABLES/2 
10. 
Bartlett CH, Mardekian J, Cotter MJ, et al. Concordance of real-world versus 
conventional progression-free survival from a phase 3 trial of endocrine therapy 
as first-line treatment for metastatic breast cancer. PLoS One. 
2020;15(4):e0227256. doi:10.1371/JOURNAL.PONE.0227256 
11. 
Ma X, Bellomo L, Magee K, et al. Characterization of a Real-World Response 
Variable and Comparison with RECIST-Based Response Rates from Clinical 
Trials in Advanced NSCLC. Adv Ther. 2021;38(4):1843-1859. 
doi:10.1007/S12325-021-01659-0/FIGURES/3 
 
 
 
 
 
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APPENDICES  
 
APPENDIX A:  Real-World Response Categories  
 
Response Category 
Definition  
Complete response 
Complete resolution of all visible disease 
Partial response 
Partial reduction in size of visible disease in some or all areas 
without any areas of increase in visible disease 
Stable Disease 
No change in overall size of visible disease (includes cases where 
some lesions increased, and some lesions decreased in size) 
Progressive Disease 
Increase in visible disease or presence of new lesions 
Indeterminate/equivocal 
Clinician specifically indicates that response is “indeterminate” or 
“uncertain” or if clinician’s interpretation of the scan(s) cannot be 
mapped to 1 of the above categories 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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