Study design concept AstraZeneca

<<DXXXYXXX>> 05-Oct-2023

Study design concept

Study code <<DXXXYXXX>>
Date 05-Oct-2023
Version V1.0

Real-world data of HER2-positivity spectrum in patients with breast cancer

and adherence to guidelines in Romania (SPECTRUM)

Observational, secondary data collection study to describe the proportion of

HER2-positive status in patients with a pathological diagnosis of breast
cancer and adherence to guidelines in 1-year interval in the real-life setting
from Romania

AstraZeneca team/function leading study: Romania

Therapy area: Oncology
Study category: SECONDARY DATA OBSERVATIONAL
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

1. RATIONALE
The human epidermal growth factor receptor 2 (HER2) is an important predictive and prognostic
marker, that is frequently identified on the primary breast cancer (BC) and/or the distant metastases
[Tapia, 2007]. The HER2 status is mainly determined through immunohistochemistry (IHC) and in
situ hybridization (ISH) [Tarantino, 2020]. In current clinical practice, HER2 status is classified
dichotomously, as either positive (IHC3+ or IHC2+ ISH+) or negative (IHC0, IHC1+ or IHC2+ ISH-)
[Holthuis, 2022]. In the last years, a potential new category has been proposed for the cases with
IHC1+ or 2+ with negative ISH, that is called HER2-low breast cancer [Tarantino, 2020]. The HER2-
low breast cancer might derive significant and meaningful survival benefit from the novel anti-HER2
antibody drug conjugates [Modi, 2020; Van der Lee, 2015; Benerji, 2019], which suggests that
HER2-positivity spectrum is expanding and it goes beyond the traditional dichotomous classification
[Won, 2022; Venetis, 2022]. Moreover, the terms HER2-ultralow for the cases where the HER2 IHC
expression is >0 but <1+, and HER2-null for HER2 0 tumors with no detectable fainting on the tissue
sections are proposed, expecting evidence in the future [Tarantino, 2023].

HER2-low breast cancer accounts for approximately 45% to 55% of all breast cancers, and
heterogeneity exists according to the expression of hormone receptor (HR) [Tarantino, 2020].
Because the proportion of patients with HER2-low breast cancer is substantial, is important to develop
precision medicine strategies in order to improve survival of these patients. This requires a thorough
understanding of breast cancer with different HER2 expression levels [Holthuis, 2022], and accurate
and reproducible testing [Schnitt, 2023]. Currently, no established guidelines exist for scoring HER2-
low status in breast cancer. The 2023 updated guidelines issued by the American Society of Clinical
Oncology (ASCO)/College of American Pathologists (CAP) for HER2 testing includes no changes to
prior recommendations (2018) or traditional terminology of positive/equivocal/negative for HER2
IHC results but calls to increased awareness for IHC 1+ or 2+ non-amplified cases that deem patients
eligible for ADC trastuzumab deruxtecan [Wolff, 2018; Wollf, 2023].

To our knowledge, this is the first study conducted in Romania to define and characterize the current
spectrum of HER2-positivity in BC patients using the histological reports of the historical HER2 fixed
tissue IHC stained slides evaluated over a pre-defined period in multiple pathology laboratories
representative at country level. We plan to assess the practice change in 1-year interval by describing
the rate of HER2-positivity in a similar number of patients with breast cancer according to guidelines
in use at that time. The insights will be used to inform the medical community about the true rate of
this new disease category and level of adoption of guidelines into real-life setting at 1-year interval.

2. OBJECTIVES AND HYPOTHESES

Primary objective(s) Outcome measure Hypothesis tested
(if relevant)
• To describe the overall proportion of • Proportion of HER2-positive (3+,
HER2-positive (3+, 2+/ISH+) in BC 2+/ISH+) BC in historically stained None
patients, based on historical HER2 HER2 IHC slides
fixed tissue IHC stained slides at site
level over a 3-month period (August
- October 2023)

2 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

Secondary objective(s) Outcome measure Hypothesis tested

1. To describe the proportion of
patients with ASCO/CAP score of
0/1+/2+/3+ reported in historical
IHC HER2 fixed tissue stained slides
(if relevant)
1. Proportion of reported ASCO/CAP
scores of 0/1+/2+/3+ in the historically None
stained HER2 IHC slides

2. To describe the proportion of 2. Proportion of ISH tests

patients for whom ISH is conducted

3. To describe the ISH method used 3. Type and proportion of ISH methods
and turnaround time used and mean (median) time from IHC
result to ISH test and time to results

4. To describe overall histopathological 4. Mean and median age, distribution by

and clinicopathological hormonal receptor status, ki67, histology
characteristics of patients overall and type, grading, primary or secondary
by type of HER2-status (metastatic) diagnosis – overall and by
type of HER2-status

5. To describe the HER2-positivity 5. Proportion of HER2-positive and HER2-

spectrum in HER2 fixed tissue IHC
stained slides at 1-year distance
(August – October 2024) using a a
similar approach and assess
adherence to guidelines in use
negative status reported in HER2 fixed
tissue IHC stained slides as per
guidelines in use at the time of second
study phase (1 year since first data
extraction)

Safety objective(s) Outcome measure Hypothesis tested

(if relevant)
None N/A
N/A

3. METHODOLOGY

3.1 Study design

This is an observational, non-interventional study to assess the HER2-positivity among all historical
HER2 fixed tissue IHC stained slides for breast cancer (all tumors) over a 3-month period at 1-year
distance in approx. 20 - 30 pathology laboratories from Romanian centers. This would allow
evaluating the HER2-low paradigm change (if any) at 1-year distance across Romania.

This is a retrospective review of histology reports led by study investigators. Approximately 150
histology reports of patients with breast cancer with HER2 status investigated in two distinct moments
at 1-year interval are expected to be included. No patient will be follow-up-ed and each group (2023
and 2024) will be independent.

3 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

SPECTRUM is conducted as a secondary data collection study, with two phases of data extraction
from histology reports, at 1-year distance. The data collection is planned to last 3 months or until the
target number of patients is reached.

3.2 Study population

For the first data collection phase, the study population is represented by patients with breast cancer
(all tumors) for whom historical IHC HER2 fixed tissue stained slides evaluated during August -
October 2023 are available at site level. The second data collection will occur at 1-year distance, and
study population will be represented by patients with breast cancer (all tumors) for whom IHC HER2
fixed tissue stained slides are evaluated during August – October 2024 at site level.

Data will be collected in a reverse, but consecutive order, starting with the last histology bulletin
meeting study criteria in the period of interest. A number of approx. 150 histology bulletins are
planned to be abstracted in each study phase. In order to reach national representativeness, a quota of
5 – 8 histology bulletins was set for each center. If the quota is not reached in the first month of
evaluation (ie, October 2023 or October 2024), the site will continue data review and collection in the
bulletins from the previous month (ie, September 2023 or September 2024, respectively) etc, until the
site quota is reached.

It is expected that a total number of 20 - 30 pathology laboratories affiliated to oncology institutes or

general hospitals, academic or non-academic medical centers, from public or private practice will
participate in this study. In the event that the data collection and sampling strategy fails to provide the
target patient numbers at country level, multiple alternatives may be considered, including raising the
quota at site level in larger sites or opening additional sites.

3.2.1 Inclusion criteria

To be included in the first data collection phase, the following criteria should be met:
• Adult men or women with breast cancer (age ≥18 years)
• Availability of histological reports with results of the assessment of HER2 fixed tissue IHC
stained slides performed between August – October 2023
To be included in the second data collection phase, the following criteria should be met:
• Adult men or women with breast cancer (age ≥18 years)
• Availability of histological reports with results of the assessment of HER2 fixed tissue IHC
stained slides performed between August– October 2024

3.2.2 Exclusion criteria

To be included in the data collection, the following criterion should not be met:
• Missing IHC results on the histology bulletins

3.3 Data source

Investigators participating in this secondary data collection study will be pathology specialists
involved in the routine pathological diagnosis of breast cancer. To ensure an accurate representativity
at country level, around 20 - 30 histology laboratories from public and private practice will be invited
to participate in the study.
4 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

All information will be extracted from the paper and/or electronic histology reports (as available in
each practice), in an anonymized manner.

3.4 Variables and epidemiological measurements (secondary use of

data)
SPECTRUM is conducted as a secondary data collection study, with two data extraction phases. The
first data extraction will collect data from all existing histology bulletins at site level reporting results
from the evaluation of historical HER2 fixed tissue IHC stained slides at site level over a 3-month
period (August-October 2023). The second data extraction will collect data at 1-year distance
following a similar approach on the IHC of HER2 fixed tissue IHC stained slides evaluated between
August-October 2024 in each participating site. The HER2-status will be assessed in a similar manner
as in the first data extraction phase, taking into account the updates of guidelines, if any.

To better describe the current and future situation of HER2 testing aross the country, details on type
of site, Investigator’s experience, and HER2 IHC testing will be collected.

To report the HER2 positivity or negativity, the following definitions will be used in this study, as per
current ASCO/CAP guidelines [Wollf, 2023]

Table 1. Groups and nomenclature of HER2 status used in the SPECTRUM study

Groups and nomenclature HER2 testing result HER2 status per ASCO/CAP
used in this study guidelines
HER2-positive IHC 2+ or 3+ HER2-positive (IHC2+ must be
ISH+)
HER2- HER2-low IHC 1+ or 2+/ISH- HER2-negative
negative HER2 ultra-low IHC >0 <1+/ISH- (IHC 2+ must be ISH-;
HER2 null IHC 0 (no stain at all) IHC 1+ and IHC zero must be ISH-
or untested)

For each patient, the investigator will collect the following study variables from the histology report
(if available).:
• Demographic caracteristics: age and gender; menopausal status
• Pathology characteristics:
o hormone receptor status,
o HER2 status,
o ki67,
o histology type,
o histological grade,
o tumor stage
o type of diagnosis (primary / metastatic)
o other mutations
5 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

o in cases where ISH was requested: type of ISH, antibody assay used, and time from
IHC result to ISH request and from ISH testing to result

The site characteristics collected will include:

• Investigator details: demographics (sex, age, county of practice), and experience (years of
practice, average number of IHC HER2 tissue slides / month )
• Institution information: institution type (academic/non-academic hospital, private or public
setting, in-house or external pathology laboratory)
• HER2 testing: typical sequence of testing, IHC HER2 information (anti-HER2 clones,
staining protocol, quality control), HER2 reporting (mode of reporting), guidelines followed
Note: HER2 testing details will be collected at every data extraction point to be able to capture
changes of practice, if any.

3.5 Study limitations

Due to the real-world, observational, retrospective nature of the SPECTRUM study and the inherent
characteristics of such designs, findings from this study may be subject to bias, such as sites and
patients selection bias, limitations in availability of data, and variability in local pathological
diagnosis practices and guidelines.

It is expected that not all histology laboratories will accept to take part in the study, therefore the
entire spectrum of diagnosis practices in BC patients may not be entirely represented. The number of
patients and the limited period allowed for patients selection are other limitations, as it will not reflect
the rate of HER2-positivity in all patients BC diagnosed yearly. Also, only data from local pathology
reports will be used, without a central confirmation of the pathological assessment. Therefore,
reporting of HER2 status may not be very specific in some cases. In consequence, the generalizability
of the results may be limited.

Data quality will depend on the information registered in the histology reports. Demographic, and
pathology characteristics will be reported as available in the histology reports. The electronic platform
for data collection will be designed to try and minimize the level of missing data collected from the
medical records.

4. STATISTICAL METHODS

4.1 Sample size and statistical power; precision assessment

This study does not involve hypothesis testing. However, in order to support a broad range of
descriptive analyses from overall general scientific questions with sufficient precision, several group
sizes were considered, based on an approx. number of 12085 new cases of breast cancer in 2020 in
Romania and around 30% of metastatic cases [Globocan 2020; Wang 2019].

Table 1. Margin of errors for various group sizes

Group size N=100 N=150 N=200 N=250 N=300

Margin of error 9.76% 7.95% 6.87% 6.13% 5.59%
[all new BC cases]
6 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

Based on these data, the real-life character of the study and preliminary feasibility with potential study
sites, the target number of patients was chosen to be 150 for an expected proportion of 50% of the
HER2-low [HER-2 positive] rate. However, in case this target sample size is not reached, this does
not imply study failure, but the precision of estimates will change. The exact binomial 95%
confidence intervals (95% CIs) for estimated proportions will be provided in the Study Protocol.

4.2 Statistical considerations

All analyses will be descriptive in nature. The distribution of continuous variables will be examined
using histograms or normality tests as appropriate. Normally distributed variables will be summarized
by providing the mean, standard deviation, minimum and maximum. Not normally distributed
variables will be summarized by providing median, IQR, minimum and maximum. Categorical
variables will be summarized by providing frequency and proportions. No comparisons between
groups at 1-year interval will be made, but comparisons of variables intra-group may be considered
using various types of statistical tests, based on type of variables: chi-squared, Student, Mann-
Whitney-Wilcoxon, Kruskal-Wallis, as applicable. Effect sizes will be reported, where possible –
Cohen’s d and Cliff’s delta. The significance level is set at p < .05.

Data will be analyzed based on observed cases only, and the extent of missing data will be reported.
Further specification on planned analyses will be provided in a separate Statistical Analysis Plan. The
data will be analyzed using R language.

5. COLLECTING AND REPORTING OF ADVERSE EVENTS

AND SPECIAL SITUATIONS

5.1 Collection of Adverse Events and Special Situations

As this is an observational study with secondary collection data only and no safety related
objective, there is no requirement for adverse events to be collected.

5.2 Reporting of Adverse Events and Special Situations

As this is a study with secondary data collection only, the adverse events are considered to
have been reported through other sources when first collected. Thus no reporting of adverse
event data is required by this study protocol.

Any adverse events (AEs) / adverse drug reactions (ADRs) or any special reportable
situations should be reported by the Investigators as spontaneous reports according to local
regulations to AstraZeneca (for AstraZeneca’s products) or national health authority.

7 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
 Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

6. REFERENCES
Banerji U, van Herpen CML, Saura C, et al, Trastuzumab duocarmazine in locally advanced and
metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-
expansion study. Lancet Oncol. 2019;20(8):1124-1135. doi: 10.1016/S1470-2045(19)30328-6.

The Global Cancer Observatory (GLOBOCAN), Romania, available at

https://gco.iarc.fr/today/data/factsheets/populations/642-romania-fact-sheets.pdf, last accessed on
December 22, 2022

Holthuis EI, Vondeling GT, Kuiper JG, et al, Real-world data of HER2-low metastatic breast cancer:
A population based cohort study. Breast. 2022;66:278-284. doi: 10.1016/j.breast.2022.11.003.

Modi S, Park H, Murthy RK, et al, Antitumor Activity and Safety of Trastuzumab Deruxtecan in
Patients With HER2-Low-Expressing Advanced Breast Cancer: Results From a Phase Ib Study. J
Clin Oncol. 2020;38(17):1887-1896. doi: 10.1200/JCO.19.02318

Schnitt SJ, Tarantino P, Collins LC. The American Society of Clinical Oncology-College of
American Pathologists Guideline Update for Human Epidermal Growth Factor Receptor 2 Testing
in Breast Cancer: How Low Can HER2 Go?. Arch Pathol Lab Med. 2023;10.5858/arpa.2023-
0187-ED. doi:10.5858/arpa.2023-0187-ED

Tarantino P, Hamilton E, Tolaney SM, et al, HER2-Low Breast Cancer: Pathological and Clinical
Landscape. J Clin Oncol. 2020;38(17):1951-1962. doi: 10.1200/JCO.19.02488.

Tarantino P, Viale G, Press MF, et al. ESMO expert consensus statements (ECS) on the definition,
diagnosis, and management of HER2-low breast cancer. Ann Oncol. 2023;34(8):645-659.
doi:10.1016/j.annonc.2023.05.008

Tapia C, Savic S, Wagner U, et al, HER2 gene status in primary breast cancers and matched distant
metastases. Breast Cancer Res. 2007;9(3):R31. doi: 10.1186/bcr1676.

Van de Lee MM et al, Groothuis PG, Ubink R, et al, The Preclinical Profile of the Duocarmycin-
Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing
Breast Cancers. Mol Cancer Ther. 2015;14(3):692-703. doi: 10.1158/1535-7163.

Venetis K, Crimini E, Sajjadi E, et al, HER2 Low, Ultra-low, and Novel Complementary Biomarkers:
Expanding the Spectrum of HER2 Positivity in Breast Cancer. Front Mol Biosci. 2022;9:834651.
doi: 10.3389/fmolb.2022.834651.

Wang R, Zhu Y, Liu X, et al, The Clinicopathological features and survival outcomes of patients with
different metastatic sites in stage IV breast cancer. BMC Cancer. 2019;19(1):1091. doi:
10.1186/s12885-019-6311-z.

Won S, Ahn J, Kim Y, et al, Clinical significance of HER2-low expression in early breast cancer: a
nationwide study from the Korean Breast Cancer Society. Breast Cancer Res. 2022;24(1):22. doi:
10.1186/s13058-022-01519-x

8 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365
Study design concept AstraZeneca
<<DXXXYXXX>> 05-Oct-2023

Wolff AC, Elizabeth Hale Hammond M, Allison KH, et al. Human epidermal growth factor receptor 2
testing in breast cancer: American society of clinical oncology/ college of American pathologists
clinical practice guideline focused update. J Clin Oncol. 2018;36(20):2105-2122.
doi:10.1200/JCO.2018.77.8738

Wolff AC, Somerfield MR, Dowsett M, et al. Human Epidermal Growth Factor Receptor 2 Testing in
Breast Cancer: ASCO–College of American Pathologists Guideline Update. J Clin Oncol.
Published online 2023. doi:10.1200/jco.22.02864

9 of 9
Evidence Study Design Concept Form
Version 2.0
Form Doc ID: TMP-0006145
Parent Doc ID: SOP-0063365