SURVIVE (Standard Surveillance vs. Intensive Surveillance in Early Breast Cancer) - a Partially Double-blinded, Multi-center, Randomized, Controlled Superiority Study
Phase: NA
Conditions: Breast Cancer
Sponsor: Prof. Wolfgang Janni

 
 
 
 
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SURVIVE (Standard Surveillance vs. Intensive Surveillance in 
Early Breast Cancer) – a partially double-blinded, multi-
center, randomized, controlled superiority study  
 
 
 
 
 
Protocol Version:   
2.0 
Protocol Version Date:  02. March 2023 
BMBF Funding code:  
01KD2202 
ClinicalTrials.gov ID: 
NCT05658172 
DRKS-ID: 
 
 
DRKS00030745 
 
 CONFIDENTIAL: 
Information and data included in this protocol contain trade secrets and privileged or confidential information 
which is the property of the University Hospital Ulm. No person is authorized to make it public without the 
permission of the University Hospital Ulm. These restrictions on disclosure will apply equally to all future 
information supplied to you which is indicated as privileged or confidential. This material may be disclosed to 
and used by your staff and associates as may be necessary to conduct the clinical study. 

 
 
 
 
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1 
PROTOCOL SYNOPSIS 
 
Study Title 
SURVIVE (Standard Surveillance vs. Intensive Surveillance in Early Breast Cancer) 
Sponsor 
University Hospital Ulm 
Rationale 
Breast cancer is the world’s most common cancer entity with a median age of 64 
at first diagnosis, affecting all age groups after puberty. While the incidence of 
breast cancer increased over the last decades, treatment options for breast 
cancer patients continue to improve, providing a growing range of therapeutic 
agents.  
After patients with early breast cancer have completed primary therapy, 
guidelines limit routine surveillance in breast cancer survivors to clinical 
surveillance and breast imaging. Screening for distant metastases is initiated only 
in patients with specific symptoms. This approach is based on two large cohort 
studies performed in the 1980s suggesting no superiority of intensified screening 
for distant metastases versus standard surveillance for overall survival (OS). 
However, patients frequently feel left-alone during this stage of their disease. 
Furthermore, diagnostic and therapeutic options for the treatment of early 
(oligo-) metastatic disease have dramatically improved over the past decades (e. 
g., HER2-neu targeted treatment, endocrine combination treatment with 
CDK4/6-inhibitors, 
cytostatic 
conjugates, 
PARP-inhibitors, 
stereotactic 
radiotherapy, etc.). We hypothesize, in consideration of modern therapeutic 
agents, that earlier detection of distant (oligo-) metastases and therefore earlier 
initiation of therapy in a pre-symptomatic stage leads to improved overall survival 
(OS) today. We suggest the evaluation of a liquid biopsy-guided follow-up 
surveillance method for the detection of distant (oligo-) metastases before the 
onset of symptoms, analyzing the tumor markers mucin-1 (MUC1)- based cancer 
antigen 27.29 (CA27.29), mucin-16 (MUC16)- based CA125 and carcinoembryonic 
antigen (CEA) serum levels as well as circulating tumor cells (CTCs) and circulating 
tumor DNA (ctDNA) in a multicenter, prospective, partially double-blinded, 
controlled randomized study for medium- and high-risk early breast cancer 
patients. This study could contribute to a paradigm shift in the current follow-up 
care of medium and high-risk early breast cancer survivors. 
Study Overview 
This is a partially double-blinded, multi-center, randomized, controlled 
superiority study to evaluate the potential benefits of intensified surveillance 
versus standard surveillance in medium-risk and high-risk early breast cancer 
patients. 
3500 patients will be randomized in a 1:1 ratio after completion of primary anti-
tumor therapy (adjuvant chemotherapy, surgery or radiotherapy, whichever 
occurs last) to receive: 
- 
Standard Surveillance according to national guidelines or 
- 
Intensive Surveillance with additional testing of blood samples for 
prospective tumor markers (CA27.29, CA125, CEA), CTC and ctDNA  

 
 
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In both study arms patients will continue to receive standard surveillance 
according to national guidelines, including clinical follow-up visits every 3 months 
for the first 3 years and every 6 months for the following 2 years. Additionally, 
blood samples will be drawn and Quality of Life (QoL) will be analyzed at these 
clinical follow-up visits in both arms. 
In the Standard Surveillance arm blood samples will be stored in a biobank. 
In the Intensive Surveillance arm blood samples will be tested for prospective 
tumor markers (CA27.29, CA125, CEA), CTCs and ctDNA. Abnormal findings of 
either marker (CA27.29 or CA125 or CEA or CTC or ctDNA) will trigger diagnostic 
imaging. Additionally, blood samples will be stored in a biobank for retrospective 
analysis. 
In both study arms detection of distant recurrence via imaging will terminate the 
surveillance protocol and treatment will be initiated according to national 
guidelines. 
Planned enrollment period is approximately 24 months, total study duration is 
approximately 144 months (2-year recruitment period, 5-year interventional 
period, 5-year follow up period). In terms of long-term follow-up after end of 
study, patients have the possibility to participate in the patient self-reporting 
registry (Patientenselbstauskunft).   
Intervention 
 
 
All participating patients with no clinical evidence of disease after primary anti-
tumor therapy (adjuvant chemotherapy, surgery or radiotherapy, whichever 
occurs last) will follow the German AGO-guideline based standard follow-up 
surveillance in addition to a blood withdrawal of 27,5 mL whole blood (2 x 10 mL 
Streck Cell-Free DNA BCT®, 1 x 7,5 mL Serum; every 3 months for the first 3 years 
after enrollment, every 6 months for the consecutive 2 years). In case of M1-
detection or local recurrence of disease, the patients will receive the guideline-
based diagnostic measures and therapy.  
Quality of Life (QoL) will be monitored using established questionnaires (EORTC 
QLQ-C30 and PA-F12) every 6 months. All patients will be asked questions 
directed to performed examinations (blood or imaging) and symptoms outside 
the study within the last months since the last visit. 
Patients will be randomized 1:1 to an intensive versus standard surveillance 
group. Randomization will be blinded for patients and treatment centers. 
However, when imaging is triggered by a positive biomarker result in patients in 
the Intensive Surveillance arm, these patients have to be unblinded.  
 
Intensive Surveillance: Prospective tumor marker (CA27.29, CA125, CEA), CTC 
and ctDNA testing of the blood samples. Abnormal findings (as defined below) of  
either marker (CA27.29 and/or CA125 and/or CEA and/or CTC and/or ctDNA) will 
be regarded as molecular relapse and trigger diagnostic imaging.  
 
Tumor marker: A sample of peripheral blood is taken after randomization for 
baseline determination (2 blood drawings 28 ± 5 days apart; mean value for 
CA27.29, CEA and CA125 determines baseline) and thereafter every 3 months for 

 
 
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3 years, thereafter every 6 months for another 2 years. Patients with an abnormal 
tumor marker serum level increase from baseline (CA27.29 +75% or CEA +100% 
or CA125 +150%) will undergo complete standard staging examinations (as a 
minimum CT scan of the chest and abdomen + SPECT bone scan). In case of M0, 
the patient will continue liquid-biopsy based tumor marker testing. If tumor 
marker elevation continues, a maximum of 3 consecutive negative staging 
examinations will be performed before another rise in tumor marker will be 
mandatory (according to the deltas stated above) to trigger further imaging. The 
minimum interval between two CT scans should be at least 3 months, and SPECT 
bone scans should be at least 6 months apart at any time point. 
 
CTC: If an abnormal tumor marker value is detected for the first time, additional 
blood for CTC-analysis will be collected at the next regular study visit. 
Furthermore, all patients of the experimental arm will receive CTC-testing 1 year 
after study enrollment. If ≥ 1 CTC is detected (irrespective of whether there is a 
confirmed abnormal tumor marker increase), patients will undergo complete 
standard staging examinations. In case of M0, the patient will continue liquid-
biopsy based testing in the same intervals as before (every 3 or 6 months). The 
minimum interval between two CT scans should be at least 3 months, and SPECT 
bone scans should be at least 6 months apart at any time point.  
 
ctDNA: Presence of ctDNA will be measured every 3 months for 3 years, 
thereafter every 6 months for another 2 years. If ctDNA is detected, patients will 
undergo complete standard staging examinations. In case of M0, the patient will 
continue liquid-biopsy based ctDNA testing. In case of persisting ctDNA, a 
maximum of 3 consecutive negative staging examinations will be performed 
before staging intervals will be extended to every 6 months for another 3 
consecutive scans. If ctDNA is still persisting and patients show no radiological 
sign of recurrence, staging intervals will be extended to once a year. The 
minimum interval between two CT scans should be at least 3 months, and SPECT 
bone scans should be at least 6 months at any time point.  
 
Standard Surveillance: Blood samples will not be analyzed immediately and will 
therefore not trigger imaging. A biobank will be established for retrospective and 
translational studies. This procedure is necessary to ensure the partially double-
blinded study design. 
 
Duration of intervention per patient: 5 years.  
 
Patients will be randomized 1:1 to the intensive versus standard surveillance 
group. Randomization will be blinded for patients and treatment centers. 
However, when imaging is triggered by a positive biomarker result in patients in 
the Intensive Surveillance arm, these patients have to be unblinded.  

 
 
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Primary Objectives 
and Endpoints  
Two Co-Primary-Endpoints will be investigated: 
 
Overall Survival (OS)  
Objective: 
To compare 5-year OS rates between patients in the Standard Surveillance arm 
versus patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
OS is defined as time from randomization until the death of the patient 
independent of cause of death. If a patient is not known to have died, OS is 
censored at the date of last contact. 
 
Overall Lead Time Effect 
Objective: 
To determine the Overall Lead Time Effect generated due to tumor 
marker/CTC/ctDNA guided Intensive Surveillance compared to Standard 
Surveillance after primary therapy in early breast cancer patients. 
Endpoint: 
This endpoint is a composite measure, defined as the median time from 
molecular to via Imaging verified Distant Recurrence Lead Time (calculated only 
for patients in the liquid-biopsy guided Intensive Surveillance arm; see secondary 
endpoint definition below) + Difference in time to distant recurrence between 
the two arms (i.e., difference between median time from randomization to 
distant recurrence for all patients with distance recurrence in the Standard 
Surveillance arm and median time from randomization to distant recurrence for 
all patients with distant recurrence in the liquid-biopsy guided Intensive 
Surveillance arm; see Figure 2). The Overall Lead Time Effect will be assessed for 
all markers in combination. 
Secondary 
Objectives and 
Endpoints 
Invasive disease-free survival (IDFS) 
Objective: 
To compare IDFS between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
IDFS is defined as time from randomization until first IDFS event, including any 
invasive ipsilateral, regional, contralateral, and distant disease recurrence, 
second primary tumors, or death from any cause as event; non-invasive, in-situ 
cancer events are excluded. If a patient has not had an event, IDFS is censored at 
the date of last adequate tumor assessment. 
 
Distant disease-free survival (DDFS) 
Objective: 
To compare DDFS between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint:  
 
 
 
 
 
 
 
       
DDFS is defined as time from randomization until first DDFS event including 

 
 
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metastasis, second primary tumors and death from any cause as event. If a 
patient has not had an event, DDFS is censored at the date of last adequate tumor 
assessment. 
 
Distant recurrence-free survival (DRFS)  
Objective: 
To compare DRFS between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
DRFS is defined as time from randomization until first DRFS event including 
metastasis and second primary tumors; death from any cause is not included as 
event. If a patient has not had an event, DRFS is censored at the date of last 
adequate tumor assessment. 
 
Breast cancer specific survival (BCSS) 
Objective: 
To compare BCSS between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
BCSS is defined as time from randomization until breast cancer associated death 
of the patient. If a patient is not known to have died, BCSS is censored at the date 
of last contact. If a patient has died for reasons not associated with breast cancer 
(by clinical assessment), BCSS is censored at the date of death. 
 
Invasive breast cancer free survival (IBCFS) 
Objective: 
To compare IBCFS between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
IBCFS is defined as time from randomization until first IBCFS event, including any 
invasive ipsilateral, regional, contralateral and distant disease recurrence or 
death from any cause as event; non-invasive, in-situ cancer events are excluded. 
If a patient has not had an event, IBCFS is censored at the date of last adequate 
tumor assessment. 
 
Overall Survival (OS) after 10 Years  
Objective: 
To compare 10-year OS rates between patients in the Standard Surveillance arm 
versus patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
 
 
 
 
 
 
 
          
OS is defined as time from randomization until the death of the patient 
independent of cause of death. If a patient is not known to have died, OS is 
censored at the date of last contact. 
 

 
 
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Molecular to via Imaging verified Distant Recurrence Lead Time in the 
Interventional arm 
Objective: 
To determine the Lead Time (median, interquartile, range, 95% confidence 
interval) generated in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
The Lead Time is defined as time from first molecular relapse (definitions see 
above and in Figure 2) to via imaging verified distant recurrence for all patients in 
the liquid-biopsy guided Intensive Surveillance arm for whom a via imaging 
verified distant recurrence is documented during the 5-year interventional period 
of the study. If a patient has a via imaging verified distant recurrence but no 
documented molecular relapse, the Lead Time for this patient is set to ‘0’. This 
secondary endpoint will be assessed for all markers in combination and, where 
applicable, for each marker separately. 
 
Quality of life (QoL) with questionnaires: EORTC QLQ-C30 and PA-F12 
Objective: 
To compare QoL between patients in the Standard Surveillance arm versus 
patients in the liquid-biopsy guided Intensive Surveillance arm. 
Endpoint: 
QoL will be monitored in both groups using 2 questionnaires which are to be 
completed every 6 months (EORTC QLQ-C30 and PA-F12) in the interventional 
phase (first five years) and once a year during the five years of follow-up. QoL 
data will be collected on paper or via a digital health application. 
  
Liquid biopsy sensitivity (CA27.29, CEA, CA125, CTC and ctDNA) 
Objective: 
To determine sensitivity (i.e., the true positive rate) of the liquid biopsy markers 
in combination (and individually where applicable) in the liquid-biopsy guided 
Intensive Surveillance arm. 
Endpoint: 
Sensitivity is defined as the proportion of all patients in the liquid-biopsy guided 
Intensive Surveillance arm with a distant recurrence as verified by imaging during  
the 5-year interventional period of the study that had a positive biomarker result 
(i.e., molecular relapse) within 36 months before the recurrence as verified by 
imaging occurred (or within 60 months before the recurrence as verified by 
imaging occurred if in the first 36 months at least once a year a positive biomarker 
is measured). 
 
Liquid biopsy specificity (CA27.29, CEA, CA125, CTC and ctDNA)  
Objective: 
To determine specificity (i.e., the true negative rate) of the liquid biopsy markers 
in combination (and individually where applicable) in the liquid-biopsy guided 
Intensive Surveillance arm. 

 
 
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Endpoint: 
Specificity is defined as the proportion of all patients in the liquid-biopsy guided 
Intensive Surveillance arm with no recurrence as verified by imaging during the 
5-year interventional period of the study that had only negative biomarker results 
(i.e., no indication of molecular relapse) within 36 months before the end of the 
5-year interventional period of the study.  
 
Liquid Biopsy False-Positive Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
Objective: 
To determine the false-positive rate of the liquid biopsy markers in combination 
(and individually where applicable) in the liquid-biopsy guided Intensive 
Surveillance arm. 
Endpoint: 
False-positive rate is defined as the proportion of all patients in the liquid-biopsy 
guided Intensive Surveillance arm with a positive biomarker result (i.e., molecular 
relapse) during the 5-year interventional period of the study that had no 
recurrence as verified by imaging within 36 months after first molecular relapse. 
 
Liquid Biopsy False-Negative Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
Objective: 
To determine the false-negative rate of the liquid biopsy markers in combination 
(and individually where applicable) in the liquid-biopsy guided Intensive 
Surveillance arm. 
Endpoint: 
False-negative rate is defined as the proportion of all patients in the liquid-biopsy 
guided Intensive Surveillance arm with only negative biomarker results (i.e., no 
indication of molecular relapse) that had a recurrence as verified by imaging 
during the 5-year interventional period of the study. 
 
Rate of liquid biopsy positivity (CA27.29, CEA, CA125, CTC and ctDNA)  
Objective: 
To determine the overall rate of positive liquid biopsy marker results in 
combination (and individually where applicable) in the liquid-biopsy guided 
Intensive Surveillance arm. 
Endpoint: 
The overall rate of liquid biopsy positivity is defined as the proportion of all 
patients in the liquid-biopsy guided Intensive Surveillance arm that had at least 
one positive biomarker result (i.e., molecular relapse) during the 5-year 
interventional period of the study.  
Translational 
Research 
Objectives and 
Endpoints 
Objective: 
To ensure the possibility of retrospective studies during and after the ongoing 
study, a biobank will be implemented.  
 
 

 
 
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Endpoint: 
A biobank will allow the storage of unused biomaterials from both, the Intensive 
Surveillance arm as well as the Standard Surveillance arm. These materials will be 
used for future retrospective studies.  
Inclusion Criteria 
Patients will be eligible for study participation only if they comply with the 
following criteria: 
1. 
Written informed consent for all study procedures according to local  
regulatory requirements prior to beginning specific protocol procedures. 
2. 
Unilateral or bilateral primary invasive carcinoma of the breast, 
confirmed histologically.  
3. 
Patients with intermediate- to high-risk early breast cancer defined as 
either  
- 
an indication for (neo-)adjuvant chemotherapy (regardless 
whether performed or not), and/or  
- 
Large tumor (> 50 mm), and/or  
- 
Positive lymph nodes (> pN1mi), and/or  
- 
High grade ( G3). 
4. 
A complete resection of the primary tumor, with resection margins free 
of invasive carcinoma. 
5. 
Completion of primary anti-tumor therapy (adjuvant chemotherapy, 
surgery or radiotherapy, whichever occurs last) no more than 24 months 
previously. Enrollment of patients during any kind of adjuvant therapy 
except chemotherapy (e.g., but not limited to endocrine therapy, 
antibody therapy, CDK4/6-inhibitors, PARP inhibitors, PI3K inhibitors, 
antibody-drug conjugates and other novel agents) is allowed. Patients 
with Luminal A/B breast cancer (ER/PGR positive, HER2 negative/low) 
may be enrolled up to 60 months after completion of primary anti-tumor 
therapy.  
6. 
Availability of primary tumor tissue from core biopsy or surgical removed 
tissue (FFPE Slide (≥ 6 mm³, min. 10 slides, thickness: 5 µm-10 µm, area 
>150 mm² and 1 H&E stained slide, minimum 20% tumor content) or FFPE 
Block (≥ 6 mm³ thickness: 100 µm, area: >150 mm² and 1 H&E stained 
slide, minimum 20% tumor content) or Genomic DNA extracted from 
FFPE slides or block (≥ 600 ng, Minimum volume: 25 µL, concentration: 
20 ng/µL, buffer: 10 mM Tris pH 8, 1 mM EDTA)) at timepoint of 
enrollment 
- 
Patients with primary systemic therapy: tissue from core biopsy 
- 
Patients receiving surgery as primary therapy: surgically removed 
cancer tissue or tissue from core biopsy. 
Important note: Tumor tissue of both sides will be needed in case of 
bilateral breast cancer.  
7. 
No current clinical evidence for distant metastases. 
8. 
Females or males ≥ 18 years and ≤ 75 years of age. 

 
 
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9. 
Performance status ≤ 1, Eastern Cooperative Oncology Group (ECOG) 
scale. 
10. 
Patient must be willing and able to comply with scheduled visits, 
treatment plans, laboratory tests, and other study procedures. 
Exclusion Criteria 
Patients will be ineligible for study participation if they comply with the following 
criteria: 
1. 
Patients with a history of any secondary primary malignancy are ineligible 
with the following exceptions: 
- 
in situ carcinoma of the cervix or  
- 
adequately treated basal cell carcinoma of the skin or 
- 
ipsi- or contralateral non-invasive carcinoma of the breast (DCIS). 
2. 
Patients in pregnancy or breastfeeding. 
3. 
History of significant neurological or psychiatric disorders including 
psychotic disorders, dementia or seizures that would prohibit the 
understanding and giving of informed consent. 
4. 
Renal insufficiency with GFR < 30 mL/min. 
5. 
Previous or concomitant cytotoxic or other systemic antineoplastic 
treatment that is not used for treating the primary breast cancer. 
Sample Size 
Determination 
 
The study is designed as a two-arm parallel, (partially) double-blinded 
randomized superiority study. To detect a 2.35% improvement in 5-year OS rate 
from 92.65% in the standard surveillance arm to 95.0% in the tumor marker, 
ctDNA and CTC guided surveillance arm (equivalent to a hazard ratio of 0.672), 
approximately 3500 patients (1750 in both arms) will be required to achieve 80% 
power at a 2-sided significance level of 5%, assuming a uniform accrual pattern 
(accrual time 2 years), a dropout rate of 15% in both arms and a total study 
duration of 10 years. The reference value of 92.65% 5-year OS rate is based on 
the recently published results from the randomized phase III Success A study 
(Clinicaltrials.gov NCT02181101) that reported 5-year OS rates for 3754 
intermediate-to-high-risk early breast cancer patients receiving standard 
chemotherapy regimen. 
Randomization  
Patients will be randomized centrally using permuted block list only if they fulfil 
the inclusion criteria and are not precluded from participation by any of the 
exclusion criteria.  
Stratification 
Factors 
 
Randomization will be stratified using block randomization by: 
• 
Hormone receptor status (negative vs positive) 
• 
HER2 status (negative vs positive) 
• 
Histological lymph node status at surgery ((y)pN0 vs (y)pN+) 
Statistical 
Methods 
 
Study Populations: 
The following study population sets will be examined: 
• 
Intention to Treat (ITT) Set: All randomized patients with signed informed 
consent 

 
 
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• 
Per Protocol (PP) Set: All patients of the ITT set who did not violate any 
inclusion or exclusion criterion and who were treated strictly according 
to protocol. 
If necessary, further data sets required for additional analyses may be specified 
in separate analysis plans. 
 
Primary endpoint analysis and population: 
The primary endpoint overall survival will be analyzed based on the ITT set, 
estimated by the Kaplan Meier product limit method, and 5-year OS rates, 95% 
confidence intervals and survival plots will be provided. Overall survival will be 
compared between patients in the two randomization arms using the log-rank 
test, and univariable cox regression as well as additional multivariable analyses 
adjusted for other factors will be performed using suitable regression models (cox 
proportional hazard regression model). Hazard ratios and the corresponding 95% 
confidence intervals will be reported as obtained by both univariable and 
adjusted multivariable analyses. 
The co-primary endpoint Overall Lead Time Effect will be calculated as described 
above. As it is a purely descriptive composite measure consisting of two unrelated 
median values calculated for different patient cohorts, no confidence intervals 
will be provided. 
 
Secondary endpoint analysis: 
All analyses regarding the secondary objectives will have exploratory character 
only. All secondary endpoints and other outcomes that are calculated based on 
frequencies/rates will be summarized using frequency tables presenting absolute 
and relative frequencies together with appropriate confidence intervals. 
 
Comparisons between groups will be performed using suitable non-parametric 
statistical tests such as Fisher’s exact test, χ2-test, or Cochran-Mantel-Haenszel 
test. Continuous variables will be summarized by descriptive statistics (number 
of valid and missing observations, mean, standard deviation, median, 
interquartiles, minimum, and maximum) and compared between arms using 
suitable non-parametric tests such as Wilcoxon-Mann-Whitney. Secondary 
survival endpoints (IDFS, DDFS, DRFS, BCSS) will be analyzed in the same way as 
the primary endpoint OS. Tumor marker (CA27.29, CEA, CA125) levels, presence 
and number of CTCs as well as presence of ctDNA measured at different time 
points will be evaluated in a descriptive way, and the temporal changes will be 
described and analyzed using appropriate statistical models (e.g., generalized 
linear mixed models). Quality of life data will be analyzed according to the 
instructions of the validated questionnaires used (EORTC QLQ-C30 and PA-F12). 
Additional exploratory analyses (e.g., subgroup analyses) will be performed as 
appropriate. 
More details regarding the statistical analyses will be provided in a Statistical 
Analysis Plan (SAP) which will be finalized prior to data base lock. 

 
 
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Interim Analyses  
 
No formal efficacy interim analysis is planned. 
A three-step futility analysis for ctDNA is planned (as per request of the 
contributing company): 
 
Step 1:  
Analysis of minimum requirement regarding ctDNA positivity rate. 
• 
Analysis is performed based on the first 150 patients enrolled in the 
experimental arm to calculate the ctDNA positivity rate.  
• 
Analysis is considered to be affirmative to continue ctDNA testing within 
the study,  
- 
if the ctDNA positivity rate measured 3 months after recruitment 
is > 5% for these 150 patients and 
- 
if the ctDNA positivity rate measured 12 months after 
recruitment is > 10% for these 150 patients. 
 
Step 2:  
Analysis of minimum requirement regarding molecular to via imaging verified 
distant recurrence lead time (from the time of ctDNA positive result – i.e., 
molecular relapse - to the time of image-verified distant relapse; see detailed 
definition in “Secondary Objectives and Endpoints”) 
• 
Analysis is performed after 30 patients in the experimental arm had a 
ctDNA positive result and a via imaging verified distant recurrence 
(without additional treatment intervention) and a minimum follow-up  
time of 6 months (as from the first ctDNA-positive result), to calculate the 
median lead time from the time of the first ctDNA positive result to the 
time of via imaging verified distant relapse for these 30 patients. 
• 
Analysis is considered to be affirmative to continue ctDNA testing within 
the study, if this lead time is > 6 months. 
Step 3:  
Analysis of minimum requirement regarding overall lead time (molecular relapse 
to via imaging verified distant recurrence lead time + Difference in median time 
to distant recurrence between the study arms; see detailed definition in “Primary 
Objectives and Endpoints” above) 
• 
Analysis is performed after 50 patients in the experimental arm had a 
ctDNA positive result and a via imaging verified distant recurrence and a 
minimum follow-up time of 12 months (as from the first ctDNA positive 
result), to calculate the overall lead time (molecular relapse to via 
imaging verified distant recurrence lead time + Difference in median time 
to distant recurrence between the study arms) for these 50 patients. 

 
 
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• 
Analysis is considered to be affirmative to continue ctDNA testing within 
the study, if this overall lead time is > 9 months. 
If these requirements for continuation of ctDNA testing are not fulfilled, 
termination of ctDNA testing in this trial might be considered pending decision of 
the steering board and the company. 
Biomaterial 
 
- 
Primary tumor tissue from core biopsy or surgical removed tissue (FFPE Slide 
(≥ 6 mm³, min. 10 slides, thickness: 5 µm-10 µm, area >150 mm² and 1 H&E 
stained slide, minimum 20% tumor content) or FFPE Block (≥ 6 mm³ thickness: 
100 µm, area: >150 mm² and 1 H&E stained slide, minimum 20% tumor 
content) or Genomic DNA extracted from FFPE slides or block (≥ 600 ng, 
Minimum volume: 25 µL, concentration: 20 ng/µL, buffer: 10 mM Tris pH 8, 
1 mM EDTA)) at timepoint of enrollment 
▪ 
Patients with primary systemic therapy: tissue from core biopsy 
▪ 
Patients receiving surgery as primary therapy: surgically removed 
cancer tissue or tissue from core biopsy. 
Important note: Tumor tissue of both sides will be needed in case of bilateral 
breast cancer. 
- 
Two Streck Cell-Free DNA BCT® tubes for ctDNA (circulating tumor DNA) 
analysis at timepoint of enrollment and every 3 months for 3 years, thereafter 
every 6 months up to 5 years.  
- 
One standard serum sampling tube (7,5 mL whole blood) for detection of 
tumor markers at timepoint of enrollment, 4 weeks after enrollment and 
every 3 months for 3 years, thereafter every 6 months up to 5 years. 
 
Additionally, another serum sampling tube will be drawn 2 weeks after 
significant increase of one of the tumor markers for confirmation. 
- 
Three whole blood samples in 10 mL CellSave preservative tubes for 
detection of CTCs (Circulating Tumor Cells) one year after study inclusion. 
Patients with detection of a tumor marker rise for the first time will have one 
additional blood sampling for CTCs (three whole blood samples in 10 mL 
CellSave preservative tubes) at the next regular study visit. If ≥ 1 CTC is 
detected and staging is M0, patients will continue liquid biopsy testing as 
before.  
Blood samples from patients in the control group will not be analyzed 
immediately but stored for later retrospective analyses in the context of 
translational research projects. 
Enrollment period 
Approximately 24 months. 
Follow-up 
After the interventional part of the study is concluded with the last liquid biopsy 
taken at the end of Year 5, follow-up of five years without any intervention is 
intended. Follow-up data is collected via digital health application, telephone 
calls or on paper. The assessments for the follow-up visits are described in the 
study protocol.  
In terms of long-term follow-up after end of study, patients have the possibility 
to participate in the patient self-reporting registry (Patientenselbstauskunft). 

 
 
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Number of sites 
 
It is planned to conduct the study with up to 150 study sites in Germany. 
These study sites have already participated in various studies within the AGO B, 
SUCCESS and DETECT study groups, and the SURVIVE study is supported by 
several German breast-cancer patient advocacy groups (mamazone e. V., PATH-
Foundation, Brustkrebs Deutschland e. V., Frauenselbsthilfe Krebs e. V., 
MammaMia!). 
Timelines: 
FPI                                                                         
Q4/2022 
 
LPI                                                                       
Q4/2024 
 
LP EOT / EOI                                                               Q4/2029 
 
 
End of Study (EOS)                                               
Q4/2035 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Figure 1: SURVIVE Study Design 

 
 
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2 
TABLE OF CONTENTS 
1 
PROTOCOL SYNOPSIS ................................................................................................................. 2 
2 
TABLE OF CONTENTS................................................................................................................ 16 
3 
INTRODUCTION ........................................................................................................................ 20 
3.1 
Prevalence, Incidence, Mortality of Breast Cancer ............................................................. 20 
3.2 
Burden of disease ............................................................................................................... 20 
3.3 
Impact of the Study ............................................................................................................. 20 
3.4 
Standard Follow-Up Surveillance in Early Breast Cancer Patients ...................................... 21 
 
Rationale for Tumor markers in Liquid biopsy-based Follow-Up ............................. 22 
 
Rationale for CTCs in Liquid biopsy-based Follow-Up .............................................. 23 
 
Rationale for ctDNA in Liquid biopsy-based Follow-Up ............................................ 23 
3.5 
Appropriateness of the Primary Endpoint .......................................................................... 24 
3.6 
Risk – Benefit Analysis for the Participants......................................................................... 25 
3.7 
Interpretation of Potential Study Results ........................................................................... 26 
4 
STUDY OBJECTIVES ................................................................................................................... 27 
4.1 
Primary Objectives .............................................................................................................. 27 
4.2 
Secondary Objectives .......................................................................................................... 27 
4.3 
Translational Research Objectives ...................................................................................... 28 
5 
STUDY ENDPOINTS ................................................................................................................... 29 
5.1 
Primary Endpoints ............................................................................................................... 29 
5.2 
Secondary Endpoints .......................................................................................................... 29 
5.3 
Translational Research Endpoints ....................................................................................... 31 
6 
STUDY DESIGN ......................................................................................................................... 32 
6.1 
Description of the Study Design .......................................................................................... 32 
6.2 
Study design ........................................................................................................................ 33 
7 
STUDY POPULATION ................................................................................................................ 34 
7.1 
Number of Patients ............................................................................................................. 34 
7.2 
Inclusion Criteria ................................................................................................................. 34 
7.3 
Exclusion Criteria................................................................................................................. 35 
8 
STUDY INTERVENTIONS ........................................................................................................... 36 
8.1 
Standard Surveillance ......................................................................................................... 36 
8.2 
Intensive Surveillance ......................................................................................................... 37 
 
Determination of Tumor markers............................................................................. 37 
 
Determination of CTC levels ..................................................................................... 38 
 
Determination of ctDNA levels ................................................................................. 39 
 
Overview of Requirements for Collection of Biomaterials ....................................... 41 
 
Blinding ..................................................................................................................... 41 

 
 
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8.3 
Staging Examinations .......................................................................................................... 41 
8.4 
Study visits .......................................................................................................................... 41 
 
Study Inclusion / Baseline Visit ................................................................................. 42 
 
Visits During Interventional Part of the Study .......................................................... 42 
 
End of Intervention (EOI) .......................................................................................... 43 
8.5 
Study Procedures ................................................................................................................ 44 
 
Patient Entry Procedures .......................................................................................... 47 
8.5.1.1 
Patient Consent Form and Screening ........................................ 47 
8.5.1.2 
Randomization and Stratification .............................................. 48 
 
Screening .................................................................................................................. 48 
 
Evaluation during Interventional Phase ................................................................... 50 
 
Evaluation after End of Intervention - Follow-up ..................................................... 51 
 
Long-Term Follow-up After End of Study ................................................................. 52 
 
Study visits table ....................................................................................................... 53 
9 
COLLECTION OF BIOMATERIALS AND TRANSLATIONAL RESEARCH ........................................ 55 
9.1 
Overview of Requirements for Collection of Biomaterials ................................................. 55 
 
Tumor Material ......................................................................................................... 56 
9.1.1.1 
Mandatory tumor material ........................................................ 56 
9.1.1.2 
Optional tumor material ............................................................ 56 
 
Blood Samples .......................................................................................................... 56 
9.1.2.1 
Mandatory blood samples for all patients ................................. 56 
 Tumor Marker Analysis ................................................................................ 57 
 CTC Analysis ................................................................................................. 58 
 ctDNA Analysis ............................................................................................. 59 
9.1.2.2 
Optional blood samples ............................................................. 60 
10 
DISCONTINUATION OF STUDY INTERVENTION, PATIENT, STUDY AND SITE ............................ 61 
10.1 Patients without Intervention ............................................................................................. 61 
10.2 Premature Intervention Discontinuation ............................................................................ 61 
10.3 Investigator-Initiated Discontinuation of Study Intervention............................................. 61 
10.4 Patient-Initiated Study Discontinuation vs Withdrawal of Consent ................................... 61 
10.5 Premature Termination of Study ........................................................................................ 62 
10.6 Premature Termination of Study at a Particular Site .......................................................... 62 
11 
END OF INTERVENTION (EOI) ................................................................................................... 63 
12 
END OF STUDY (EOS) ................................................................................................................ 64 
13 
ASSESSMENT OF OUTCOME .................................................................................................... 65 
13.1 Evaluation of Local Recurrence ........................................................................................... 65 
13.2 Evaluation of Distant Metastases ....................................................................................... 65 

 
 
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13.3 Evaluation of Biomarkers .................................................................................................... 65 
14 
ASSESSMENT OF HEALTH-RELATED QUALITY OF LIFE ............................................................. 67 
14.1 Rationale ............................................................................................................................. 67 
14.2 Questionnaires .................................................................................................................... 68 
14.3 Digital Health Application ................................................................................................... 68 
14.4 Analysis ............................................................................................................................... 69 
15 
DATA HANDLING AND DATA QUALITY ASSURANCE ................................................................ 70 
15.1 Data Management and Documentation ............................................................................. 70 
 Data Entry and Queries ............................................................................................. 70 
 Data Validation ......................................................................................................... 70 
 Registration / Recruitment Stop ............................................................................... 70 
 Database Lock ........................................................................................................... 70 
15.2 Privacy Protection and Data Safety..................................................................................... 71 
 Pseudonymization .................................................................................................... 71 
 User Access Control .................................................................................................. 71 
15.3 Record retention after study completion ........................................................................... 71 
16 
STATISTICS ................................................................................................................................ 72 
16.1 Randomization and stratification........................................................................................ 72 
16.2 Analysis Sets ........................................................................................................................ 72 
 Intent-to-Treat (ITT) Set ............................................................................................ 72 
 Per Protocol (PP) Set ................................................................................................. 72 
16.3 Sample Size Determination ................................................................................................. 72 
16.4 Patient disposition, data re-coding and display .................................................................. 73 
16.5 Analysis of Primary Endpoint(s) .......................................................................................... 73 
16.6 Analysis of Secondary Endpoints ........................................................................................ 73 
16.7 Pre-planned subgroup analyses .......................................................................................... 74 
16.8 Interim/Futility Analyses ..................................................................................................... 74 
 Interim analyses for efficacy ..................................................................................... 74 
 Futility analyses ........................................................................................................ 74 
 Interim analysis for Quality of Life (QoL) .................................................................. 75 
17 
INDEPENDENT DATA MONITORING COMMITTEE (IDMC) ....................................................... 76 
17.1 IDMC Members and Mission .............................................................................................. 76 
17.2 Documentation Provided to the IDMC ............................................................................... 76 
17.3 Recommendations of the IDMC .......................................................................................... 76 
18 
ADMINISTRATIVE EXECUTION.................................................................................................. 77 
18.1 Monitoring and Auditing ..................................................................................................... 77 
18.2 Sponsor’s Responsibilities ................................................................................................... 77 

 
 
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18.3 Investigator’s Responsibilities ............................................................................................. 77 
18.4 Patient Informed Consent ................................................................................................... 78 
18.5 Ethics and Regulatory Considerations ................................................................................ 78 
18.6 Declaration of Helsinki ........................................................................................................ 79 
18.7 Study Conduct ..................................................................................................................... 79 
18.8 Modification of the Protocol ............................................................................................... 79 
18.9 Study Documents ................................................................................................................ 79 
18.10 Case Report Forms .............................................................................................................. 79 
18.11 Necessary Documents ......................................................................................................... 80 
18.12 Archiving ............................................................................................................................. 80 
18.13 Use of Information and Publication .................................................................................... 80 
18.14 Finance and Insurance ........................................................................................................ 80 
19 
INSURANCE COVER .................................................................................................................. 81 
20 
GLOSSARY OF ABBREVIATIONS AND ACRONYMS .................................................................... 82 
21 
INVESTIGATOR SIGNATURE PAGE ............................... FEHLER! TEXTMARKE NICHT DEFINIERT. 
22 
APPENDICES ............................................................................................................................. 85 
22.1 Appendix A:  Quality of Life Questionnaires ....................................................................... 85 
 EORTC-QLQ C30 ........................................................................................................ 85 
 PA-F12 ....................................................................................................................... 85 
22.2 Appendix B: Assessment of Performance status and activities of daily living .................... 85 
 Determination of Performance Status ..................................................................... 85 
22.3 Appendix C: Deutsche Synopse ....................................... Fehler! Textmarke nicht definiert. 
23 
REFERENCES ............................................................................................................................. 86 
 

 
 
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3 
INTRODUCTION 
Breast cancer is the world’s most common cancer entity. After primary therapy of early breast cancer, 
guidelines limit routine surveillance in breast cancer survivors to clinical surveillance in combination 
with breast imaging. This approach is based on two large, but outdated cohort studies conducted in 
the 1980s suggesting no superiority of intensified screening for distant metastasis versus standard 
surveillance for OS. To date, no other large prospective studies have been conducted addressing the 
matter. Thus, screening for distant metastases is initiated only in patients with specific symptoms.  
We aim to establish a liquid biopsy guided follow-up surveillance method for the sensitive and specific 
detection of distant metastases before the onset of symptoms. We hypothesize, in consideration of 
modern therapeutic agents, that earlier detection of distant (oligo-) metastasis and therefore earlier 
initiation of therapy in a pre-symptomatic stage will improve OS. Furthermore, we expect patient’s 
Quality of Life (QoL) to increase due to intensified follow-up examinations exceeding clinical 
evaluation. 
3.1 
Prevalence, Incidence, Mortality of Breast Cancer 
Newly diagnosed per year – worldwide: ~ 2.26 million; in Germany: ~ 69 700. 
5-year prevalence – worldwide: ~ 7.79 million; in Germany: ~ 300 000. 
Incidence Rate – worldwide: 47.8 per 100 000; in Germany: 82.2 per 100 000. 
Mean age at first diagnosis: 64 years, affecting all age groups after puberty. 
Mortality Rate – worldwide: 13.6 per 100 000 (~ 685 000); in Germany: 16.0 per 100 000 (~ 20 600) 
(1). 
3.2 
Burden of disease 
On the global incidence of 1.7 million newly diagnosed patients in 2016, breast cancer caused 15.1 
million (95% CI, 14.3-16.2 million) DALYs (disability-adjusted life-years), of which 95% came from YLLs 
(years of life lost) and 5% from YLDs (years lived with disability) (2). Treatment costs and therefore the 
socioeconomical burden of breast cancer generally increases with higher disease stages at diagnosis. 
The mean treatment costs of FIGO stage I disease were $29,724. At stages II, III and IV mean costs 
were 32%, 95%, and 109% higher than those of stage I disease, and the mean treatment costs of 
locoregional advanced disease and distant breast cancer were 41% and 165% higher than those of 
local disease. Earlier detection of breast cancer increases the likelihood of cure and therefore reduces 
treatment costs  (3,4).  
3.3 
Impact of the Study 
Routine surveillance in breast cancer survivors is limited to clinical examinations every three months 
and breast imaging once a year. Screening for distant metastases is only performed in patients with 
metastasis-specific symptoms. To address patients’ needs and to improve OS, we propose a liquid 
biopsy guided follow-up surveillance method for the detection of distant (oligo-)metastases to enable 
earlier initiation of therapy in a pre-symptomatic stage.  

 
 
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If liquid biopsy-guided follow-up is found to detect metastatic disease with high sensitivity and 
specificity, this study could contribute to a paradigm shift in the current follow-up of breast cancer 
survivors. Such an intensified follow-up could, in turn, lead to an improvement in the QoL of patients 
as they are regularly screened for a relapse and therefore, if the screening is negative, have the 
assurance of being disease free. On the other hand, if screening is positive, early detection of distant 
recurrence before the onset of symptoms would allow earlier initiation of therapy, thereby delaying 
the onset of symptoms, prolonging survival, and potentially still curing patients. We therefore expect 
the patients’ QoL to improve by the intervention alone, and not only in case of an increased OS. 
Furthermore, earlier detection of distant recurrence and (oligo-)metastatic disease might reduce the 
need for cytotoxic chemotherapy and may provide a chance of full recovery in the future, even at the 
stage of secondary disease. In the long term, this could contribute to a reduction of treatment costs 
and to more cost-efficient therapies as well as proposing an important link between breast centers 
and usually resident follow-up doctors. 
3.4 
Standard Follow-Up Surveillance in Early Breast Cancer Patients 
Follow-up surveillance in patients with early breast cancer today is based on two large prospective 
randomized studies conducted in the 1980s in Italy (5,6). Both aimed to assess different follow-up 
policies and their impact on survival. In the GIVIO trial (published in 1995), 1320 women with breast 
cancer stage I-III were randomized to an intensive surveillance including bone scans, liver sonograms 
and chest X-rays, or to a control regimen. After a median follow-up of 71 months, OS did not differ 
between groups with 20% and 18% deaths in the intensive and the control group, respectively. No 
significant differences were apparent in time to detection of recurrence between the two groups (5). 
In the other trial, conducted by Del Turco et al. and Palli et al. (published in 1994), 1243 women with 
early breast cancer were also randomized to conventional or intensive follow-up including bone scans 
and chest X-rays. Increased detection of isolated pulmonary and bone metastases was evident in the 
intensive compared to the clinical follow-up group (112 vs. 71 cases), while no difference was 
observed for other sites and for local and/or regional recurrences. The 5-year relapse-free survival 
rate was significantly higher for the clinical follow-up group, with patients in the intensive follow-up 
group showing earlier detection of recurrences, whereas no difference in 5-year OS (81,4% (intensive) 
vs. 80,5% (conventional)) was observed (6).  
Based on this evidence, national and international guidelines recommend standard follow-up without 
imaging (except for mammography) for asymptomatic breast cancer patients. In Germany patients are 
encouraged to visit their doctors every three months for the first three years after diagnosis and every 
six months for the consecutive two years. Here, a clinical evaluation on the patients’ health will be 
performed, adherence to any adjuvant therapy will be checked, follow-up subscriptions will be issued 
and yearly mammograms will be scheduled.  
However, these trials have been conducted in an era of outdated technology and limited therapeutic 
options. It is possible that recent improvements in diagnostics and early, target-oriented treatments 
could lead to long-term remissions and improve both, survival and QoL outcomes. Some of the most 
important improvements in breast cancer treatment include: enhancement of endocrine therapy 
(aromatase-inhibitors, GnRH-analogues, new SERMs, CDK 4/6 inhibitors, mTOR inhibitors, PI3K-
inhibitors), modern cytostatic treatment regimens (including taxane-based chemotherapy, 
Capecitabine, Gemcitabine, Vinorelbine, Eribulin) and reduced cytotoxicity due to supporting agents 

 
 
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(especially modern antiemetics), biological targeted therapies like monoclonal antibodies 
(Trastuzumab, Pertuzumab, Atezolizumab) and antibody-drug-conjugates (Trastuzumab-emtansine, 
Trastuzumab-deruxtecan, 
Sacituzumab-govitecan), 
immunotherapies 
(Atezolizumab, 
Pembrolizumab) other targeted treatments like tyrosine-kinase-inhibition or PARP-inhibition as well 
as improved locoregional therapy. Many of these procedures have shown to improve overall survival, 
especially when conducted in early phases of metastatic disease (7–12). In addition, survival depends 
on when treatment is started after diagnosis of metastatic disease (13). 
Despite the feasibility of these new therapies, there have not been any new studies about the impact 
of intensified follow-up for breast cancer patients yet, with the last Cochrane review dating back to 
2016, lacking additional recommendations (14). Thus, research is necessary to improve breast cancer 
follow-up strategies, as aftercare visits today are heterogeneous. We aim to establish a concept to 
improve the follow-up surveillance for patients with early breast cancer and to change the current 
standard of care for this patient group. As cancer therapies become increasingly individualized, 
molecular biomarkers represent a key technique in aiming to predict the prognosis or monitor 
treatment response. 
 
Rationale for Tumor markers in Liquid biopsy-based Follow-Up 
Soluble MUC1 has been discussed as a biomarker for predicting prognosis, treatment efficacy, and 
monitoring disease activity in breast cancer patients, as clear associations were found between tumor 
size/burden and soluble MUC1 levels (15–19). Immunoassays for CA15.3 and CA27.29 target the 
epitopes on the same MUC1 and are considered identical. They are well-known assays that allow the 
detection of circulating MUC1 antigen in peripheral blood. CEA levels are usually less commonly 
elevated than the MUC1-assay, but studies suggest an additional value through gaining sensitivity by 
testing CEA levels in addition to CA27.29/CA15.3 (20). As with MUC1 levels, a clear correlation 
between tumor size/burden and CEA levels is reported (15,21). While the American Society of Clinical 
Oncology (ASCO) recommends using CA27.29/CA15.3 and CEA only in conjunction with diagnostic 
imaging for treatment monitoring of patients with metastatic disease, their role during follow-up of 
early-stage breast cancer remains unclear (20,22). Several studies could show that an increase in 
CA27.29/CA15.3 and CEA after primary and/or adjuvant therapy may predict distant recurrence 
before the onset of symptoms or (imaging) tests (23,17). However, prospective randomized clinical 
trials to demonstrate whether detection and treatment of these asymptomatic metastases impact on 
the most significant outcomes (OS, QoL) are lacking. MUC16-based CA125 is commonly used to 
monitor ovarian cancer, but has rarely been studied in detecting breast cancer recurrence. Yet, it has 
shown sensitivity in breast cancer (24,25), especially when used in addition to MUC1 based 
CA27.29/CA15.3 and CEA, through which sensitivity of detecting metastasis could be increased by 
21.3%, while maintaining a high specificity (26). Heterogeneity of CA125 in breast cancer is mainly 
associated with the sites of metastasis as high CA125 levels are associated with visceral or pleural, 
rather than with bone or soft tissue involvement. In general, elevated levels of these tumor markers 
are associated with worse prognosis (27,28), and retrospective studies suggest that not a cut-off based 
evaluation, but a kinetics based approach (increase-delta from an individual baseline) should be used 
to increase sensitivity and specificity in detecting metastatic disease or disease progression (26,29,30). 
The most feasible cut-off values for increasing tumor markers have been reported as 75% for CA15.3, 
100% for CEA and 150% for CA125 (29), where a sensitivity of 72.3% and a specificity of 99% could be 

 
 
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achieved (data from the “Münchner Nachsorgestudie Großhadern”, Dr. P. Stieber, personal 
communication).  
While interchanging CA15.3 and CA27.29 testing is not advised, results are usually similar and 
concordant (20,31,32). An application of the presented principles for CA15.3 on CA27.29 testing 
appears unproblematic.  
 
Rationale for CTCs in Liquid biopsy-based Follow-Up 
An early hematogenic spread of tumor cells is observed in some patients with early breast cancer. 
These cells can be detected in the peripheral blood, are known as circulating tumor cells (CTCs) and 
are reported widely as a valuable prognostic factor, both at time of primary diagnosis and in the 
context of distant metastases (33–35). In metastasized patients, the detection of CTCs itself and 
especially over 5 CTCs in 7,5 mL of blood is reportedly associated with poor prognosis and a lower life 
expectancy (36,37). Currently, the AGO-guidelines assess the detection of CTCs in metastasized 
patients as useful in particular cases to assist clinical decision-making. In the SUCCESS-trial conducted 
by our study group, we demonstrated the prognostic relevance of circulating tumor cells in peripheral 
blood of early breast cancer patients before and after adjuvant treatment. Persistence of CTCs after 
adjuvant chemotherapy demonstrated an unfavorable prognosis (38), which confirmed the data of 
other studies suggesting CTCs as an independent prognostic factor for early relapses and aggressive 
tumor behavior (39–41). 
 
Rationale for ctDNA in Liquid biopsy-based Follow-Up 
Breast cancer genomes may harbor from few to hundreds of rearrangements and mutations per 
tumor (42). Whereas somatic point mutations can be present in various tumor types and individuals, 
chromosomal rearrangements are individual and highly tumor specific, serving as a distinctive genetic 
signature of a tumor (43). ctDNA has been shown to mirror the mutational signature of the primary 
tumor (analyzed via exome sequencing), is detectable the in blood plasma of patients with advanced 
malignancies and shows increasing potential as a non-invasive biomarker for monitoring tumor 
growth and response to treatment (44). During follow-up, where patients’ plasma usually contains 
very few tumor-derived DNA fragments, detection of ctDNA can be challenging. However, by using 
information from primary tumor tissue, it is possible to design personalized sequencing panels to track 
patient-specific tumor mutations and to highly increase sensitivity (45)(46). The use of ctDNA during 
breast cancer follow-up via personalized ctDNA profiling has been described as highly sensitive, 
generating a lead-time in the ctDNA detection preceding the clinical detection of metastasis up to two 
(47), respectively three years (48). Both studies showed a specificity of 100%, with no non-relapsing 
patients being ctDNA-positive at any timepoint. Nevertheless, there are to date no prospective, 
randomized clinical trials to analyze the clinical validity of conventional tumor markers, CTCs and 
ctDNA combined with diagnostic imaging that will eventually lead to a therapeutic intervention, which 
is where our proposed study will close the gap. 
To support our approach, we conducted a blinded pilot study during the development phase of this 
study. During this pilot study, a retrospective analysis of tumor and blood samples from early breast 
cancer patients with known (but blinded) clinical outcome was conducted using the RaDaR™ (Residual 
Disease and Recurrence) assay by Inivata Inc. It was shown that in 15 of 21 patients with confirmed 

 
 
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clinical distant recurrence, ctDNA could be detected. Out of the 6 remaining patients, 5 suffered from 
local recurrence without evidence of distant metastasis. These results indicate that the RaDaR™ assay 
is well suited as a ctDNA-based approach for early detection of disease relapse (49). 
3.5 
Appropriateness of the Primary Endpoint  
Overall Survival is defined as the time from randomization in this study until death of any cause. OS is 
considered to be a gold-standard endpoint in oncological studies leading to a good comparability to 
other studies on a global level. Furthermore, it is easily measured, unbiased and clinically significant. 
To evaluate the benefit of an intensified, liquid-biopsy based surveillance, the OS seems to be 
appropriate. Recurrence- or disease-free survival (DFS) as well as distant disease-free survival (DDFS) 
as a surrogate marker for overall survival are not adequate in this study, as in case of positive lead 
time, DFS and DDFS are shortened in the experimental group, while OS might be positively influenced 
at the same time because of early treatment intervention. 
As our second primary objective, we will determine whether an Overall Lead-Time effect can be 
generated in the follow-up of early breast cancer patients due to tumor marker/CTC/ctDNA guided 
surveillance compared to standard surveillance after primary therapy. Overall Lead Time Effect is a 
composite measure, defined as the time from molecular to via imaging verified distant recurrence 
lead time plus the difference in time to distant recurrence between the two arms (i.e., difference 
between median time from randomization to distant recurrence for all patients with distant 
recurrence in the Standard Surveillance arm and median time from randomization to distant 
recurrence for all patients with distant recurrence in the liquid-biopsy guided Intensive Surveillance 
arm; see Figure 2). This will be assessed only for all markers in combination (as this composite 
endpoint cannot be calculated for each marker separately). The Overall Lead Time Effect will be a 
surrogate parameter for the time gained by using liquid biopsy to detect a recurrence. As we hope to 
detect distant recurrence of disease earlier, these patients will be treated earlier with the applicable 
therapy regimen. It has been shown that a delay in treatment can result in poorer overall survival in 
invasive non-metastatic as well as metastatic breast cancer. Delayed first treatment (>90 vs ≤30 days 
postdiagnosis) was associated with worse OS in patients with both invasive nonmetastatic and 
metastatic breast cancer (50), while another study in metastatic breast cancer patients found that a 
treatment delay of 12-24 weeks compared to a treatment delay of 4-12 weeks was associated with 
greater risk of death from first treatment (13). Thus, in both of these studies, the time lost due to later 
start of therapy was associated with a poorer OS. By investigating and evaluating the Overall Lead 
Time Effect, we aim to quantify the time gained through intensified liquid biopsy-based monitoring to 
treat the recurrent breast cancer earlier and therefore improve OS. 
 

 
 
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Figure 2: SURVIVE Study Design with indicated Overall Lead Time and other relevant timepoints for 
calculation 
3.6 
Risk – Benefit Analysis for the Participants 
In addition to routine aftercare practice, patients participating in this study will receive regular blood-
drawals to have the chance of being monitored by liquid-biopsy based procedures. If imaging is 
triggered and metastases are detected, the time to receive treatment is potentially shorter compared 
to the Standard Surveillance arm. This may have an impact on OS on the one hand, but will introduce 
anticancer therapy at an earlier timepoint, causing side effects and possibly a deterioration of QoL.  
In the Standard Surveillance arm, the patients will have no direct benefit from participating in this 
study. Nevertheless, the standard of current aftercare in breast cancer patients is always met, thus 
they have no disadvantage to patients not participating in this study, receiving standard aftercare.  
Risks participating in this study mainly arise from drawing blood, where swelling, pain, bleeding, 
hematoma, infection or fainting may be commonly observed. However, standard procedures of care 
e.g., disinfection of the site and using pressure to prevent bleeding and consecutive hematoma will 
be taken by the participating study centers. If imaging is triggered, a CT scan will be performed. The 
American Food and Drug Administration (FDA) describes the risks of a CT scan as the possibility of 
finding something benign that could lead to unnecessary diagnostics and as a source of radiation (51). 
The risk of developing radiation-induced cancer is said to be very low in standard CT scans (52) and 
widely discussed. As a preemptive measure, we limit the number of consecutive CT scans to a 
maximum of four CT scans per year. Further disadvantages by participating in this study may include: 
Extra commitment and time spent by additional appointments at the study center and constant 
reminders of a potentially threatening illness at a stage in life, where most patients have carried on 
with their lives and would otherwise solely see their personal Ob/Gyn for aftercare visits. This may 
result in a deterioration of QoL compared to patients not participating in this study.  
 
 

 
 
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Reasons for discontinuation of the study are outlined in section 10.   
Balancing the risk of interventions and potential benefits, we conclude that the anticipated benefit of 
earlier detection of recurrence outweighs the risks and is medically justified.  
3.7 
Interpretation of Potential Study Results  
Different scenarios of results may lead to the following differential conclusions. 
A higher overall survival could be seen in the Intensive Surveillance arm which would lead to a change 
in standard surveillance for breast cancer and make way to establish liquid biopsy as a screening tool. 
If a higher or equal overall survival could be seen in the Standard Surveillance arm, standard of care 
will be proven and nothing will change in the current standard.  This interpretation is also valid for an 
observed difference in the secondary objectives such as Invasive disease-free survival (IDFS), distant 
disease-free survival (DDFS) and distant recurrence-free survival (DRFS). If a higher Overall Lead Time 
Effect can be shown in the Intensive Surveillance group, the use of liquid-biopsy can be proven as a 
useful tool to detect recurrence of disease earlier and might lead to earlier treatment intervention. If 
no Overall Lead Time Effect can be seen, standard of care will persist. If sensitivity and specificity of 
the liquid biopsy markers used (CA27.29, CEA, CA125, CTC, ctDNA) is seen as high for one marker or a 
combination of markers, establishing these specific markers in the aftercare as an extra tool to 
monitor patients is a possibility. If sensitivity and/or specificity of one or multiple markers is seen as 
low, the concerning marker may be discarded as a screening tool. Depending on the Molecular to via 
Imaging verified Recurrence Lead Time in the Intensive Surveillance arm, possible timepoints for 
future treatments of the concerning patients can be hypothesized and establish further grounds for 
future interventional drug studies.  
Regarding QoL, the influence of blood sampling, liquid biopsy results and imaging on quality of live 
has to be studied and monitored thoroughly. If a higher QoL is seen in the Intensive Surveillance arm, 
further encouragement to implement liquid biopsy could be given, as liquid biopsy and consecutive 
imaging if necessary, could elevate the patients’ feeling of being closely watched and not left alone. If 
QoL is lower in the Intensive Surveillance arm, liquid biopsy and consecutive imaging could be seen as 
a source of frightening patients and reminding them that they had cancer instead of reassuring them 
of their health. The potential influence has to be balanced with potential benefits and risks outside 
the spectrum of QoL. 
As the field of research regarding liquid-biopsy based decision-making on therapeutic interventions is 
rapidly evolving, a therapeutic intervention for patients with positive findings in their liquid biopsy 
during this study could be a possibly scenario and would require an amendment of this protocol. 

 
 
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4 
STUDY OBJECTIVES 
4.1 
Primary Objectives 
Overall Survival (OS) 
To compare 5-year OS rates between patients in the Standard Surveillance arm versus patients in the 
liquid-biopsy guided Intensive Surveillance arm. 
 
Overall Lead Time Effect 
To determine the Overall Lead Time Effect generated due to tumor marker/CTC/ctDNA guided 
Intensive Surveillance compared to Standard Surveillance after primary therapy in early breast cancer 
patients. 
4.2 
Secondary Objectives 
Invasive disease-free survival (IDFS) 
To compare IDFS between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Distant disease-free survival (DDFS) 
To compare DDFS between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Distant recurrence-free survival (DRFS)  
To compare DRFS between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Breast cancer specific survival (BCSS) 
To compare BCSS between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Invasive breast cancer free survival (IBCFS) 
To compare IBCFS between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Overall Survival (OS) after 10 Years  
To compare 10-year OS rates between patients in the Standard Surveillance arm versus patients in 
the liquid-biopsy guided Intensive Surveillance arm. 
 
Molecular to via Imaging verified Recurrence Lead Time in the Interventional Arm 
To determine the Lead Time (median, interquartile, range, 95% confidence interval) generated in the 
liquid-biopsy guided Intensive Surveillance arm. 
 
 

 
 
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Quality of life (QoL) with questionnaires: EORTC QLQ-C30 and PA-F12 
To compare QoL between patients in the Standard Surveillance arm versus patients in the liquid-
biopsy guided Intensive Surveillance arm. 
 
Liquid biopsy sensitivity (CA27.29, CEA, CA125, CTC and ctDNA) 
To determine sensitivity (i.e., the true positive rate) of the liquid biopsy markers in combination (and 
individually where applicable) in the liquid-biopsy guided Intensive Surveillance arm. 
 
Liquid biopsy specificity (CA27.29, CEA, CA125, CTC and ctDNA)  
To determine specificity (i.e., the true negative rate) of the liquid biopsy markers in combination (and 
individually where applicable) in the liquid-biopsy guided Intensive Surveillance arm. 
 
Liquid Biopsy False-Positive Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
To determine the false-positive rate of the liquid biopsy markers in combination (and individually 
where applicable) in the liquid-biopsy guided Intensive Surveillance arm. 
 
Liquid Biopsy False-Negative Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
To determine the false-negative rate of the liquid biopsy markers in combination (and individually 
where applicable) in the liquid-biopsy guided Intensive Surveillance arm. 
 
Rate of liquid biopsy positivity (CA27.29, CEA, CA125, CTC and ctDNA)  
To determine the overall rate of positive liquid biopsy marker results in combination (and individually 
where applicable) in the liquid-biopsy guided Intensive Surveillance arm. 
4.3 
Translational Research Objectives 
To ensure the possibility of retrospective studies during and after the ongoing study, a biobank will be 
implemented.  
 

 
 
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5 
STUDY ENDPOINTS 
5.1 
Primary Endpoints 
Overall Survival (OS)  
OS is defined as time from randomization until the death of the patient independent of cause of death. 
If a patient is not known to have died, OS is censored at the date of last contact. 
 
Overall Lead Time Effect 
This endpoint is a composite measure, defined as the median time from molecular to via Imaging 
verified Recurrence Lead Time (calculated only for patients in the liquid-biopsy guided Intensive 
Surveillance arm; see secondary endpoint definition below) + Difference in time to distant recurrence 
between the two arms (i.e., difference between median time from randomization to distant 
recurrence for all patients with distant recurrence in the Standard Surveillance arm and median time 
from randomization to distant recurrence for all patients with distant recurrence in the liquid-biopsy 
guided Intensive Surveillance arm; see Figure 2). The Overall Lead Time Effect will be assessed for all 
markers in combination. 
5.2 
Secondary Endpoints 
Invasive disease-free survival (IDFS) 
IDFS is defined as time from randomization until first IDFS event, including any invasive ipsilateral, 
regional, contralateral, and distant disease recurrence, second primary tumors, or death from any 
cause as event; non-invasive, in-situ cancer events are excluded. If a patient has not had an event, 
IDFS is censored at the date of last adequate tumor assessment. 
 
Distant disease-free survival (DDFS) 
DDFS is defined as time from randomization until first DDFS event including metastasis, second 
primary tumors and death from any cause as event. If a patient has not had an event, DDFS is censored 
at the date of last adequate tumor assessment. 
 
Distant recurrence-free survival (DRFS)  
DRFS is defined as time from randomization until first DRFS event including metastasis and second 
primary tumors; death from any cause is not included as event. If a patient has not had an event, DRFS 
is censored at the date of last adequate tumor assessment. 
 
Breast cancer specific survival (BCSS) 
BCSS is defined as time from randomization until breast cancer associated death of the patient. If a 
patient is not known to have died, BCSS is censored at the date of last contact. If a patient has died 
for reasons not associated with breast cancer (by clinical assessment), BCSS is censored at the date of 
death. 
 
 
 

 
 
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Invasive breast cancer free survival (IBCFS) 
IBCFS is defined as time from randomization until first IBCFS event, including any invasive ipsilateral, 
regional, contralateral and distant disease recurrence or death from any cause as event; non-invasive, 
in-situ cancer events are excluded. If a patient has not had an event, IBCFS is censored at the date of 
last adequate tumor assessment. 
 
Overall Survival (OS) after 10 Years  
OS is defined as time from randomization until the death of the patient independent of cause of death. 
If a patient is not known to have died, OS is censored at the date of last contact. 
 
Molecular to via Imaging verified Recurrence Lead Time in the Interventional arm 
The Lead Time is defined as time from first molecular relapse (definitions see above and in Figure 2) 
to via imaging verified recurrence for all patients in the liquid-biopsy guided Intensive Surveillance 
arm for whom a via imaging verified recurrence is documented during the 5-year interventional period 
of the study. If a patient has a via imaging verified recurrence but no documented molecular relapse, 
the Lead Time for this patient is set to ‘0’. This secondary endpoint will be assessed for all markers in 
combination and, where applicable, for each marker separately. 
 
Quality of life (QoL) with questionnaires: EORTC QLQ-C30 and PA-F12 
QoL will be monitored in both groups using 2 questionnaires which are to be completed every 6 
months (EORTC QLQ-C30 and PA-F12) in the interventional phase (first five years) and once a year 
during the five years of follow-up. QoL data will be collected on paper or via a digital health application. 
  
Liquid biopsy sensitivity (CA27.29, CEA, CA125, CTC and ctDNA) 
Sensitivity is defined as the proportion of all patients in the liquid-biopsy guided Intensive Surveillance 
arm with a recurrence as verified by imaging during the 5-year interventional period of the study that 
had a positive biomarker result (i.e., molecular relapse) within 36 months before the recurrence as 
verified by imaging occurred (or within 60 months before the recurrence as verified by imaging 
occurred if in the first 36 months at least once a year a positive biomarker is measured). 
 
Liquid biopsy specificity (CA27.29, CEA, CA125, CTC and ctDNA)  
Specificity is defined as the proportion of all patients in the liquid-biopsy guided Intensive Surveillance 
arm with no recurrence as verified by imaging during the 5-year interventional period of the study 
that had only negative biomarker results (i.e., no indication of molecular relapse) within 36 months 
before the end of the 5-year interventional period of the study.  
 
Liquid Biopsy False-Positive Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
False-positive rate is defined as the proportion of all patients in the liquid-biopsy guided Intensive 
Surveillance arm with a positive biomarker result (i.e., molecular relapse) during the 5-year 
interventional period of the study that had no recurrence as verified by imaging within 36 months 
after first molecular relapse. 
 
 
 
 

 
 
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Liquid Biopsy False-Negative Rate (CA27.29, CEA, CA125, CTC and ctDNA)  
False-negative rate is defined as the proportion of all patients in the liquid-biopsy guided Intensive 
Surveillance arm with only negative biomarker results (i.e., no indication of molecular relapse) that 
had a recurrence as verified by imaging during the 5-year interventional period of the study. 
 
Rate of liquid biopsy positivity (CA27.29, CEA, CA125, CTC and ctDNA)  
The overall rate of liquid biopsy positivity is defined as the proportion of all patients in the liquid-
biopsy guided Intensive Surveillance arm that had at least one positive biomarker result (i.e., 
molecular relapse) during the 5-year interventional period of the study. 
5.3 
Translational Research Endpoints 
A biobank will allow the storage of unused biomaterials from both, the Intensive Surveillance arm as 
well as the Standard Surveillance arm. These materials will be used for future retrospective studies. 
 

 
 
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6 
STUDY DESIGN 
6.1 
Description of the Study Design 
This is a partially double-blinded, multi-center, randomized, controlled superiority study to evaluate 
the potential benefits of intensified surveillance versus standard surveillance in medium-risk and high-
risk early breast cancer patients. 
3500 patients will be randomized after completion of primary anti-tumor therapy (adjuvant 
chemotherapy, surgery or radiotherapy, whichever occurs last) in a 1:1 ratio to receive: 
- 
Standard Surveillance according to national guidelines or 
- 
Intensive Surveillance with additional testing of blood samples for prospective tumor markers 
(CA27.29, CA125, CEA), CTC and ctDNA  
In both study arms, patients will continue to receive standard surveillance according to national 
guidelines, including clinical follow-up visits every 3 months for the first 3 years and every 6 months 
for the following 2 years after study enrollment. Additionally, blood samples will be drawn and QoL 
will be analyzed at these clinical follow-up visits in both arms. 
In the Standard Surveillance group all blood samples will be stored in a biobank. 
In the Intensive Surveillance group blood samples will be tested for prospective tumor markers 
(CA27.29, CA125, CEA), CTCs and ctDNA. Abnormal findings of either marker (CA27.29 or CA125 or 
CEA or CTC or ctDNA) will trigger diagnostic imaging. Additionally, blood samples will be stored in a 
biobank for retrospective analysis. In both study arms detection of distant recurrence will terminate 
the surveillance protocol and treatment will be initiated according to national guidelines. 
Blinding will be partial, as individual patients in the Intensive Surveillance arm, where the liquid biopsy 
shows an indication for molecular residual disease (MRD), will receive further assessments and must 
therefore be unblinded.   
After the 5-year interventional period, 5 years of follow-up will follow where data is collected once a 
year (questionnaire directed towards the patient’s living status, wellbeing and QoL). 
Planned enrollment period is approximately 24 months, total study duration is approximately 148 
months (2-year recruitment period, 5-year interventional period, 5-year follow up period). In terms of 
long-term follow-up after end of study, patients have the possibility to participate in the patient self-
reporting registry (Patientenselbstauskunft). 
 
Randomization will be stratified using block randomization by: 
• 
Hormone receptor status (negative vs positive) 
• 
HER2 status (negative vs positive) 
• 
Histological lymph node status at surgery ((y)pN0 vs (y)pN+) 

 
 
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6.2 
Study design 
 
 
Figure 3: Study Design 
 
 

 
 
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7 
STUDY POPULATION 
Investigators should consider each of the criteria outlined below when selecting a patient for this 
study. In order to participate in the study, a patient must fulfill all inclusion criteria and must not meet 
any of the exclusion criteria. Investigators should also consider all other relevant medical and non-
medical factors, as well as the risks and benefits of the study, when deciding if a patient is an 
appropriate candidate for this study.  
7.1 
Number of Patients 
Target accrual for this study will be 3500 randomized patients with a hypothesized number of 3850 
patients assessed for eligibility, corresponding to an expected screening failure rate of approximately 
10%. Patients who have been randomized but did not complete all follow-up visits with the 
appropriate assessments are considered drop-outs. 
If a personalized ctDNA assay cannot be established for a patient in the Intensive Surveillance arm due 
to problems related to quality and/or quantity of the collected primary tumor tissue, a second primary 
tumor tissue sample from this patient will be obtained to facilitate another try at establishing a 
personalized ctDNA assay. If it still is not possible, this already randomized patient will be accounted 
for as drop-out.  
7.2 
Inclusion Criteria 
Patients will be eligible for study participation only if they comply with the following criteria: 
 
1. Written informed consent for all study procedures according to local regulatory requirements 
prior to beginning specific protocol procedures. 
2. Unilateral or bilateral primary invasive carcinoma of the breast, confirmed histologically.  
3. Patients with intermediate- to high-risk early breast cancer defined as either  
- 
an indication for (neo-)adjuvant chemotherapy (regardless whether performed or 
not), and/or  
- 
Large tumor (> 50 mm), and/or  
- 
Positive lymph nodes (> pN1mi), and/or  
- 
High grade ( G3). 
Indication to (neo-)adjuvant chemotherapy is seen as stated in the German S3 guideline for 
breast cancer (53) as well as stated in the guidelines from the AGO (54). 
4. A complete resection of the primary tumor, with resection margins free of invasive carcinoma. 
5. Completion of primary anti-tumor therapy (adjuvant chemotherapy, surgery or radiotherapy, 
whichever occurs last) no more than 24 months previously. Enrollment of patients during any 
kind of adjuvant therapy except chemotherapy (e.g., but not limited to endocrine therapy, 
antibody therapy, CDK4/6-inhibitors, PARP inhibitors, PI3K inhibitors, antibody-drug 
conjugates and other novel agents) is allowed. Patients with Luminal A/B breast cancer 
(ER/PGR positive, HER2 negative/low) may be enrolled up to 60 months after completion of 
primary anti-tumor therapy. 

 
 
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6. Availability of primary tumor tissue from core biopsy or surgical removed tissue (FFPE Slide (≥ 
6 mm³, min. 10 slides, thickness: 5 µm-10 µm, area >150 mm² and 1 H&E stained slide, 
minimum 20% tumor content) or FFPE Block (≥ 6 mm³ thickness: 100 µm, area: >150 mm² and 
1 H&E stained slide, minimum 20% tumor content) or Genomic DNA extracted from FFPE 
slides or block (≥ 600 ng, Minimum volume: 25 µL, concentration: 20 ng/µL, buffer: 10 mM 
Tris pH 8, 1 mM EDTA)) at timepoint of enrollment. 
- 
Patients with primary systemic therapy: tissue from core biopsy 
- 
Patients receiving surgery as primary therapy: surgically removed cancer tissue or 
tissue from core biopsy. 
Important note: Tumor tissue of both sides will be needed in case of bilateral breast cancer. 
7. No current clinical evidence for distant metastases. 
8. Females or males ≥ 18 years and ≤ 75 years of age. 
9. Performance status ≤ 1, Eastern Cooperative Oncology Group (ECOG) scale. 
10. Patient must be willing and able to comply with scheduled visits, treatment plans, laboratory 
tests, and other study procedures. 
It is possible for patients to participate in adjuvant interventional breast cancer trials and in 
interventional trials that are based on MRD detection without metastases being confirmed via 
imaging. These interventional trials should be defined therapy-intervention trials that are evidence-
based and have to be approved by the SURVIVE Steering Committee. 
7.3 
Exclusion Criteria 
Patients will be ineligible for study participation if they comply with the following criteria: 
1. 
Patients with a history of any secondary primary malignancy are ineligible with the following 
exceptions: 
- 
in situ carcinoma of the cervix or  
- 
adequately treated basal cell carcinoma of the skin or 
- 
ipsi- or contralateral non-invasive carcinoma of the breast (DCIS). 
2. 
Patients in pregnancy or breastfeeding. 
If a patient gets pregnant during the participation in the interventional phase of the study 
(Year 1-5), an end of intervention visit will be scheduled and the patient will enter the follow-
up phase of the study (see section 11 for further information). Pregnancy during the follow-
up phase of the study is to be reported but does not lead to an exclusion of the study. 
3. 
History of significant neurological or psychiatric disorders including psychotic disorders, 
dementia or seizures that would prohibit the understanding and giving of informed consent. 
4. 
Renal insufficiency with GFR < 30 mL/min. 
5. 
Previous or concomitant cytotoxic or other systemic antineoplastic treatment that is not used 
for treating the primary breast cancer. 
 

 
 
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8 
STUDY INTERVENTIONS 
This is a partially double-blinded, multi-center, randomized, controlled superiority study. Patients will 
be randomly assigned to one of the two groups (Intensive Surveillance versus Standard Surveillance) 
in a 1:1 ratio. 
We will acquire peripheral blood samples from patients in both groups as a tertiary diagnostic 
intervention every 3 months for the first 3 years after study inclusion, thereafter every 6 months for 
another 2 years. Peripheral blood samples from patients in the Standard Surveillance group will be 
stored in a biobank for translational studies and retrospective analyses. Peripheral blood samples from 
patients in the Intensive Surveillance group are collected to test CA27.29, CEA and CA125 serum levels 
as well as ctDNA and CTC levels. As an abnormal increase in these tumor marker levels or detection of 
ctDNA or CTC correlate closely with tumor load, further examinations will be initiated for patients with 
abnormal tests in the Intensive Surveillance group. Patients with standard follow-up on the other hand 
will have access to further examinations if metastasis-specific symptoms occur, according to current 
German guidelines. 
8.1 
Standard Surveillance 
Current aftercare for early breast cancer in Germany begins after completion of primary anti-tumor 
therapy, which can be adjuvant chemotherapy, surgery or radiotherapy, whichever occurs last. For 
the first 3 years of aftercare, visits at the aftercare specialist (gynecologist/family 
physician/oncologist) should be scheduled every 3 months. For the following 2 years, visits every 6 
months are recommended. After five years, the frequency of visits is usually reduced to once a year, 
noting that no official guidelines currently address intervals after the first 5 years after primary 
diagnosis. In these visits, patients should be asked about their well-being, about possible symptoms 
for distant metastases and about their therapy adherence if still under therapy with e.g., endocrine 
treatment. Recommendations about lifestyle should be given, i.e., preferring a healthy diet, reduction 
of alcohol consumption, abstention from nicotine and practising physical activity on a regular basis.  
Furthermore, a clinical examination of the breasts should be performed including inspection and 
palpation of the breasts and locoregional lymph nodes. Imaging in the form of mammography with or 
without supplementary sonography of the breasts is recommended every 12 months. Magnet 
resonance imaging (MRI) is only to be considered, if mammography and sonography are inconclusive. 
Monthly self-palpation of the breasts is recommended for patients. 
Other imaging modalities, including CT scans, are only recommended if there are symptoms suspicious 
of local recurrence or distant metastases. Blood tests should only be performed to assess side effects, 
e.g., when the patient is under endocrine treatment. 
Determining tumor markers and imaging other than breast imaging is explicitly not advised in 
asymptomatic patients. 
The aims of current standard aftercare are stated by the AGO in Germany to be the early detection of 
locoregional recurrence, of contralateral breast malignancy, and of symptomatic metastases. In 
addition, aftercare should try to improve overall QoL, ensure adherence to adjuvant treatment, 
manage side effects and include psychosocial care (55). Detection of asymptomatic metastases is 
currently not of interest. 
 

 
 
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8.2 
Intensive Surveillance 
All measures are taken in addition to standard surveillance.  
Staging examinations will be performed if one or more of the markers (tumor markers, CTC, ctDNA) 
show an abnormal result. A normal result of one marker cannot prevent further measures, if not 
indicated differently below.  
 
Determination of Tumor markers 
For tumor marker analysis, no absolute cut-offs will be used, but a delta in tumor marker increase 
(CA27.29 +75% or CEA +100% or CA125 +150%) from baseline. The baseline will be defined as the 
mean value of two blood drawings four weeks apart after randomization for each patient individually 
(28  5 days). After determination of the individual baseline, blood samples will be drawn every three 
months for three years, thereafter every six months for another two years.  
Abnormal tumor marker serum level increase from baseline (CA27.29 +75% or CEA +100% or CA125 
+150%) will trigger complete standard staging examinations (CT scan of the chest and abdomen as 
well as SPECT bone scan). In case of M0, the patient will continue liquid-biopsy based tumor marker 
testing. If tumor marker elevation continues, a maximum of three consecutive negative staging 
examinations will be performed before another rise in tumor marker will be mandatory (according to 
the deltas stated above) to trigger further imaging (compare the complete workflow in Figure 4). If 
repeated, the staging examination will be performed via CT scan +/- a SPECT bone scan. The minimum 
interval between two CT scans should be at least 3 months, and the minimum interval between two 
SPECT bone scans should be at least 6 months at any time point. 
All staging examinations are done according the national guidelines for breast cancer. 
CA27.29, CEA and CA125 will be measured with the AIA®-CL1200 by TOSOH BIOSCIENCE (TOSOH 
CORPORATION, Tokyo, Japan). CA125 corresponds to OVCA TEST CUP. Specimen will be collected in 
one standard serum sampling tube (7,5 mL whole blood), and shipped for centralized evaluation. 
Serum probes will be stable for 48h at 20-25°C, for 7 days at 2-8°C and thereafter at -20°C. Principles 
of analysis and probe handling is explained further in 13.1.2.1.1 and in the according leaflets (56).  
 

 
 
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Figure 4: Pathway for Tumor Marker Analysis and Consequences 
 
Determination of CTC levels 
If an abnormal tumor marker value is detected in a patient in the Intensive Surveillance arm for the 
first time, CTC analysis will be initiated at the next regular study visit. Furthermore, all patients in the 
Intensive Surveillance arm will receive CTC testing one year after study enrollment. If ≥ 1 CTC is 
detected, patients will undergo complete standard staging examinations. In case of M0, the patient 
will continue standard liquid-biopsy based testing.  
CTCs will be analyzed using the FDA-approved, standardized semiautomatic CellSearch® System 
(Menarini Silicon Biosystems). Whole blood samples will be collected in 3 x 10 mL CellSave 
preservative tubes and analyzed centrally within 96 h of collection (stable at room temperature) 
aiming for a numeration of CTCs – nucleated cells of epithelial origin (CD45-, EpCAM+, cytokeratin 8+ 
/ 18+ and/or 19+) (41).  
 
 

 
 
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Figure 5: Pathway for CTC Analysis 
 
Determination of ctDNA levels 
If ctDNA is detected, patients will undergo complete standard staging examinations. In case of M0, 
the patient will continue liquid-biopsy based ctDNA testing. In case of persisting ctDNA, a maximum 
of three consecutive negative staging examinations will be performed before staging intervals will be 
extended to every six months for another three consecutive scans. If ctDNA is persisting and patients 
show no radiological sign of recurrence, staging intervals will be extended to once a year (compare 
the complete workflow in Figure 6). If repeated, the staging examination will be performed via CT scan 
+/- a SPECT bone scan. If repeated, the minimum interval between two CT scans should be at least 3 
months, and the minimum interval between two SPECT bone scans should be at least 6 months at any 
time point. 
All staging examinations are done according the national guidelines for breast cancer. 
Peripheral blood specimens will be collected in two Streck Cell-Free DNA BCT® tubes. ctDNA will be 
stable for up to 14 days at 6 °C to 37 °C. Presence of ctDNA will be analyzed centrally at Inivata Inc. 
using the RaDaRTM assay. Therefore, primary tumor tissue and peripheral blood specimens will be 
shipped for centralized analysis to Inivata Inc. RaDaRTM is a tumor-informed approach, beginning with 
whole exome sequencing (WES) of a tumor specimen from a patient’s biopsy or surgical resection. 

 
 
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Somatic variants [single nucleotide variants (SNVs) and small insertions and deletions (indels)] 
identified from the exome sequencing are prioritized to build a patient specific primer panel of up to 
48 tumor-specific somatic variants. This design is unique to each patient and is used as a molecular 
signature that can be tracked from diagnosis, in order to monitor presence of tumor DNA throughout 
the course of the patient’s disease. Patient specific primers are combined with common Single-
Nucleotide Polymorphism (SNP) primers for Next Generation Sequencing (NGS) for quality control 
purposes. To detect patient specific ctDNA, NGS testing is performed with the RaDaRTM assay using a 
multiplex PCR based on the InVision® platform (57,58). 
Primary tumor tissue is mandatory for WES (FFPE Slide (≥ 6 mm³, min. 10 slides, thickness: 5 µm-10 
µm, area >150 mm² and 1 H&E-stained slide, minimum 20% tumor content) or FFPE Block (≥ 6 mm³ 
thickness: 100 µm, area: >150 mm² and 1 H&E-stained slide, minimum 20% tumor content) or 
Genomic DNA extracted from FFPE slides or block (≥ 600 ng, Minimum volume: 25 µL, concentration: 
20 ng/µL, buffer: 10 mM Tris pH 8, 1 mM EDTA)). If this cannot be provided, the patient will be 
accounted for as a screening failure. 
 
 
Figure 6: Pathway for ctDNA Analysis 
 
 

 
 
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Overview of Requirements for Collection of Biomaterials 
Details for collection and handling of biomaterials are outlined in the Lab Manual/Sample 
Management Guide and in Table 4. 
 
 
Blinding 
This study will be performed as partially double-blinded. This means, all patients and doctors are 
blinded initially as blood sampling is done in all patients, irrespective of randomization to the Standard 
Surveillance arm or the Intensive Surveillance arm. If one of the biomarkers (CA27.29, CEA, CA125, 
CTC, ctDNA) is abnormal and triggers imaging, unblinding is the consequence as these patients will be 
asked to undergo further assessments and the responsible doctor will arrange these. Unblinding in 
this case is the ethical consequence of not letting all patients undergo additional imaging without 
elevated biomarkers. If no imaging is necessary, patients in the Intensive Surveillance arm will not be 
unblinded as there is no purpose to serve. Patients in the Standard Surveillance arm will not be 
unblinded altogether.  
8.3 
Staging Examinations 
Staging will be triggered by symptoms of metastases as indicated by physician’s choice (i.e., 
pathological fractures and associated symptoms, icterus, hepatic capsule pain, dyspnea, etc.) in both 
arms, as intended by current standard of care. Moreover, in the Intensive Surveillance arm staging 
will be triggered by biomarker findings indicating MRD as defined in 8.2.1, 8.2.2 and 8.2.3.  
Staging examinations should as a minimum include a CT scan of the chest and abdomen as well as a 
SPECT bone scan. In cases of consecutive negative staging examinations or severe allergy to the 
contrast agent used for the CT scan, a PET scan (Positron emission tomography) or whole-body MRI 
may be considered as well and present a suitable addition. Additional body imaging like sonography 
can be used for further examination but should not replace a CT scan. 
The minimum interval between two CT scans should be at least 3 months, and two SPECT bone scans 
should be at least 6 months apart at any time point. 
The examinations will be performed in the radiological practice or hospital of the patients’ choice. A 
medical radiological report with RECIST finding is not required. 
8.4 
Study visits 
All study visits requiring blood sampling should be performed at the participating study centers. The 
standard aftercare examination with the recommended intervals of every three months for the first 
three years and every six months for the consecutive two years should take place at the patients’ 
aftercare specialist (gynecologist/family physician/oncologist). 

 
 
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Study Inclusion / Baseline Visit 
Patients should be thoroughly informed and need to give (written) consent to participate in the study. 
Furthermore the patient history, current health status and physical examination should be noted (see 
section 12.5.2). Eligible patients should show no clinical signs of metastatic disease.  
At study inclusion, primary tumor tissue is required in form of a FFPE Slide, FFPE Block or Genomic 
DNA extracted from FFPE slides or block for sending to Inivata Inc. to establish the patient-specific 
RaDaRTM assay for later ctDNA testing. Tumor tissue of both sides will be needed in case of bilateral 
breast cancer. As the study is blinded, tissue is required from all patients in both arms. After 
randomization, tissue not used for establishing the RaDaRTM will be stored in a biobank. If a 
personalized ctDNA assay cannot be established for a patient in the Intensive Surveillance arm due to 
problems related to quality and/or quantity of the collected primary tumor tissue, a second primary 
tumor tissue sample from this patient will be obtained to facilitate another try at establishing a 
personalized ctDNA assay. If it still is not possible, this already randomized patient will be accounted 
for as drop-out.  
Baseline tumor marker blood sampling of whole blood in 1 x 7,5 mL Serum tube will be done at 
randomization (Baseline visit 1) and 28 ± 5 days later (Baseline visit 2). Baseline ctDNA blood sampling 
of whole blood in 2 x 10 mL Streck Cell-Free DNA BCT® tubes will be done at randomization (Baseline 
visit 1).  
Again, in order to maintain a double-blinded approach, biomarkers will only be analyzed in the 
Intensive Surveillance arm, while the biomaterial from the Standard Surveillance arm will be stored in 
a biobank. 
Besides blood sampling, patients in both arms will get two questionnaires to assess their current QoL 
(EORTC QLQ-C30 and PA-F12). 
 
Visits During Interventional Part of the Study 
In the five years of interventional study participation, blood sampling will be done every three months 
for the first three years and every six months for the following two years after randomization in both 
arms. Blood sampling for all patients will contain whole blood in one 7,5 mL Serum tube as well as two 
10 mL Streck Cell-Free DNA BCT® tubes in both arms (total of 27,5 mL whole blood). After one year, 
all patients will have an additional blood sampling of three 10 mL CellSave tubes (at study center visit 
for month 12 – a total of 57,5 mL whole blood). 
Biomarkers will only be analyzed in the Intensive Surveillance arm, the biomaterial from the Standard 
Surveillance arm will be stored in a biobank. 
If tumor marker, CTC or ctDNA testing is abnormal in the Intensive Surveillance arm, the study center 
will be notified to initiate a complete staging (compare to section 8.3). 
Besides blood sampling, patient’s QoL will be assessed in both arms with two questionnaires (EORTC 
QLQ-C30 and PA-F12) every six months. Furthermore, patients will be asked questions addressing 
examinations outside this study. 
 

 
 
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End of Intervention (EOI) 
At the end of intervention, patients’ current health status should be noted. 
If the detection of recurrence is the reason for the end of intervention, the site of recurrence, the site 
of metastases and – if possible – the planned therapy should be noted. In this case, biomaterial 
collection and submission are highly endorsed. If possible, tumor tissue in form of a FFPE Slide, FFPE 
Block or Genomic DNA extracted from FFPE slides or block for sending to Inivata Inc. would be 
appreciated.  
 
One last blood sampling of whole blood in form of one 7,5 mL Serum tube as well as two 10 mL Streck 
Cell-Free DNA BCT® tubes, (± 3 x 10 mL CellSave tubes, if applicable, see section 12.2.2) is mandatory. 
Besides blood sampling, patients in both arms will get two questionnaires to assess their current QoL 
(EORTC QLQ-C30 and PA-F12). 
If the end of the follow-up visits is the reason for the end of intervention, the EOI corresponds to Visit 
16 (see Figure 8). 

 
 
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8.5 
Study Procedures 
 
 
Figure 7: Study Procedures 
 
 
Figure 8: Timeline for Study Procedures during the interventional Phase 
 

 
 
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Every effort should be made to ensure that protocol required tests and procedures are performed as 
described. 
Blood sampling and performing questionnaire will be done as seen in the flow chart (see Figure 7). 
The whole study timeline can be observed in Figure 9.  
 
 
Figure 9: Study Timeline 
To ensure standardization of biomarker testing, all tubes of blood samples will be sent from the study 
centers to the Central Study Center at University Hospital Ulm where the respective tubes will be 
distributed to the corresponding laboratories. All tumor marker and CTC testing will take place 
centrally with standardized assays. Blood for ctDNA-testing will be sent to Inivata Inc. for further 
analysis. 
Unused and excess biomaterial will be stored in a biobank. 
In case of an abnormal biomarker result, the Central Study Center will be informed by the 
corresponding institution. The Central Study Center will then inform the patient’s respective study 
center which measures are to be taken (see Figure 10 + Figure 11). 
If imaging is triggered, staging examinations (see section 8.3) will be performed in the radiological 
practice or hospital of the patients’ choice as we trust in the professional knowledge of trained doctors 
to find metastases. A medical radiological report with RECIST finding is not required. 
 

 
 
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Figure 10: Processing of Biomaterial 
  
 
 
 

 
 
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Figure 11: Responsibilities in Case of Positive Findings in Biomarkers 
 
Patient Entry Procedures 
8.5.1.1 
Patient Consent Form and Screening 
Prior to study entry, the consent form including any addenda must be signed and dated by the patient 
and the person obtaining informed consent. After written informed consent has been obtained, the 
patient has to be registered via an electronic data capturing (EDC) system (see section 8.5.1.2). The 
study site will obtain a unique patient number which will stay the same throughout the entire study 
covering all study periods. At this time point, the patient is enrolled into the study. 
After a patient has completed the necessary screening visit procedures, the corresponding baseline 
case report forms (CRFs) have to be completed by the patient’s study center using the EDC system. 
Eligibility of the patient will be automatically and visually checked in the EDC system. 
It is possible for patients to participate in adjuvant interventional breast cancer trials and in 
interventional trials that are based on MRD detection without metastases being confirmed via 
imaging. These interventional trials should be defined therapy-intervention trials that are evidence-
based and have to be approved by the SURVIVE Steering Committee. 
 

 
 
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8.5.1.2 
Randomization and Stratification 
Once eligibility has been established and the primary tumor tissue has been obtained, patients will be 
randomized in the EDC system in a 1:1 ratio, and the site will obtain the confirmation of the patient’s 
randomization (without knowledge of the study arm) from the EDC system. Neither the 
investigators/co-investigators nor the patient can influence the randomization. A partially double-
blinded study design will be used, where only patients in the Intensive Surveillance arm with detection 
of MRD resulting in imaging or additional blood sampling will be informed about their randomization 
arm. Correspondingly, the patient’s study center will also be informed about the randomization arm 
in case of positive biomarker findings as they will be the link between the Central Study Center and 
the patient and have to organize required imaging and/or additional blood sampling. 
Patients will be randomized centrally only if they fulfil the inclusion criteria and are not precluded 
from participation by any of the exclusion criteria. The randomization list will be generated by the IFG 
GmbH Erlangen using adequate and validated algorithms.  
 
Randomization will be stratified using block randomization by: 
• 
Hormone receptor status (negative vs positive) 
• 
HER2 status (negative vs positive) 
• 
Histological lymph node status at surgery ((y)pN0 vs (y)pN+) 
 
 
Screening 
Table 1: Screening exams and tests prior randomization 
 
Investigations at Screeninga 
Timing 
(prior to randomization) 
Patient informed 
consent  
Obtained yes/no 
Prior to any protocol 
investigations or 
procedures 
History and 
physical 
examination 
History including:  
• Gender / Sex 
• ethnicity 
• diagnosis of breast cancer 
• menopausal status 
• therapy history of breast cancer 
• current aftercare status 
• family history of cancer 
• known genomic mutation 
• general medical history including cardiac history 
and allergy 
• concurrent illnesses  
• concomitant medications (any prescription 
medications or over-the-counter medications),  
 
Within 84 days prior to 
randomization 

 
 
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Investigations at Screeninga 
Timing 
(prior to randomization) 
and their indication, used within one month prior 
to study entry 
• current pregnancy 
 
Physical examination including: 
• ECOG performance status 
• Weight 
• Height 
• Palpation of breasts, axillary, supra- and 
infraclavicular region 
• Pre-existing signs and symptoms as in no clinical 
signs of distant metastases 
Biomaterialsb 
Primary tumor tissue as either of the following: 
• FFPE Slide (≥ 6 mm³, min. 10 slides, thickness: 5 
µm-10 µm, area >150 mm² and 1 H&E stained 
slide, minimum 20 % tumor content) or  
• FFPE Block (≥ 6 mm³ thickness: 100 µm, area: >150 
mm² and 1 H&E stained slide, minimum 20 % 
tumor content) or  
• Genomic DNA extracted from FFPE slides or block 
(≥ 600 ng, minimum volume: 25 µL, concentration: 
20 ng/µL, buffer: 10 mM Tris pH 8, 1 mM EDTA) 
 
 
20 mL whole blood (2 x 10 mL Streck tubes)  
7,5 mL whole blood (1 Serum tube) 
 
 
7,5 mL whole blood (1 Serum tube) 
  
After ICF signature  
 
 
 
 
 
 
 
 
 
 
baseline visit 1 
(randomization visit) 
 
 
 
baseline visit 2: 28 days 
after baseline visit 1 (± 5 
days) 
Biochemistry 
Serum creatinine and resulting renal clearance 
Within 84 days prior to 
randomization 
Breast Imagingc 
Bilateral mammography and supplementary bilateral 
breast ultrasound with axillary lymph nodes (where 
applicable) 
and/or MRI of the breasts 
 
 
Within 1 year prior to 
randomization 

 
 
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Investigations at Screeninga 
Timing 
(prior to randomization) 
a. Results of screening tests or examinations performed as standard of care prior to obtaining informed 
consent but within the timelines may be used rather than repeating required test. 
b. Materials for blood collection and transportation devices will be provided by the Central Study 
Center at Dep. Ob/Gyn University Hospital Ulm. 
c. Examination is performed externally, no study physician needed. 
 
Evaluation during Interventional Phase 
The investigator may schedule visits (unplanned visits) in addition to those listed on the schedule of 
activities, in order to conduct evaluations or assessments required to protect the wellbeing of the 
patient. 
Patients who stop study interventions due to breast cancer recurrence (distant or local), second 
primary breast cancer, or diagnosis of another cancer or who discontinue all protocol requirements 
for reasons other than disease recurrence must complete the end of intervention (EOI) assessments. 
In case of recurrence or other primary malignancy, biomaterial collection and submission are strongly 
endorsed.  
Table 2: Requirements during Interventional Phase  
 
Evaluation during Interventional Phase 
Timing 
Historyd 
• Last aftercare visits at the aftercare specialist 
(gynecologist/family physician/oncologist). 
• Current adjuvant therapy regarding breast cancer. 
• Last breast imaging (mammography/ ultrasound) 
• Any test for tumor markers or imaging done 
outside of study. 
Every 3 months  
(year 1–3)  
 
Every 6 months  
(year 4–5) 
 
EOI 
Breast Cancer 
Aftercaree 
• Contents of interview depending on the external 
aftercare specialist’s (gynecologist/family 
physician/oncologist) approach 
• Breast examination with palpation of breasts and 
locoregional lymph nodes 
• Laboratory tests as needed 
• Initiation/Scheduling of yearly breast imaging 
Every 3 months  
(year 1–3)  
 
Every 6 months  
(year 4–5) 
 
Breast Imaginge 
Bilateral mammography and supplementary bilateral 
breast ultrasound with axillary lymph nodes (where 
applicable) 
and/or MRI of the breasts 
Once a year 

 
 
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Evaluation during Interventional Phase 
Timing 
Biomaterialsa, b 
7,5 mL whole blood (1 Serum tube) 
 
20 mL whole blood (2 x 10 mL Streck tubes) 
 
Every 3 months  
(year 1–3)  
 
Every 6 months  
(year 4–5) 
 
EOI 
30 mL whole blood (3 x 10 mL CellSave tubes) 
Visit month 12 
 
In case of first abnormal 
increase in tumor 
markerc 
Questionnairesd 
EORTC QLQ-C30 
PA-F12 
Every 6 months (year 1-5) 
Imaging tests for 
staginge 
• 
CT scan of chest and abdomen 
• 
SPECT bone scan 
• 
If not possible due to e.g., allergy to the contrast 
agent: PET scan or whole-body MRI 
• 
Complementary imaging tests if indicated 
• Symptomatic 
patients: physician’s 
choice 
• Asymptomatic 
patients: only if 
center is advised to 
a. Materials for blood collection and transportation devices will be provided by the Central Study 
Center at Dep. Ob/Gyn University Hospital Ulm. 
b. The corresponding blood kit will be sent to the study center by the Central Study Center. 
c. Only if CTC assessment was not done at regular visit. 
d. Done by questionnaire. 
e. Examination is performed externally, no study physician needed. 
 
 
Evaluation after End of Intervention - Follow-up 
The investigator may schedule visits (unplanned visits) in addition to those listed on the schedule of 
activities, in order to conduct evaluations or assessments required to protect the wellbeing of the 
patient. 
Patients who discontinue all protocol interventions for reasons other than disease recurrence must 
follow the exams and assessments outlined in this section. 
 
Follow-up data will be collected via questionnaires which will be provided in paper form or digitally 
(via E-Mail / Digital Health Application or collected via telephone interview.  
 
 
 

 
 
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Table 3: Evaluation during Follow-up 
 
Evaluation during Follow-up 
Timing 
Historyb 
• Aftercare visits at the aftercare specialist 
(gynecologist/family physician/oncologist).  
• Current status of breast cancer (recurrence?) 
• Current (adjuvant) therapy regarding breast 
cancer 
• Last breast imaging (mammography/ultrasound) 
• Any test for tumor markers or imaging done 
Once a year (year 6-10) 
Breast Cancer 
Aftercarea 
• Contents of interview depending on the external 
aftercare specialist’s (gynecologist/family 
physician/oncologist) approach 
• Breast examination with palpation of breasts and 
locoregional lymph nodes 
• Laboratory tests as needed 
• Initiation of yearly breast imaging 
At least once a year (year 
6-10) 
Breast Imaginga 
Bilateral mammography and supplementary bilateral 
breast ultrasound with axillary lymph nodes (where 
applicable) 
and/or MRI of the breasts 
Once a year (year 6-10) 
Questionnairesb 
EORTC QLQ-C30 
PA-F12 
Once a year (year 6-10) 
a. Examination is performed externally, no study physician needed. 
b. Done by questionnaire. 
 
Patients who stop study therapy due to breast cancer progression/relapse, second breast primary 
cancer, or diagnosis of another cancer or who discontinue all protocol requirements for reasons other 
than disease recurrence must complete the end of intervention (EOI) assessments.  
Follow-up visits after the EOI are once yearly and include no biomaterial sampling. 
In case of recurrence or other primary malignancy, biomaterial collections in form of blood sampling 
and tumor tissue are endorsed. 
 
Long-Term Follow-up After End of Study 
In terms of long-term follow-up after end of study, patients have the possibility to participate in the 
patient self-reporting registry (Patientenselbstauskunft)

 
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Study visits table 
Trial Period 
Screeninga 
Randomisation 
visit 
Baseline visit 
Surveillance Phase 
End of 
Intervention 
Follow Up 
End of 
Study 
Baseline 1 
Baseline 2 
Year 1-3 
Year 4-5 
Month 60 or 
premature 
discontinuationb 
Year 6-10 
 
 
Visit schedule 
Prior to 
randomisation 
Tumorblock in 
central pathology 
28 d post 
randomisation 
Every 3 months 
Every 6 months 
 
Every 12 
months 
 
Day/Month 
-84 d to 0 d 
0 
28 d ± 5 d 
 
3 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
42 
48 
54 
60 
72 to 120 
120 
APPLICABLE FOR BOTH ARMS 
Registration 
requirements 
Informed Consent 
Form signed 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusio
n criteria 
evaluation 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Documentation 
Disease Baseline 
Characteristics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical 
examination 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Primary archived 
(FFPE) tumor 
tissuec 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistryd 
Serum creatinine 
and renal 
clearance 
X 
 
 
 
 
 
 
 
 
 
 
Biomaterialse 
Tumormarkers 
1x 7.5 mL serum 
tube 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
ctDNA  
2x 10 mL Streck 
tubes 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
CTCsf 
3x 10 mL cell save 
tubes 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Archived (FFPE) 
Tumor tissue 
upon progressiong 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 

 
 
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a: 
Results of screening tests or examinations performed as standard of care prior to obtaining informed consent but within the timelines may be used rather than repeating required test 
b: 
Premature study intervention discontinuation due to either of the following: 
1. via imaging verified recurrence 
2. pregnancy 
3. safety or medical reasons 
4. patients’ withdrawal of consent 
c: 
Primary tumor tissue according to the requirements stated in the study protocol section 11.2. Important note: in case of bilateral breast cancer, tissue from both sides is needed. 
d: 
Results of serum creatinine and renal clearance performed within 84 days prior to obtaining informed consent may be used rather than repeating required test 
e: 
Materials for blood collection and transportation devices will be provided by the Central Study Center at Dep. Ob/Gyn University Hospital Ulm 
f: 
Mandatory at Month 12, additionally required at visit after first verified tumormarker increase, whenever that occurs 
g: 
Highly endorsed in case of detection of recurrence/metastatic disease 
h: 
Referral to radiologist from investigator or aftercare specialist (gynecologist/family physician/oncologist) - Can be triggered by either of the following:  
1. clinical indication (both arms) 
2. increase in tumormarkers (only intensive surveillance arm) 
3. if presence of ctDNA was detected (only intensive surveillance arm) 
i: 
If CT and SPECT not possible due to e.g., allergy to the contrast agent: PET scan or whole-body MRI, Complementary imaging tests if indicated  
j: 
Available on paper, handed out at study visits 
 
Trial Period 
Screeninga 
Randomisation 
visit 
Baseline visit 
Surveillance Phase 
End of 
Intervention 
Follow Up 
End of 
Study 
 
 
 
Baseline 1 
Baseline 2 
Year 1-3 
Year 4-5 
Month 60 or 
premature 
discontinuationb 
Year 6-10 
 
 
Visit schedule 
Prior to 
randomisation 
Tumorblock in 
central pathology 
28 d post 
randomisation 
Every 3 months 
Every 6 months 
 
Every 12 
months 
 
 
Day/Month 
-84 d to 0 d 
0 
28 d ± 5 d 
3 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
42 
48 
54 
60 
 
72 to 120 
120 
APPLICABLE FOR BOTH ARMS 
Staging 
examinationsh,i 
CT scan of chest 
and abdomen and 
SPECT bone scan 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Questionnairesj 
EORTC QLQ-C30 
(QOL) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PA-F12 (QOL) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
History 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Follow-up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 

 
 
 
 
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9 
COLLECTION OF BIOMATERIALS AND TRANSLATIONAL RESEARCH  
By signing the informed consent form, the patient agrees to collection and submission of required 
sample collections as outlined below. The tumor and blood samples will be used for testing at the 
institutions stated and/or translational research as described in the protocol.  
Material (i.e., test tubes) for blood collection and transportation devices in the study centers will be 
provided by the Central Study Center. 
9.1 
Overview of Requirements for Collection of Biomaterials 
Details for collection and handling of biomaterial are outlined in the Lab Manual/Sample Management 
Guide and in the table below. 
 
Table 4: Schedule of biomaterial collection 
Biomaterial 
Screening Phase 
Surveillance Phase 
 
Before 
randomization 
Baseline 
(after randomization) 
Year 1-3 
(every 3 months) 
Year 4-5 
(every 6 months) 
EOI 
Visit month 12 
Primary Tumor 
Tissue 
Xa 
 
 
 
Whole Blood  
(7,5 mL Serum) 
 
Xb 
X 
 
Whole Blood  
(3 x 10 mL CellSave) 
 
 
Xc 
X 
Whole Blood  
(2 x 10 mL Streck) 
 
Xd 
X 
 
a. FFPE Slide, FFPE Block or Genomic DNA extracted from FFPE slides or block (technical requirement for 
establishing ctDNA testing). Tumor tissue of both sides will be needed in case of bilateral breast cancer.  
b. Two blood samples with four weeks apart 
c. 
In case of first abnormal tumor marker increase 
d. At Randomization visit/Baseline visit 1 only 
 
 

 
 
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Tumor Material 
9.1.1.1 
Mandatory tumor material 
Primary tumor tissue in form of a FFPE or a FFPE Block or genomic DNA extracted from FFPE slides or 
block will be shipped for centralized analysis to Inivata Inc. for the establishment of the patient-specific 
RaDaRTM assay that is necessary for further detection of ctDNA during the time of the study. In case of 
bilateral breast cancer, tumor tissue of both sides is mandatory. For establishing this assay, whole 
exome sequencing (WES) of the patient’s tumor tissue is performed. Somatic variants [single 
nucleotide variants (SNVs) and small insertions and deletions (indels)] identified from the exome 
sequencing are prioritized to build a patient specific primer panel of up to 48 tumor-specific somatic 
variants. This design is unique to each patient and is used as a molecular signature that can be tracked 
from diagnosis, in order to monitor presence of tumor DNA throughout the course of the patient’s 
disease. Patient specific primers are combined with common Single-Nucleotide Polymorphism (SNP) 
primers for Next Generation Sequencing (NGS) for quality control purposes. This creation process is 
only necessary at the beginning of the study as the assay can then be used for the whole duration of 
the study. 
Tumor tissue which is not used will be stored in a biobank.  
9.1.1.2 
Optional tumor material 
In case of local or distant recurrence or in case of a second primary malignancy, biomaterial collection 
and submission is highly endorsed. Biomaterial in form of biopsy of tumor or metastasis would be 
appreciated (FFPE or a FFPE Block or genomic DNA extracted from FFPE slides or block). 
 
Blood Samples 
Blood sampling will be performed by the established standard of care in the corresponding study 
centers and the biomaterial will then be sent to the Central Study Center for further distribution to 
the respective institutions who will perform the further analysis specified in this study protocol (tumor 
markers, CTC and ctDNA). Excess blood samples and blood samples not used for analysis will be stored 
in a biobank. 
9.1.2.1 
Mandatory blood samples for all patients 
All patients in both arms will receive blood sampling every three months for the first three years and 
every six months for the following two years after randomization. The sampling will at least include 
an amount of 27,5 mL whole blood (1 x 7,5 mL Serum and 2 x 10 mL Streck) for regular visits for 
patients in both arms. As a one-time event, at Visit month 12 (after one year of study participation), 
an extra 30 mL whole blood will be taken for CTC-testing (3 x 10 mL CellSave) in the intensive 
surveillance arm. 
In the Intensive Surveillance arm, an extra 30 mL whole blood (3 x 10 mL CellSave) is required if a 
tumor marker increase is detected for the first time and if no CTC testing was done at the regular visit. 

 
 
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 Tumor Marker Analysis 
As no cut-offs but a delta in increase from baseline (CA27.29 +75% or CEA +100% or CA125 +150%) 
will be used to determine significant tumor marker elevation, two blood samplings for tumor marker 
testing will be performed in a 28 ± 5 days interval to establish a baseline. The baseline will be defined 
as the mean tumor marker value of two blood drawings four weeks apart (28 ± 5 days) at the timepoint 
of randomization for each patient individually. After determination of the baseline, blood samples will 
be drawn in the intervals described above.  
Depending on the result of this blood test and the result of the imaging, if necessary, planned intensive 
surveillance will be continued or not (for details see section 12.2.1). 
CA27.29, CEA and CA125 will be measured with the AIA®-CL1200 by TOSOH BIOSCIENCE (TOSOH 
CORPORATION, Tokyo, Japan). CA125 corresponds to OVCA TEST CUP. Specimen will be collected in 
one standard serum sampling tube (7,5 mL whole blood), and shipped for centralized evaluation. 
Serum probes will be stable for 48h at 20-25°C, for 7 days at 2-8°C and thereafter at -20°C. For each 
analysis, the sample volume needed is 5, 10 and 20 µL respectively for CA27.29, CEA and OVCA and 
up to 120 results per hour may be generated.  
The CL AIA-PACK CA27.29/CEA/OVCA assays are two-step chemiluminescence enzyme immunoassay 
(CLEIA) kits.  The assays are performed in the CL AIA-PACK CA27.29/CEA/OVCA TEST CUP respectively.  
CA27.29/CEA/CA125 present in a test sample is bound to anti- CA27.29/CEA/CA125 mouse 
monoclonal antibody immobilized on the magnetic microparticles in one cell (Cell-I).  After first 
incubation, the magnetic microparticles are washed to remove unbound materials and then a specific 
volume of the enzyme-labeled anti- CA27.29/CEA/CA125 mouse monoclonal antibody that has been 
reconstituted in another cell (Cell-II) is dispensed into Cell-I.  After second incubation, the magnetic 
microparticles are washed again to remove unbound enzyme-labeled monoclonal antibody and are 
incubated with a chemiluminescent substrate, DIFURAT® (3-(5-tert-Butyl-4,4-dimethyl-2,6,7-
trioxabicyclo[3.2.0]hept-1-yl) phenylphosphate disodium salt). The amount of enzyme-labeled 
antibodies that bind to the magnetic microparticles is directly proportional to the CA27.29/CEA/CA125 
concentration in the test sample.  A standard curve is constructed, unknown sample concentrations 
are calculated by using this curve. 
Principles of analysis and probe handling is explained detailed in the according leaflets (56).  
 
 

 
 
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 CTC Analysis 
If an abnormal tumor marker value is detected for the first time, CTC analysis will be initiated. 
Furthermore, all patients in the Intensive Surveillance arm will receive CTC testing one year after 
study-enrollment.  
Whole blood samples will be collected in 3 x 10 mL CellSave preservative tubes at the study centers at 
specific timepoints and transported with the other biomaterial to the Central Study Center.  
 
 
Figure 12: Principle of the CA27.29, CEA and CA125 (OVCA) Analysis Workflow (provided by Tosoh 
Bioscience) 

 
 
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CTCs in CellSave tubes will be stable for 96 h at 20-25°C.  
CTCs will be analyzed using the FDA-approved, standardized semiautomatic CellSearch® System by 
Menarini Silicon Biosystems.  
The CellSearch® system is designed to enumerate CTCs of epithelial origin (CD45-, EpCAM+, 
cytokeratin 8+ / 18+ and/or 19+)(59). The basic principle is linking a magnetic ferrofluid reagent that 
contains i. a. antibodies targeting the EpCAM antigen to CTCs. After steps of immunomagnetic capture 
and enrichment as well as addition of fluorescent reagents (that contain anti-CK-PE, DAPI and anti-
CD45-APC), the automatic dispersion to a magnetic cartridge holder takes place. Via strong magnetic 
field, the magnetically labeled epithelial cells are attracted to the surface of the cartridge where they 
can be scanned automatically. 
Images of events where CK-PE and DAPI fluorescence are co-located are presented to the user for final 
classification. An event is classified as a tumor cell when its morphological features are consistent with 
that of a tumor cell and it exhibits the phenotype EpCAM+, CK+, DAPI+ and CD45- (59). 
 ctDNA Analysis 
ctDNA will be measured every three months for three years, thereafter every six months for another 
two years. Peripheral blood specimens will be collected in two Streck Cell-Free DNA BCT® tubes by the 
study centers and transported with the other biomaterial to the Central Study Center. At this point, 
distribution to Inivata Inc. will take place for further analysis. ctDNA will be stable for up to 14 days at 
6 °C to 37 °C. Depending on the result of the test and the result of the imaging, further controls may 
be necessary in the regular interval (for details see section 8.2.3). 
Presence of ctDNA will be analyzed using the personified RaDaRTM assay consisting of the patient 
specific-primer panel that was designed with the primary tumor tissue send at baseline. 
Plasma cell- free DNA and buffy coat DNA will be acquired from the whole blood sample. 
To detect patient specific ctDNA, NGS testing is performed with the RaDaRTM assay using a multiplex 
PCR based on the InVision® platform (57,58). 
 

 
 
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Figure 13: Principle of the RaDaRTM Workflow (provided by Inivata Ltd.) 
9.1.2.2 
Optional blood samples 
Optional blood samples in form of 57,5 mL whole blood (1 x 7,5 mL Serum, 3 x 10 mL CellSave, 2 x 10 
mL Streck) would be highly endorsed when breast cancer recurrence is observed. The blood samples 
will be sent to the Central Study Center for further distribution to the respective institutions who will 
perform further analysis. 

 
 
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10 DISCONTINUATION OF STUDY INTERVENTION, PATIENT, STUDY AND SITE 
10.1 Patients without Intervention 
This study is conducted according to the principle of intent-to-treat (ITT). 
Patients who have been randomized in the study but withdrew their consent or are withdrawn by the 
investigator from the study prior to first blood sampling are to be included in the intent-to-treat 
analysis and will not be replaced. The reasons for not starting blood sampling are collected. 
If a personalized ctDNA assay cannot be established for a patient in the Intensive Surveillance arm due 
to problems related to quality and/or quantity of the collected primary tumor tissue, a second primary 
tumor tissue sample from this patient will be obtained to facilitate another try at establishing a 
personalized ctDNA assay. If it still is not possible, this already randomized patient will be accounted 
for as drop-out.  
10.2 Premature Intervention Discontinuation  
Patients who have been randomized and have received study intervention and, for whatever reason, 
did not participate throughout the entire study are classified as premature intervention 
discontinuation. 
Investigators must attempt to contact patients who fail to attend scheduled visits by telephone, letter, 
visit, etc. Reasons for discontinuation must be documented in the CRF and in the patient’s medical 
records. 
10.3 Investigator-Initiated Discontinuation of Study Intervention 
In addition to the conditions outlined in the protocol, the investigator may require a patient to 
discontinue study participation if one of the following occurs: 
• 
The patient’s health deteriorates. 
• 
The patient is unable to meet the study requirements. 
• 
New information about the study interventions for breast cancer becomes available. 
• 
Any medical condition that may jeopardize the patient’s safety if he or she continues receiving 
study intervention. 
10.4 Patient-Initiated Study Discontinuation vs Withdrawal of Consent  
If a patient chooses to have no further interaction regarding the study, reason of discontinuation 
should be documented. The Investigator should not urge the patient to withdraw their consent. If a 
patient wants to discontinue all study related activities and withdraws her consent actively, 
withdrawal of consent should be documented. Biomaterials which have already been collected will be 
kept at the biobank and results from analyzed blood samples will be analyzed in final analysis unless 
the patient disagrees.  

 
 
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10.5 Premature Termination of Study 
The University Hospital Ulm as the sponsor has the right to close this study due to, but not limited to 
the following reasons: 
• 
If risk-benefit ratio becomes unacceptable owing to, for example: 
o safety findings from this study 
o results of interim analysis 
o results of futility analysis 
o results of parallel/other clinical studies 
• 
If the study conduct (e.g., recruitment rate; drop-out rate; data quality; protocol 
compliance) does not suggest a proper completion of the study within a reasonable and 
expected time frame 
The Independent Data Monitoring Committee (IDMC) and the Protocol Board will provide advice. The 
decision to premature termination of the study is binding to all investigators of all study sites. 
Responsible ethics committees will be informed about the reason(s) and time of termination 
according to the applicable laws and regulations. 
If the study is terminated prematurely, all investigators have to inform their patients and take care of 
appropriate follow-up and further treatment of patients. 
10.6 Premature Termination of Study at a Particular Site  
The University Hospital Ulm as the sponsor has the right to close this study at a particular study site 
which may be due but not limited to the following reasons: 
• 
Non-compliance with the protocol, GCP and/or regulatory requirements 
• 
Insufficient number of recruited patients 
• 
False documentation in the CRF due to carelessness or deliberately 
• 
Inadequate co-operation with University Hospital Ulm or its representatives 
• 
The Investigator request to close of his/her study site 
The reasons will be discussed with the investigator. If the study is prematurely terminated in a study 
site, the responsible investigators have to inform their patients and take care of appropriate follow-
up and ensure further treatment. The responsible ethics committee will be informed about the reason 
and time of termination according to the applicable laws and regulations. 

 
 
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11 END OF INTERVENTION (EOI) 
EOI is defined as last withdrawal and analysis of blood samples and - if staging is triggered - last staging 
examination (whichever comes last).  
EOI is possible in different scenarios: 
• 
The patient was diagnosed with local recurrence of breast cancer, breast cancer 
metastases or a secondary malignancy. Guideline-based diagnostics and treatments 
will take place outside of the study. 
• 
The patient becomes pregnant. Care at an obstetrician is advised. 
• 
Patient or study center discontinues intervention. Patient is willing to participate in 
follow-up phase. 
• 
All scheduled visits have been accomplished (end of Year 5). 
• 
Death of the patient. 
Afterwards, patients will enter the follow-up phase which will last for five years and does not contain 
any interventions in form of blood sampling or staging examinations triggered by biomarkers.  
Follow-up data will be collected once a year via Digital Health Application, via Telephone interviews 
or in written via post and include the assessments mentioned in section 8.5.4. 
 
If a patient shows a relevant finding in any biomarker (CA27.29, CEA, CA125, CTC, ctDNA) at the end 
of intervention but the consecutive staging shows no evidence of disease, the result of the biomarker 
will be told to the patient’s study center so that further staging modalities can be decided per 
Investigator’s discretion. 
 

 
 
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12 END OF STUDY (EOS) 
Planned end of study is the last follow-up visit of the last patient being in the 5-year follow-up phase.  
 
After end of study, final analysis of endpoints, publications and distribution of results will take place if 
not already happened. 
 
In terms of long-term follow-up after end of study, patients have the possibility to participate in the 
patient self-reporting registry (Patientenselbstauskunft). 
 

 
 
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13 ASSESSMENT OF OUTCOME 
13.1 Evaluation of Local Recurrence 
Breast examination with palpation including locoregional lymph nodes will be performed at each 
aftercare visit at the aftercare specialist (gynecologist/family physician/oncologist; every three 
months for the first three years of aftercare, every six months for the following two years). 
Additionally, patients should be advised to perform monthly self-palpation of the breasts and lymph 
nodes to detect any potential tumor. As such clinical examination cannot be 100% accurate and also 
benign breast tumors can be palpable, additional mammography and, where needed, additional 
breast ultrasound is done when a recurrence is suspected. If palpation is inconspicuous during 
aftercare, mammography and ultrasound of both breasts are advised once a year. If mammography 
and/or ultrasound is inconclusive, an additional MRI of the breasts may be necessary.  
Clinical Examinations and performance of mammography and breast ultrasound are recorded in the 
eCRF at every scheduled visit. If a recurrence of local breast cancer is diagnosed, the study center has 
to be notified immediately and the study center in turn reports to the IFG on eCRF. 
13.2 Evaluation of Distant Metastases 
Distant metastases can be diagnosed with any imaging available (CT scan, MRI, SPECT bone scan, 
sonography, etc.). In some cases, these examinations may be initiated due to other causes like back 
pain or elevated liver enzymes with the accidental finding of metastases of breast cancer. 
In symptomatic patients with suspected metastases, a CT scan of chest and abdomen as well as a 
SPECT bone scan is advised. If another type of imaging was performed before that does not depict the 
whole patient, these examinations should be performed additionally. 
In patients randomized to the Intensive Surveillance arm, a complete staging examination with CT scan 
of chest and abdomen as well as a SPECT bone scan will also be initiated in asymptomatic patients 
when a significant increase of tumor markers from baseline (CA27.29 +75% or CEA +100% or CA125 
+150%) is verified after confirmatory blood sampling or when CTCs or ctDNA are detected. 
Staging examinations done outside of the study are recorded with the patient questionnaire 
completed at every scheduled visit. If distant metastases are diagnosed, the study center has to be 
notified immediately and the study center in turn reports to the IFG GmbH Erlangen on CRF. 
13.3 Evaluation of Biomarkers 
The biomarkers used in this study (CA27.29, CEA, CA125, CTC, ctDNA) are in part well-known and in 
part new as diagnostic measures. Tumor markers are in use for monitoring metastatic breast cancer 
patients, usually in the combination of CA27.29 (or other MUC1-epitope CA15.3) and CEA as 
recommended by the ASCO. Hints of the additional tumor marker CA125 as a source of information 
have been seen so that we added it in the combination of tumor markers used in this study (24). 
However, absolute levels of tumor markers are only an indirect hint at possible tumor spread and can 
be elevated due to other reasons (e.g., renal insufficiency) which is why the kinetics of these markers 
instead of absolute numbers are of importance. CTCs as well as ctDNA are evidence that tumor cells 
can be found in the peripheral blood and therefore are a more direct way of detecting tumor 

 
 
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recurrence. As no comparison between these markers has been made so far, we chose to examine all 
three types to identify the most reliable one.  
 
One of our primary endpoints is the Overall Lead Time defined as molecular to via imaging verified 
recurrence lead time in addition to the DFS-difference between the two arms (see Figure 2). This will 
be analyzed for each marker separately and also - depending on the biomarker - in combination. 
For the calculation of overall lead time, a positive biomarker result is defined as an abnormal finding 
for any of the biomarkers used (i.e., either CA27.29 or CA125 or CEA or CTC or ctDNA). However, to 
specifically evaluate the performance with regard to early detection of MRD for single biomarkers, 
overall lead time will also be calculated separately for the conventional tumor markers (CA27.29, 
CA125, CEA), for CTCs and for ctDNA. In addition to that, the secondary endpoint of Molecular to via 
Imaging verified Recurrence Lead Time in the Intensive Surveillance arm is analyzed to determine 
whether a lead time effect can be generated between molecular to via imaging verified recurrence 
through liquid biopsy. 
For another measurement of efficacy of these biomarkers, we analyze as secondary endpoints 
specificity and sensitivity of all the markers individually and also in combination regarding the tumor 
markers. The reason for the latter is the evidence that the combination of tumor markers may show 
a higher sensitivity than seen individually (26). As an additional secondary objective, the False-Positive 
Liquid Biopsy Rate being defined as no clinical relapse 36 months after first and persistent molecular 
relapse shall be analyzed. 
We expect the availability of reliable data from all patients shortly after the end of study. 

 
 
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14 ASSESSMENT OF HEALTH-RELATED QUALITY OF LIFE 
14.1 Rationale 
QoL may be influenced in different ways in both arms of the study as patients and doctors are blinded 
with the exception of positive findings in the Intensive Surveillance arm. 
In the Standard Surveillance arm, QoL could either be influenced by the assurance of being closely 
monitored by being in a study or by the uncertainty of the possibility of being in the Intensive 
Surveillance arm and possibly have a positive finding in the biomarkers. 
In the Intensive Surveillance arm – in case of unblinding due to positive findings – patients could show 
an increase in QoL by the reassurance that they are free of disease. 
Furthermore, QoL may be increased hypothetically by detecting disease before development of 
symptoms, by virtue of early intervention, by delaying the time to the onset of symptoms, by 
prolonging survival and potentially even cure patients. QoL might improve by the intervention alone, 
but also in case of an increased OS. Moreover, earlier detection of distant recurrence and (oligo-
)metastatic disease might reduce the need for aggressive cytotoxic chemotherapy regiments and may 
provide a chance of full recovery in the future, even at the stage of secondary disease.  
However, a positive finding in biomarkers and consecutive staging examinations, which - in case of a 
molecular lead time - show no evidence of disease, could lead to an increase in fear of progression 
and therefore a deterioration of QoL.  
Thus, QoL is to be evaluated in both groups with two different questionnaires: EORTC QLQ-C30 asking 
about general wellbeing in breast cancer patients and PA-F12 focusing on the fear of disease 
progression. 
The questionnaires will be completed every six months during the interventional phase as well as 
every year during the follow-up phase.  
 An interim analysis will be performed one year after 50% of patients (n = 1750) have been enrolled in 
to the study. 
 
 
Figure 14: Timeline for QoL-Assessment 
 

 
 
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Figure 15: Timeline for QoL-Assessment respective to study visits 
14.2 Questionnaires 
QoL will be assessed every six months over the five years of interventional phase and every year in 
the five year follow-up phase in both arms. The validated and well-evaluated questionnaires EORTC 
QLQ-C30 and PA-F12 are used to analyze QoL with a focus on fear of progression as well as to evaluate 
any deterioration due to additional measures in the Intensive Surveillance arm at an early stage. The 
main issues affecting QoL in breast cancer follow-up will be fear (of progression/recurrence) and 
depression. The EORTC QLQ-C30 is a standardized and widely used questionnaire specifically for 
cancer patients with very good comparability, used since 1993. It consists of 30 questions to evaluate 
QoL in a multidimensional way with 10 different sub scales (60). The fear of progression will also be 
evaluated in the PA-F12 (German for FoP-Q-SF), which is a validated and reliable instrument and has 
been widely studied in breast cancer patients as well. In this short form of the PA-F, 12 items are 
inquired which form five sub scales. The general value for fear of progression can be seen as a sum of 
all items with a higher value corresponding to a higher level of fear of progression (61).  
The questionnaires will be supplied to patients on paper and should be answered at the study center 
appointment. After submission, data will be entered in the eCRF. 
14.3 Digital Health Application 
The Digital Health Application may be used for Follow-Up questionnaires and may be used for study-
related appointment planning.  
If the participants decide to use the Digital Health Application, it is required to install the specific 
application on the participant’s smart phone and consent to the data processing through the 
application. An individual username and password will be generated for each patient. 
With the Digital Health Application, the participant will get a reminder on the due date of the Follow-
Up questionnaire and the questionnaires can be answered digitally. If the questionnaires are not 
answered, the participant will get another friendly reminder. If the participant does not want to 
continue to use the application, it can be deleted any time from the smart phone in use. The study 
center should be informed by the patient if paper-based questionnaires are needed. 

 
 
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14.4 Analysis 
QoL data will be analyzed according to the instructions of the validated questionnaires used. 
 

 
 
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15 DATA HANDLING AND DATA QUALITY ASSURANCE 
15.1 Data Management and Documentation  
Data management, which comprises CRF design, database creation, data processing and data 
validation will be performed by the IFG GmbH Erlangen for all study sites. 
The IFG GmbH Erlangen will provide the investigator site with a web-based EDC system that is fully 
validated and conforms to 21 CFR Part 11 requirements. Investigator site staff will not be given access 
until they have been trained on the EDC system. 
 Data Entry and Queries 
All CRF data will be entered into the study database by trained members of the investigator site using 
the web-based EDC system, which will perform automated plausibility and value range checks before 
accepting the data into the database. 
Data that doesn’t match the study guidelines will be queried and followed up on with the sites until 
satisfactory resolution. 
 Data Validation 
Visual and computerized methods of data validation are applied in order to ensure accurate, 
consistent and reliable data for the subsequent analyses. 
 Registration / Recruitment Stop 
At the end of recruitment, new patient registration and randomization functionality via EDC system is 
stopped and locked, although documentation for registered patients is possible until database lock. 
 Database Lock 
Database lock for the primary analysis will be initiated at EOS, after all data has been entered and 
queries have been handled. After database lock no data can be changed. The database is locked to 
any kind of manipulation and then handed over to the Statistics Department. Further database locks 
for exploratory analyses or quality control measures will be done when necessary. 
 

 
 
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15.2 Privacy Protection and Data Safety  
 Pseudonymization 
In order to protect patient data confidentiality and for safeguarding the privileged doctor patient 
relationship, each participating patient is assigned a unique patient number. Instead of the true 
patient identity the pseudonym is used in all communication. 
 User Access Control 
Every user is provided with a unique personal username and unique password which defines their 
access to the web-based EDC system. Access control is based on the users’ role in the web-based EDC 
system. Therefore, users can only access and amend those datasets necessary for them to fulfil their 
tasks. 
15.3 Record retention after study completion 
After completion of the study, all documents and data relating to the study will be kept in an orderly 
manner by the investigator in a secure study file. This file will be available for inspection by the sponsor 
or their representatives. Essential documents must be retained for twenty-five (25) years after 
completion of the study (here: End of Follow-Up). 

 
 
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16 STATISTICS  
The statistical analysis of the present study is performed in accordance with the principles stated in 
the Consensus Guideline E9 (Statistical Principles for Clinical Trials) of the International Conference on 
Harmonization (ICH) including E9 (R1) addendum. 
16.1 Randomization and stratification  
Patients will be randomized centrally using permuted block list only if they fulfil the inclusion criteria 
and are not precluded from participation by any of the exclusion criteria.  
Randomization will be stratified using block randomization by: 
• 
Hormone receptor status (negative vs positive) 
• 
HER2 status (negative vs positive) 
• 
Histological lymph node status at surgery ((y)pN0 vs (y)pN+) 
16.2 Analysis Sets 
The following study population sets will be examined. 
If necessary, further data sets required for additional analyses may be specified in separate analysis 
plans. 
 Intent-to-Treat (ITT) Set 
The ITT set includes all patients who are randomized with signed informed consent. Patients will be 
analyzed according to the group they were randomized to. 
 Per Protocol (PP) Set 
The PP set includes all patients of the ITT set who did not violate any inclusion or exclusion criterion 
and who were treated strictly according to protocol. 
16.3 Sample Size Determination  
The study is designed as a two-arm parallel, (partially) double-blinded randomized superiority study. 
To detect a 2.35% improvement in 5-year OS rate from 92.65% in the standard surveillance arm to 
95.0% in the tumor marker, ctDNA and CTC guided surveillance arm (equivalent to a hazard ratio of 
0.672), approximately 3500 randomized patients (1750 in both arms) will be required to achieve 80% 
power at a 2-sided significance level of 5%, assuming a uniform accrual pattern (accrual time 2 years), 
a dropout rate of 15% in both arms and a total study duration of 10 years. The reference value of 
92.65% 5-year OS rate is based on the recently published results from the randomized phase III Success 
A study (Clinicaltrials.gov NCT02181101) that reported 5-year OS rates for 3754 intermediate-to-high-
risk early breast cancer patients receiving standard chemotherapy regimen. 

 
 
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16.4 Patient disposition, data re-coding and display  
Patients’ demographics, baseline characteristics, protocol compliance and study withdrawals will be 
summarized by randomization arm in the intent-to-treat population. For categorical variables, 
frequency tables with absolute and relative frequencies will be presented with descriptive listings of 
details specified in text fields, when appropriate. Continuous variables will be reported using mean 
and standard deviation and/or median, range and interquartile range when appropriate. A CONSORT 
diagram will be used to document the flow of patients through the various stages of the study. The 
number of patients included in the various analysis populations will be presented in a table and 
reasons for exclusions will be detailed in listings.  
16.5 Analysis of Primary Endpoint(s) 
The primary endpoint overall survival will be analyzed based on the ITT set, estimated by the Kaplan 
Meier product limit method, and 5-year OS rates, 95% confidence intervals and survival plots will be 
provided. Overall survival will be compared between patients in the two randomization arms using 
the log-rank test, and univariable cox regression as well as additional multivariable analyses adjusted 
for other factors will be performed using suitable regression models (cox proportional hazard 
regression model). Hazard ratios and the corresponding 95% confidence intervals will be reported as 
obtained by both univariable and adjusted multivariable analyses. 
The co-primary endpoint Overall Lead Time Effect will be calculated as described in chapter 5.1. As it 
is a purely descriptive composite measure consisting of two unrelated median values calculated for 
different patient cohorts, no confidence intervals will be provided.  
16.6 Analysis of Secondary Endpoints 
All analyses regarding the secondary objectives will have exploratory character only. All secondary 
endpoints and other outcomes that are calculated based on frequencies/rates will be summarized 
using frequency tables presenting absolute and relative frequencies together with appropriate 
confidence intervals. Comparisons between groups will be performed using suitable non-parametric 
statistical tests such as Fisher’s exact test, χ2-test, or Cochran-Mantel-Haenszel test. Continuous 
variables will be summarized by descriptive statistics (number of valid and missing observations, 
mean, standard deviation, median, interquartiles, minimum, and maximum) and compared between 
arms using suitable non-parametric tests such as Wilcoxon-Mann-Whitney. Secondary survival 
endpoints (IDFS, DDFS, DRFS, BCSS) will be analyzed in the same way as the primary endpoint OS. 
Tumor marker (CA27.29, CEA, CA125) levels, presence and number of CTCs as well as presence of 
ctDNA measured at different time points will be evaluated in a descriptive way, and the temporal 
changes will be described and analyzed using appropriate statistical models (e.g., generalized linear 
mixed models). Quality of life data will be analyzed according to the instructions of the validated 
questionnaires used (EORTC QLQ-C30 and PA-F12).  
More details regarding the statistical analyses will be provided in a Statistical Analysis Plan (SAP) which 
will be finalized prior to data base lock. 
 

 
 
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16.7 Pre-planned subgroup analyses  
Subgroup analyses will be conducted to assess the consistency of the results (primary and secondary 
endpoints) across the following prespecified key subgroups: Hormone receptor status (negative vs 
positive), HER2 status (negative vs positive), histological lymph node status at surgery ((y)pN0 vs 
(y)pN+). These subgroup analyses will be exploratory. The presence of treatment heterogeneity 
regarding the primary endpoint OS will be examined by testing the presence of an interaction in a Cox 
model with the key variable of interest, treatment indicator and the interaction term, stratifying for 
the other stratification factors. The hazard ratio and associated confidence interval for the treatment 
effect will be calculated within the subgroups of interest from the model. The same level of confidence 
as for the overall population will be used for the confidence intervals in the subgroups. Furthermore, 
the estimated hazard ratios in all subgroups and the p-value for interaction will be displayed on a 
forest plot. Furthermore, possible heterogeneity of the results between patients below and above 70 
years old will be similarly investigated. Additional exploratory subgroup analyses will be performed as 
appropriate. 
The results of these subgroup analyses should be interpreted with caution. No type I error adjustment 
will be made for multiple testing. 
16.8 Interim/Futility Analyses 
 Interim analyses for efficacy 
No formal efficacy interim analysis is planned. 
 Futility analyses 
No formal efficacy interim analysis is planned. 
A one-step futility analysis for ctDNA is planned (as per request of the contributing company): 
 
Step 1:  
Analysis of minimum requirement regarding ctDNA positivity rate. 
• 
Analysis is performed based on the first 150 patients enrolled in the experimental arm to 
calculate the ctDNA positivity rate.  
• 
Analysis is considered to be affirmative to continue ctDNA testing within the study,  
- 
if the ctDNA positivity rate measured 3 months after recruitment is > 5% for these 150 
patients and 
- 
if the ctDNA positivity rate measured 12 months after recruitment is > 10% for these 
150 patients. 
If these requirements for continuation of ctDNA testing are not fulfilled, termination of ctDNA testing 
in this trial might be considered pending decision of the steering board and the company 

 
 
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 Interim analysis for Quality of Life (QoL) 
An informal, nonbinding interim analyses of QoL will be performed one year after 50% of patients (n 
= 1750) have been enrolled. Based on the individual data from the PA-F12 and EORTC QLQ C30 
questionnaires obtained from these patients 12 months after enrollment, differences in QoL between 
the two randomization arms will be evaluated (with the main focus being fear of progression and 
depression) to ensure that patients in the Intensive Surveillance arm do not suffer from unacceptable 
emotional burden as compared to the standard arm. The findings will be presented to the IDMC to 
decide if (and if yes, which) measures should be taken to lessen the emotional burden for patients in 
the Intensive Surveillance arm. This analysis will be repeated at regular intervals according to DSMB 
requirements to ensure that there is no long-term impairment on patients’ emotional well-being 
because of the study. Regular study monitoring regarding compliance of blood sampling and 
evaluation of the reasons for incompliance or even drop-outs will complement the QoL assessments. 

 
 
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17 INDEPENDENT DATA MONITORING COMMITTEE (IDMC) 
17.1 IDMC Members and Mission 
In addition to the Protocol Board and Steering Committee, the IDMC and the IFG GmbH Erlangen 
review and observe the conduct of the study. 
The members are independent of the study and familiar with the methodology of oncology trials. They 
are aware of the risks of conclusions based on immature data and have agreed with the design and 
the goals of this protocol. IDMC meetings are held regularly; if necessary (for example, if results of an 
interim safety analysis become available between regular meetings), ad hoc IDMC meetings may be 
organized. The mission of the IDMC is to ensure the ethical conduct of the study and to protect 
patients' safety interests in this study. 
17.2 Documentation Provided to the IDMC 
Before any meeting of the IDMC, the statistician should provide the IDMC with at least the following 
key documents: 
• 
Study protocol 
• 
Recruitment summary 
• 
Patient baseline characteristics 
• 
Narratives of deaths 
• 
(Distant) disease recurrence rates 
• 
Summary of follow-up 
All data will be broken down by treatment arm, participating institution and patient (whenever 
necessary). 
17.3 Recommendations of the IDMC 
After each meeting, the IDMC will provide the Protocol Board/Steering Committee with a written 
recommendation to either modify the study (with reasons), or discontinue the study (with reasons), 
or continue the study unchanged. The final decision to amend the protocol or to discontinue the study 
will be taken only by the Protocol Board.  
Early termination of the study will be considered by the Protocol Board/Steering Committee based on 
the suggestion of the IDMC if less than 500 patients are recruited within 12 months. 
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werden.. 

 
 
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18 ADMINISTRATIVE EXECUTION 
18.1 Monitoring and Auditing 
All source data verification (SDV) and source data review (SDR) is conducted according to ICH-GCP 
guidelines, the and the Trial Monitoring Plan (TMP). The investigator must permit study monitors, the 
sponsor’s auditors to inspect all study-related documents and pertinent hospital or medical records 
for confirmation of data contained within the CRFs. 
Source documentation will be reviewed and verified in a risk-based approach for adequacy and 
consistency. 
18.2 Sponsor’s Responsibilities 
The University Hospital Ulm as the sponsor 
• 
agrees to provide the investigator with sufficient material and support to permit the 
investigator to conduct the study according to the agreed protocol. 
• 
reserves the right to request the withdrawal of a patient due to protocol deviations, 
administrative or other reasons. 
• 
reserves the right to terminate the study prematurely due to persistent protocol deviations of 
the site, administrative or other reasons. Should this be necessary, the procedures will be 
arranged after review and after consultation by both parties to ensure protection of the 
patients’ interests. 
18.3 Investigator’s Responsibilities 
The investigator agrees to conduct the study in accordance with and the procedures and requirements 
laid out in this protocol and by ICH-GCP guidelines. In particular, the investigator agrees to conduct 
the study in accordance with strict ethical principles. Any modification to the agreed protocol must be 
approved in writing by both University Hospital Ulm as the sponsor and, if appropriate, the ethics 
committee(s) approving the original protocol before any modifications are put into effect. 
It is the responsibility of the investigator to complete the CRFs for each patient in the study, and when 
a patient completes the study, the investigator must (electronically) sign all CRFs. 
In addition to the CRFs, the investigator will maintain adequate records that fully document the 
progress of the study. The investigator has to state that the patient has taken part in a study and 
record the study number in the patient’s medical records. The exact dates of the beginning and the 
end of intervention should be given as well. 
Copies of these study records (and all study-related documents) shall be kept by the investigator for 
25 years by the hospital, institution or private practice. All documentation and materials provided by 
IFG GmbH Erlangen and the University Hospital Ulm for this study are to be retained in a secure place 
and treated as confidential material. 
 

 
 
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The investigator has the right to request termination of the study for administrative or other reasons. 
Should this be necessary and agreed upon, the procedures will be arranged after review and after 
consultation by both parties, to ensure protection of the patients’ interests. 
By signing this document, the investigator indicates that he/she has read the protocol, fully 
understands the requirements and agrees to abide by all protocol requirements. 
Further obligations of the investigator are agreed on in the investigator’s contract with the University 
Hospital Ulm as the sponsor. 
The investigator will be responsible for retaining sufficient information about each patient (e.g., 
informed consent form, name, address, phone number, and identity in the study) so that the auditor 
of the University Hospital Ulm as the sponsor may access this information should the need to do so 
arise. These records should be retained in a confidential manner for as long as legally mandated 
according to local requirements. 
18.4 Patient Informed Consent 
Prior to the beginning of specific protocol procedures, the patient is informed about the nature of the 
study intervention and is given pertinent information as to the intended purpose, possible benefits, 
and possible adverse experiences. The procedures and possible hazards to which the patient will be 
exposed are explained. Patient insurance for the compensation of patients for possible study-related 
injury as well as a commuting accidents insurance policy is provided by the University Hospital Ulm as 
the sponsor according to local law. 
An approved informed consent (ICF) statement will then be read and signed by the patient, and, if 
required, a witness, as well as the investigator. The patient will be provided with a copy of the signed 
ICF. The patient may withdraw from the study at any time in any way without prejudicing future 
medical treatment.  
Patients are informed that pseudonymized data from their case may be stored electronically and that 
such data will not be revealed to any unauthorized third party. Data will be reviewed by the monitor, 
an independent auditor and possibly by representatives of ethics committees. The terms of the local 
data protection legislation will be applied as appropriate. 
18.5 Ethics and Regulatory Considerations 
This study will be conducted according to the ICH-GCP guideline in compliance with the 
recommendation of the German task force of medical ethical committees regarding studies that are 
neither part of the German drug law nor part of the German law on medical products (Arbeitskreis 
Medizinischer Ethikkommissionen; „Empfehlung für den Umgang mit multizentrischen Studien 
außerhalb von AMG oder MPG durch Ethik-Kommissionen“) and applicable regulations in all aspects 
of preparation, monitoring, reporting, auditing, and archiving along the § 15 Musterberufsordnung 
(MBO). The final approved protocol and the ICF is reviewed by a properly constituted Ethics 
Committee. The decision of Ethics Committee concerning the conduct of the study is made in writing 
to the investigator. 
 

 
 
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18.6 Declaration of Helsinki 
This study is to be performed in accordance with the Declaration of Helsinki (62). 
18.7 Study Conduct 
This study will be conducted according to the ICH-GCP in compliance with the recommendation of the 
German task force of medical ethical committees regarding studies that are neither part of the 
German drug law nor part of the German law on medical products (Arbeitskreis Medizinischer 
Ethikkommissionen; „Empfehlung für den Umgang mit multizentrischen Studien außerhalb von AMG 
oder MPG durch Ethik-Kommissionen“) and their regulatory requirements.  
18.8 Modification of the Protocol 
Any modifications to the protocol which may impact on the conduct of the study, on the potential 
benefit of the patient or may affect patient safety, including changes of study objectives, study design, 
patient population, sample sizes, study procedures, or significant administrative aspects, will require 
a formal amendment to the protocol. Such an amendment will be agreed upon by the Protocol Board, 
and approved by the Ethics Committee prior to implementation. 
Administrative changes of the protocol are minor corrections and/or clarifications that have no effect 
on the way the study is to be conducted. These administrative changes will be agreed upon by the 
Protocol Board and will be documented in a memorandum. The Ethics Committee may be notified of 
administrative changes at the discretion of the investigator. 
18.9 Study Documents 
All information concerning the intervention and study conduction, such as scientific data and material 
not previously published are considered confidential and shall remain the sole property of University 
Hospital Ulm. 
The investigator agrees to use the information provided for the conduct of this study only and to use 
it for no other purposes unless she/he obtains the written consent of the University Hospital Ulm as 
the sponsor. 
18.10 Case Report Forms  
All key study information must be recorded in the patient’s hospital chart.  
Study procedures will be fully online documented on the electronic CRFs (eCRF) provided through the 
web-based EDC system. The data from the questionnaires of the study (study visit questionnaires, QoL 
questionnaires) will also be fully documented and the data collected via Digital Health Application will 
be transmitted to the IFG GmbH Erlangen. Both, data from questionnaires via Digital Health 
Application and on-paper questionnaires will be transferred into the eCRF. 
 

 
 
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Relevant laboratory reports with study number, patient number and medical interpretation of results 
have to be signed and dated by the investigator. 
The CRFs remain in the property of the University Hospital Ulm and the IFG GmbH Erlangen at all 
times. On the CRFs, patients should be identified only by their patient number. 
Patients’ data entries may only be made by the persons registered on the form “Delegation of 
responsibilities and signature list of investigators and medical staff”. 
For details concerning the CRF submission process, please refer to the application manual and training 
material. 
18.11 Necessary Documents 
The following documents are collected from the investigator’s site: 
● Signed Investigator’s Agreement 
● Scientific Curriculum vitae of Principal Investigator 
● Form “Delegation of responsibilities and signature list of investigators and medical staff” 
● Responsibility of data handling and protection according to DSGVO 
18.12 Archiving 
Any records and documents relating to the conduct of this study, including ICFs, eCRFs, QoL data, 
laboratory test results, and imaging records, must be retained by the Principal Investigator for a 
minimum of twenty-five (25) years after completion (here: End of Follow-Up) or discontinuation of 
the study, or for the length of time required by relevant national or local health authorities, whichever 
is longer. After that period of time, the documents may be destroyed, subject to local regulations. 
Notification should be provided to the Sponsor prior to transferring any records to another party or 
moving them to another location. 
18.13 Use of Information and Publication 
To allow for the use of the information derived from this clinical study and to ensure compliance to 
current regulations, the investigator is obliged to provide the sponsor with complete test results and 
all data obtained in this study. The final statistical study report will be prepared by the responsible 
biostatistician and the final medical report by the coordinating investigator and the sponsor. 
The final study report will be a publication in a peer-reviewed journal under the responsibility of the 
Sponsor according to publication regulations. 
The Protocol Board will review the manuscript to prevent forfeiture of patent rights to data not in the 
public domain. The authorship list will be agreed upon by University Hospital Ulm and the investigators 
prior to publication. 
18.14 Finance and Insurance 
Details on finance and insurance will be outlined in a separate agreement between the investigator 
and the sponsor. 

 
 
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19 INSURANCE COVER 
The sponsor takes out an insurance policy for all study participants covering all injuries patients suffer 
from due to the study participation. Furthermore, the sponsor takes out a commuting accidents 
insurance policy for all study participants covering all injuries patients suffer on their direct commute 
to and from study centers. A certificate of insurance will be provided to the investigator.  
Compensation is limited to 500.000,00 Euro at most per patient and 5.000.000,00 Euro at most for 
the whole study. Additional limits for travel insurance cover 100.000,00 Euro at most per person for 
invalidity or death.  
Insurance provider:  
Chubb European Group SE, Direktion für Deutschland, Baseler Straße 10, 
60329 Frankfurt am Main, Germany 
Police-No.:  
 
DELSCA45932 
Policy Holder:   
Universitätsklinikum Ulm, Albert-Einstein-Allee 29, 89081 Ulm, Germany 
 
The investigator informs each patient on his responsibilities resulting from the terms of insurance.  

 
 
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20 GLOSSARY OF ABBREVIATIONS AND ACRONYMS 
°C 
 
 
degree Celsius 
µg  
 
 
microgram 
AGO 
Arbeitsgemeinschaft für Gynäkologische Onkologie (German study group for  
APC 
 
Allophycocyanin (APC) 
ASCO 
 
American Society of Clinical Oncology 
BCSS 
 
Breast cancer specific survival 
CA 
 
Cancer antigen 
CD 
 
Cluster of differentiation 
CDK  
 
cyclin-dependent kinase 
CEA 
 
Carcinoembryonic antigen 
CFR 
 
 
Code of Federal Regulations 
CI 
 
confidence interval 
CK-PE 
 
cytokeratin-phycoerythrin 
CRF 
 
Case Report Form 
CT 
 
computed tomography 
CTC 
 
Circulating tumor cell 
ctDNA  
 
Circulating tumor deoxyribonucleic acid 
DALYs 
 
disability-adjusted life-years 
DAPI 
 
4′,6-diamidino-2-phenylindole 
DCIS 
 
ductal carcinoma in situ 
DDFS 
 
distant disease-free survival 
DFS 
 
disease-free survival 
DNA 
 
deoxyribonucleic acid 
DRFS 
 
distant recurrence-free survival 
DSGVO 
 
Datenschutzgrundverordnung der Europäischen Union 
DSMB 
 
Data Safety Monitoring Board 
DSUR  
 
Development Safety Update Report 
e. g. 
 
 
exempli gratia 
e. V. 
 
 
eingetragener Verein (registered organization) 
ECLIA   
 
ElectroChemiLuminescence-ImmunoAssay 
ECOG 
 
Eastern Cooperative Oncology Group 
eCRF 
 
electronic Case Report Form 
EDC 
 
electronic data capture 
EDTA 
 
Ethylenediaminetetraacetic acid 
EpCAM 
 
epithelial cellular adhesion molecule 
EOI 
 
End of Intervention 
EORTC 
 
European Organisation for Research and Treatment of Cancer 
EORTC QLQ-C30 
EORTC Core Quality of Life questionnaire with 30 question 
EOS 
 
end of study 
EOT 
 
end of treatment 
ER 
 
estrogen receptor 
ET 
 
endocrine therapy 
Etc. 
 
etcetera 

 
 
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EudraCT 
 
European Union Drug Regulating Authorities Clinical Trials 
FDA 
 
Food and Drug Administration 
FFPE 
 
Formalin-fixed, paraffin-embedded 
FIGO 
 
The International Federation of Gynecology and Obstetrics 
FoP-Q-SF  
 
Fear of Progression Questionnaire-Short Form  
FPI 
 
first patient in 
FU 
 
 
Follow up 
GCP 
 
Good Clinical Practice 
GmbH  
 
Gesellschaft mit beschränkter Haftung (Limited Company) 
GnRH 
 
Gonadotropin-releasing Hormone 
H 
 
hour 
H&E 
 
Hematoxylin and eosin 
HER 
 
human epidermal growth factor receptor 
HR 
 
hormone receptor 
HTTP 
 
 
Hypertext Transfer Protocol 
i. a. 
 
 
inter alia 
i. e. 
 
 
id est 
IBCFS   
 
invasive breast cancer free survival 
IB 
 
Investigator's Brochure 
ICF 
 
informed consent form 
ICH 
 
International Conference on Harmonization of Technical Requirements for 
 
Registration of Pharmaceuticals for Human Use 
iDFS 
 
invasive disease-free survival 
IDMC  
 
Independent Data Monitoring Committee 
IFG GmbH Erlangen 
Institut für Frauengesundheit GmbH Erlangen 
LP 
 
last patient 
LPI 
 
last patient in 
mL 
 
milliliter 
MRD               
 
molecular residual disease 
MRI 
 
magnetic resonance imaging 
MUC 
 
mucin 
mTOR  
 
mechanistic Target of Rapamycin 
NCI 
 
National Cancer Institute 
ng  
 
nanogram 
NGS 
 
Next Generation Sequencing  
Ob/Gyn 
 
Obstetrics and Gynecology 
OS 
 
overall survival 
PA-F12 
Progredienzangstfragebogen 
mit 
12 
Fragen 
(fear 
of 
progression 
questionnaire with 12 questions) 
PARP 
 
Poly ADP (Adenosine Diphosphate)-Ribose Polymerase 
PCR 
 
polymerase chain reaction 
PET 
 
positron emission tomography 
PgR 
 
progesterone receptor 
PI 
 
 
principle investigator 
PI3K 
 
phosphatidylinositol 3-kinase  

 
 
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PP 
 
Per Protocol 
PRO 
 
Patient reported outcome 
QoL 
 
 
Quality of life 
RaDaR™  
 
Residual Disease and Recurrence 
SAP 
 
 
Statistical analysis plan 
SDR 
 
 
source data review 
SDV 
 
 
source data verification 
SERMs  
 
Selective estrogen receptor modulators 
SNP 
 
 
Single-nucleotide polymorphism 
SNV 
 
 
single nucleotide variants 
SOP 
 
 
standard operations procedures 
SPECT  
 
single-photon emission computerized tomography 
TDM-1 
 
Trastuzumab emtansine 
T-Dx  
 
 
Trastuzumab deruxtecan 
TM 
 
Tumor marker 
TMP 
 
 
Trial Monitoring Plan 
TNBC 
 
triple-negative breast cancer 
U 
 
Unit 
USA 
 
United States of America 
vs. 
 
versus 
WES 
 
whole exome sequencing 
WMA  
 
World Medical Association  
YLD 
 
 
years lived with disability 
YLL 
 
 
years of life lost 

 
 
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21 APPENDICES 
21.1 Appendix A:  Quality of Life Questionnaires 
 EORTC-QLQ C30 
 PA-F12 
21.2 Appendix B: Assessment of Performance status and activities of daily living 
 Determination of Performance Status 
ECOG or Zubrod Scale 
 
Karnofsky Score 
0 
Fully active; able to carry on 
all pre-disease performance 
without restriction 
90–100% 
1 
Restricted in physically 
strenuous activity but 
ambulatory 
70–80% 
2 
Ambulatory and capable of 
self-care, but unable to carry 
out any work activities 
50–60% 
3 
Capable of only limited self-
care; confined to bed or chair 
more than 50% of waking 
hours 
30–40% 
4 
Completely disabled 
10–20% 
 
 
 

 
 
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