A Real-world Study on the Treatment of HR+/HER2- Advanced Breast Cancer Patients With CDK4/6 Inhibitors and Treatment Options After Drug Resistance.
Conditions: Breast Cancer
Sponsor: Tianjin Medical University Cancer Institute and Hospital

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Investigator-Initiated Retrospective Real-World Study 
 
 
 
 
A Real-World Study on CDK4/6 
Inhibitors for HR+/HER2- Advanced 
Breast Cancer and Subsequent 
Therapeutic Options After Drug 
Resistance 
 
 
Study Protocol 
 
 
 
 
 
 
 
Principal Investigator: Shi Yehui

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Investigator Signature Page 
 
Study Title: 
 
A Real-World Study on CDK4/6 Inhibitors for HR+/HER2- Advanced Breast Cancer 
and Subsequent Therapeutic Options After Drug Resistance 
 
 
 
 
I will faithfully perform the investigator's responsibilities in accordance with the 
provisions of the Good Clinical Practice (GCP) of China, and personally participate in 
or directly supervise this clinical study. We have read and confirmed this protocol, and 
agree with its scientificity and ethicality. We will fulfill the relevant responsibilities in 
accordance with the laws of China, the Declaration of Helsinki, the GCP of China and 
the provisions of this study protocol. Any modification to the protocol will only be 
made after notifying the sponsor, and can be implemented only after being approved by 
the Institutional Review Board (IRB), unless measures must be taken to protect the 
safety, rights and interests of the subjects. 
We will keep this study protocol confidential. 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator  
 
Signature:   
 
Date: 
/ 
/  
 

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Table of Contents 
1. Study Background ......................................................................................................................... 3 
2. Study Objectives ........................................................................................................................... 4 
3. Study Design ................................................................................................................................. 5 
4. Technical Roadmap ...................................................................................................................... 6 
5. Inclusion and Exclusion Criteria ................................................................................................... 6 
6. Data Collection and Statistical Analysis ....................................................................................... 6 
References: ........................................................................................................................................ 9 
  Appendix: ...................................................................................................................................... 10 

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1. Study Background 
 
Since 2020, breast cancer has surpassed lung cancer to become the most incident 
malignant tumor worldwide [1]. Advanced breast cancer is almost incurable, so delaying 
disease progression and improving quality of life are of vital importance. Among 
advanced breast cancer cases, nearly 75% are hormone receptor-positive (HR+) and 
human epidermal growth factor receptor 2-negative (HER2−) molecular subtypes. 
According to clinical guidelines, the standard first-line treatment regimen is cyclin-
dependent kinase 4/6 inhibitors (CDK4/6 inhibitors) combined with endocrine therapy. 
To date, based on the results of multiple phase III clinical trials [2-5], palbociclib, 
abemaciclib, ribociclib and dalpiciclib have been approved by the U.S. Food and Drug 
Administration (FDA) and the National Medical Products Administration (NMPA) of 
China respectively, for the first-line treatment of HR+/HER2− advanced breast cancer 
patients and those with disease progression after prior endocrine therapy. However, for 
different HR+/HER2− advanced breast cancer patients, which CDK4/6 inhibitor is the 
optimal first-line treatment option? Which CDK4/6 inhibitor is more suitable for patients 
with progression after prior endocrine therapy? In addition, the real-world adverse 
reactions of different CDK4/6 inhibitors in clinical practice are all worthy of exploration 
as clinical issues. The number of Asian populations in most landmark clinical trials of 
CDK4/6 inhibitors is limited [6], and the incidence of potential grade≥3 adverse reactions 
is higher in Asian populations [7], which also poses challenges for selecting the most 
appropriate CDK4/6 inhibitor for the Chinese population. Moreover, despite the 
significant clinical benefits of these drugs, patients will eventually develop drug 
resistance. At present, there are no high-level guideline recommendations for therapeutic 
options after CDK4/6 inhibitor resistance. Therefore, the optimal treatment strategy after 
progression on CDK4/6 inhibitors has become an urgent clinical problem to be solved. 
In recent years, real-world evidence derived from the analysis of real-world data 
including treatment patterns and outcomes has attracted increasing attention. Real-world 
studies usually cover a broader and more realistic patient population, and can generate 
new analyses and insights after phase III studies and the approval of treatment regimens. 
A real-world study based on the Flatiron Health database published by Rugo et al. 
indicated that in a real-world setting, first-line palbociclib combined with endocrine 
therapy, rather than endocrine therapy alone, can significantly prolong the overall 

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survival of HR+/HER2− advanced breast cancer patients [8]. In addition, real-world 
studies can often include populations that are overlooked in phase III studies, provide 
important clinical evidence, and reflect treatment patterns in real-world practice. For 
example, some real-world studies have focused on populations that are usually 
underrepresented in phase III studies, such as elderly patients [9-10]. Therefore, we 
designed this study to evaluate the real-world data of CDK4/6 inhibitors (including but 
not limited to the four currently marketed drugs: palbociclib, abemaciclib, ribociclib and 
dalpiciclib) in HR+/HER2− advanced breast cancer patients and the real-world clinical 
treatment options after drug resistance, so as to provide real-world practice insights for 
clinical decision-making in HR+/HER2− advanced breast cancer patients. 
 
2. Study Objectives 
 
This study aims to collect clinical data and case materials of HR+/HER2- advanced breast 
cancer patients treated with CDK4/6 inhibitors combined with endocrine therapy in real-
world clinical practice, evaluate the real-world data of CDK4/6 inhibitors (including but 
not limited to the four currently marketed drugs: palbociclib, abemaciclib, ribociclib and 
dalpiciclib) in HR+/HER2− advanced breast cancer patients and the real-world clinical 
treatment options after drug resistance, so as to provide real-world practice insights for 
clinical decision-making in HR+/HER2− advanced breast cancer patients. 
 
Primary Objectives:  
1. To evaluate the differences in efficacy among different CDK4/6 inhibitors 
combined with endocrine therapy regimens in advanced patients. 
Endpoint: Real-world progression-free survival in the treatment line with 
CDK4/6 inhibitors (rwPFS1)； 
2. To investigate the subsequent treatment regimens and their efficacy received by 
patients in the real world after progression on CDK4/6 inhibitors. 
Endpoint: Real-world progression-free survival in the treatment line after 
CDK4/6 inhibitor resistance (rwPFS2)； 
 Secondary Objectives: 
1. To truly reflect the differences in adverse reactions of different CDK4/6 
inhibitors in clinical practice among the Chinese population through the 
collection of laboratory test results, imaging findings and other examination data； 

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2. To collect tumor samples or peripheral blood specimens from some patients for 
genomics and transcriptomics analyses, so as to lay the foundation for 
translational research on the resistance mechanisms of CDK4/6 inhibitors； 
 
3. Study Design 
 
This study is a single-center, retrospective, observational cohort study. It plans to enroll 
all HR+/HER2- advanced breast cancer patients who were treated in our hospital from 
March 2022 to March 2025 and received CDK4/6 inhibitors during the treatment of 
advanced stage. According to different CDK4/6 inhibitors, the patients will be divided 
into five cohorts: palbociclib cohort, abemaciclib cohort, ribociclib cohort, dalpiciclib 
cohort and other investigational CDK4/6 inhibitor cohort. 

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4. Technical Roadmap 
 
 
 
5. Inclusion and Exclusion Criteria 
 
Inclusion Criteria 
1. Aged ≥ 18 years old. 
2. Patients diagnosed with HR+/HER2- (defined according to ASCO/CAP 
guidelines) advanced breast cancer who were treated in our hospital from 
March 2022 to March 2025. 
3. Received CDK4/6 inhibitors (including but not limited to the four currently 
marketed drugs: palbociclib, abemaciclib, ribociclib and dalpiciclib) for at least 
one cycle as the treatment for advanced stage. 
4. Have complete medical records, including demographic information, 
pathological reports, treatment records, laboratory test results, imaging 
examination reports, etc. 
Exclusion Criteria 
1. Incomplete medical records. 
2. Received only CDK4/6 inhibitors monotherapy. 
3. Received CDK4/6 inhibitors as neoadjuvant therapy or postoperative adjuvant 
consolidation therapy. 
 
6. Data Collection and Statistical Analysis 
 
Referring to the results of previous similar real-world studies, assuming that the median 
progression-free survival (PFS) of CDK4/6 inhibitor treatment is 12 months, the 
expected absolute error is 1.5 months, α=0.05 (two-sided), β=0.2 (statistical power 
80%), the minimum sample size required is calculated to be 220 cases using the sample 
size estimation formula for survival analysis. Considering the loss to follow-up rate 

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during the follow-up period (expected 10%), the final minimum sample size is 
determined to be 245 cases. According to different CDK4/6 inhibitors, the study will be 
divided into five cohorts: palbociclib cohort, abemaciclib cohort, ribociclib cohort, 
dalpiciclib cohort and other investigational CDK4/6 inhibitor cohort. The clinical 
treatment regimens combined with CDK4/6 inhibitors and the treatment regimens after 
CDK4/6 inhibitor resistance in clinical practice will be recorded truthfully.。 
Data Source: The hospital's Hospital Information System (HIS) electronic medical 
records, combined with the imaging department, laboratory department, follow-up 
center and other systems to obtain patients' case data.  
Specimen Source: Residual blood, urine and preserved tumor tissue sections stored 
in our hospital will be collected from patients, and no additional biological sample 
collection will be performed on patients. 
Data and Specimen Collection Methods: A unified data collection form will be 
formulated, and personnel involved in the study will be trained to ensure the accuracy 
and consistency of data and specimen information collection. Data will be entered into 
an electronic database using double data entry and cross-checking methods, and logical 
data verification rules will be set to detect and correct entry errors in a timely manner. 
A data management specialist will be appointed to be responsible for the daily 
management of data, quality verification and communication of doubts. All patient data 
will be anonymized, and access permissions will be set for the database to strictly 
protect patient privacy. Unauthorized disclosure is prohibited. 
Statistical Analysis Methods: SPSS 26.0 and R 4.0.0 statistical software will be used 
for data analysis. 
1. Descriptive Statistics: Measurement data conforming to normal distribution will 
be expressed as mean ± standard deviation (x±s), while those not conforming to normal 
distribution will be expressed as median (interquartile range) [M (Q1, Q3)]. Count data 
will be expressed as frequency (percentage) [n (%)]. 
2. Survival Analysis: Kaplan-Meier method will be used to draw survival curves, 
and the median PFS1, PFS2 and their 95% confidence intervals (CI) will be calculated. 
Log-rank test will be used to compare survival differences among different subgroups 
(such as different age groups, different comorbidities, different treatment regimens after 
drug resistance, etc.). Cox proportional hazards regression model will be used for 
multivariate analysis of independent risk factors affecting survival. 
3. Correlation Analysis: Spearman or Pearson correlation analysis will be used to 
explore the correlation between biomarkers and efficacy as well as drug resistance. 

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4. Safety Analysis: The incidence of adverse reactions and their 95% CI will be 
calculated. Chi-square test or Fisher's exact test will be used to compare the differences 
in the incidence of adverse reactions among different subgroups. 
5. All statistical tests are two-sided, and a P value < 0.05 will be considered 
statisticallysignificant.

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References: 
[1] Siegel RL, Kratzer TB, Wagle NS, Sung H, Jemal A. Cancer statistics, 2026. CA Cancer J 
Clin. 2026 Jan-Feb;76(1):e70043. doi: 10.3322/caac.70043. PMID: 41528114; PMCID: 
PMC12798275. 
[2] Hortobagyi GN, Stemmer SM, Burris HA, et al. Ribociclib as first-line therapy for HR-
positive, advanced breast cancer. N Engl J Med 2016; 375:1738–1748. 
[3] Goetz MP, Toi M, Campone M, et al. MONARCH 3: abemaciclib as initial therapy for 
advanced breast cancer. J Clin Oncol 2017; 35: 3638–3646. 
[4] Finn RS, Martin M, Rugo HS, et al. Palbociclib and letrozole in advanced breast cancer. N 
Engl J Med 2016; 375: 1925–1936. 
[5] Zhang P, Zhang Q, Tong Z, et al. Dalpiciclib plus letrozole or anastrozole versus placebo 
plus letrozole or anastrozole as first-line treatment in patients with hormone receptor-positive, 
HER2- negative advanced breast cancer (DAWNA-2): a multicentre, randomised, double-blind, 
placebo controlled, phase 3 trial. Lancet Oncol 2023; 24: 646–657. 
[6] O’Sullivan CC, Clarke R, Goetz MP, et al. Cyclin-dependent kinase 4/6 inhibitors for 
treatment of hormone receptor-positive, ERBB2-negative breast cancer: a review. JAMA Oncol 
2023; 9: 1273–1282. 
[7] Braal CL, Jongbloed EM, Wilting SM, et al. Inhibiting CDK4/6 in breast cancer with 
palbociclib, ribociclib, and abemaciclib: similarities and differences. Drugs 2021; 81: 317–331. 
[8] Rugo HS, Brufsky A, Liu X, et al. Real-world study of overall survival with palbociclib plus 
aromatase inhibitor in HR+/HER2− metastatic breast cancer. NPJ Breast Cancer 2022; 8: 114. 
[9] Pla H, Felip E, Obadia V, et al. Elderly patients with hormone receptor-positive HER2-
negative metastatic breast cancer treated with CDK4/6 inhibitors in a multicentre cohort. Clin 
Transl Oncol 2024; 26: 1748–1758. 
[10] Rugo HS, Liu X, Li B, et al. Real-world effectiveness of palbociclib plus aromatase 
inhibitors in African American patients with metastatic breast cancer. Oncologist 2023; 28: 866–
87

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Appendix: 
Informed Consent Form  
Dear Patient, 
We sincerely invite you to participate in a study entitled "A Real-World Study on 
CDK4/6 Inhibitors for HR+/HER2- Advanced Breast Cancer and Subsequent 
Therapeutic Options After Drug Resistance". This study will be conducted at Tianjin 
Medical University Cancer Institute and Hospital, with Professor Shi Yehui as the 
principal investigator. After full discussion by an expert panel, the clinical trial 
protocol has been formulated. It has been reviewed and approved by the Institutional 
Review Board of Tianjin Medical University Cancer Institute and Hospital, and the 
clinical study is allowed to be carried out in accordance with the formulated trial 
protocol. This study will strictly comply with the Declaration of Helsinki and the 
relevant laws and regulations of China. 
Before you decide whether to participate in this clinical study, please carefully read 
the following content, which can help you understand the purpose of this study, the 
procedures and duration of the study, as well as the potential benefits, risks and 
discomforts that may be brought to you after participating in the study. You can 
discuss it with your relatives and friends, or consult the doctor in charge of the study, 
who will explain the relevant questions to you and help you make a final decision. 
 
1. Background and Purpose of the Study:  
Since 2020, breast cancer has surpassed lung cancer to become the most incident 
malignant tumor worldwide. Advanced breast cancer is almost incurable, so delaying 
disease progression and improving quality of life are of vital importance. Among 
advanced breast cancer cases, nearly 75% are hormone receptor-positive (HR+) and 
human epidermal growth factor receptor 2-negative (HER2−) molecular subtypes. 
According to clinical guidelines, the standard first-line treatment regimen is cyclin-
dependent kinase 4/6 inhibitors (CDK4/6 inhibitors) combined with endocrine therapy. 

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To date, based on the results of multiple phase III clinical trials, palbociclib, 
abemaciclib, ribociclib and dalpiciclib have been approved by the U.S. Food and 
Drug Administration (FDA) and the National Medical Products Administration 
(NMPA) of China respectively, for the first-line treatment of HR+/HER2− advanced 
breast cancer patients and those with disease progression after prior endocrine therapy. 
However, for different HR+/HER2− advanced breast cancer patients, which CDK4/6 
inhibitor is the optimal first-line treatment option? Which CDK4/6 inhibitor is more 
suitable for patients with progression after prior endocrine therapy? In addition, the 
real-world adverse reactions of different CDK4/6 inhibitors in clinical practice are all 
worthy of exploration as clinical issues. The number of Asian populations in most 
landmark clinical trials of CDK4/6 inhibitors is limited, and the incidence of potential 
grade ≥3 adverse reactions is higher in Asian populations, which also poses 
challenges for selecting the most appropriate CDK4/6 inhibitor for the Chinese 
population. Moreover, despite the significant clinical benefits of these drugs, patients 
will eventually develop drug resistance. At present, there are no high-level guideline 
recommendations for therapeutic options after CDK4/6 inhibitor resistance. Therefore, 
the optimal treatment strategy after progression on CDK4/6 inhibitors has become an 
urgent clinical problem to be solved. In recent years, real-world evidence derived 
from the analysis of real-world data including treatment patterns and outcomes has 
attracted increasing attention. Real-world studies usually cover a broader and more 
realistic patient population, and can generate new analyses and insights after phase III 
studies and the approval of treatment regimens. 
This study aims to collect clinical data and case materials of HR+/HER2- advanced 
breast cancer patients treated with CDK4/6 inhibitors combined with endocrine 
therapy in real-world clinical practice, evaluate the real-world data of CDK4/6 
inhibitors (including but not limited to the four currently marketed drugs: palbociclib, 
abemaciclib, ribociclib and dalpiciclib) in HR+/HER2− advanced breast cancer 
patients and the real-world clinical treatment options after drug resistance, so as to 
provide real-world practice insights for clinical decision-making in HR+/HER2− 

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advanced breast cancer patients. 
 
2. Content and Process of Participating in the Study:  
If you agree to participate in this study, each subject will be assigned a unique 
identification number and a medical record file will be established. During the study, 
we need to collect the imaging data, laboratory test results, pathological specimens 
and other materials stored in our hospital. Your specimens will only be used for the 
clinical research of this project. 
 
3. Potential Benefits of the Study 
The analysis of your clinical case data and specimens will provide real-world data for 
Chinese HR+/HER2− advanced breast cancer patients, as well as the real-world 
clinical treatment options after drug resistance, so as to provide real-world practice 
insights for clinical decision-making in HR+/HER2− advanced breast cancer patients. 
 
4. Risks and Compensation Measures of Participating in the Study: 
For your participation in this study, we will use the residual specimens and tissues 
preserved in our hospital, and no additional sample collection will be performed on 
you. This study will not cause harm to your psychology and social relations, nor will 
it have a negative impact on the diagnosis and treatment of your disease. The entire 
study process will be supervised by the Institutional Review Board of Tianjin Medical 
University Cancer Institute and Hospital. If you have any questions during the study, 
you can consult the researchers. 
 
5. Privacy Issues: 
If you decide to participate in this study, your participation in the trial and your 

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personal data during the trial will be kept confidential. Your medical records will be 
stored in the hospital. To ensure that the study is conducted in accordance with the 
regulations, the competent government departments or members of the Institutional 
Review Board may be allowed to review your personal data at the research site in 
accordance with the regulations when necessary. Any public report on the results of 
this study will not disclose your personal identity. We will make every effort to 
protect the privacy of your personal medical data to the extent permitted by law. At 
any time, you can request to access your personal information and modify it if 
necessary. 
Your signature on this informed consent form indicates that you agree to your 
personal and medical information being used for the purposes described above. Your 
files will be stored in the filing cabinet of Tianjin Medical University Cancer Institute 
and Hospital, and will only be accessible to the researchers. 
Your personal medical data will not be disclosed when the results of this study are 
published. 
 
6. Your Rights: 
Your participation in the trial is completely voluntary. You can withdraw from the 
trial at any time without giving any reason, and it will never affect your relationship 
with medical staff and your future diagnosis and treatment. All your personal data 
and observation records are confidential and will only be used for this study. During 
the trial, you can obtain relevant information at any time. If you have any problems 
or need to consult relevant questions during the trial, you can contact the attending 
physician. 
 
7. Contact Information of the Institutional Review Board: 
If you have any questions related to the rights and interests of the subjects, or want to 

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provide opinions and suggestions related to this study, you can also contact the 
Institutional Review Board of Tianjin Medical University Cancer Institute and 
Hospital. Contact telephone number: ____________. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Signature Page of the Informed Consent Form for  
A Real-World Study on CDK4/6 Inhibitors for HR+/HER2- Advanced Breast 
Cancer and Subsequent Therapeutic Options After Drug Resistance 
Subject's Statement 
The researchers have explained the purpose, process, potential risks and benefits of 
this study to me in detail. I have carefully read the informed consent form, all my 
questions have been answered satisfactorily, and I have fully understood all the 
contents. 
I agree to the researchers collecting and using my medical data. I agree that Tianjin 
Medical University Cancer Institute and Hospital can review my medical data and 
research results in this study for scientific research purposes. I agree that members of 
the Institutional Review Board and representatives of the competent government 
departments can review my medical data in accordance with their respective 
authorities on the premise of abiding by the principle of confidentiality. I understand 
that the review of these records is to ensure that the data collected from this study is 
true, complete and reliable. 
I clearly understand that my participation in this study is voluntary. I can withdraw 
from the study at any time and it will not affect my subsequent medical treatment or 
legitimate rights and interests. 
I have received a signed copy of the informed consent form. By signing this consent 
form, I do not waive any of my legitimate rights and interests. 
I agree to the collection of my imaging data, laboratory test results, pathological 
specimens and other materials stored in Tianjin Medical University Cancer Institute 
and Hospital for this study: 
Yes (Please tick the box) □  
No (Please tick the box) □  

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Subject's Signature: ‗‗‗‗‗‗‗‗‗‗‗ Date: ‗‗‗‗‗‗‗‗‗‗  
Legal Representative's Signature*: ‗‗‗‗‗‗‗‗‗‗‗ Date: ‗‗‗‗‗‗‗‗‗‗Relationship 
with the Subject: ‗‗‗‗‗‗‗  
*A legal representative's signature is not required unless the subject is unable to read 
(e.g., illiterate or blind) or unable to sign by himself/herself for other reasons. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Investigator's Statement 
I promise to strictly abide by the GCP principles, relevant regulations of the state and 
Tianjin Medical University Cancer Institute and Hospital in the clinical study, protect 
the rights and interests of the subjects, and ensure the research time and the 
truthfulness, completeness and reliability of the research data. I agree that Tianjin 
Medical University Cancer Institute and Hospital can review the medical data and 
research results of this study for scientific research purposes. I agree to return the 
remaining biological specimens of the study to Tianjin Medical University Cancer 
Institute and Hospital for preservation.  
I have fully explained the purpose, process, potential risks and benefits of this study 
to the patient. The patient has obtained sufficient information to make a decision to 
participate in this study. I will provide the patient or his/her legal representative with 
a signed and dated copy of the informed consent form.  
  
  
Investigator's Signature: ‗‗‗‗‗‗‗‗‗‗‗‗‗‗ Date: ‗‗‗‗‗‗‗‗‗      
Investigator's Contact Information: ‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗