Patient Derived Organoids (PDOs) to Observe the Clinical Consistency of Personalized Neoadjuvant Therapy for Resectable Esophageal Squamous Cell Carcinoma
Conditions: Esophageal Cancer
Sponsor: Shanghai Zhongshan Hospital

Zhongshan Hospital Fudan University
Observational clinical research protocol initiated by
researchers
Project Name: Patient Derived Organoids (PDOs) to Observe the Clinical Consistency
of Personalized Neoadjuvant Therapy for Resectable Esophageal Squamous Cell
Carcinoma
Research Institution: Zhongshan Hospital affiliated to Fudan
University
Principal Investigator: Lijie Tan
Sponsor: Zhongshan Hospital affiliated to Fudan University
Version Number: V1.3
Version Date: 2025-06-20.

Zhongshan Hospital Fudan University
Investigator's Statement
I
will
conscientiously
fulfill
my
responsibilities
as
a
researcher
by
personally
participating in or directly supervising this clinical study. We have read and confirmed
this protocol, agreeing with its scientific and ethical aspects. We will adhere to relevant
responsibilities as prescribed by Chinese laws and regulations, the Helsinki Declaration,
Chinese GCP, and this study protocol, and it can only be implemented after approval by
the academic committee and ethics committee, unless measures must be taken to protect
the safety, rights, and interests of the subjects. We will keep this study protocol
confidential.
Research Institution: Zhongshan Hospital affiliated to Fudan University
Principal Investigator: Lijie Tan
Summary of the Plan
Project Name
Patient Derived Organoids (PDOs) to Observe the
Clinical Consistency of Personalized Neoadjuvant
Therapy for Resectable Esophageal Squamous Cell
Carcinoma
Research background
Esophageal squamous cell carcinoma is one of the
common malignant digestive tract tumors in China. In
recent years, neoadjuvant therapy for esophageal

squamous cell carcinoma has developed rapidly, but the
efficacy varies significantly among individuals. To further
improve the benefit rate of neoadjuvant therapy, it is
necessary to explore more efficient efficacy prediction
models.
Human tumor organoid models (patient derived
organoids, PDO) are biological models obtained by
culturing patient tumor samples, which can better retain
the original tumor characteristics and interpatient
variability. Existing studies have confirmed the feasibility
of PDO immune co-culture models and their potential for
predicting the efficacy of neoadjuvant personalized
therapy.
Research Objective
Establish a human tumor organoid (PDO) model for
ESCC patients, explore the consistency between patient-
derived PDO models and the clinical efficacy of
neoadjuvant personalized therapy for resectable
esophageal cancer, and evaluate their clinical application
value.
Study Endpoints
Primary Endpoint: Culture formation indicators and
pathological identification indicators of esophageal
squamous cell carcinoma; interpretation of drug
sensitivity results.
Secondary Endpoint: Clinical efficacy evaluation
(reference RECIST1.1)
Research Design
This is a single-center, prospective, observational study
that uses patient-derived human tumor organoid models
(PDO) for drug sensitivity tests and compares them with
actual clinical efficacy to evaluate consistency and
potential clinical application value.
Sample size estimation
A preliminary plan to enroll 30 patients
Inclusion
criteria/Exclusion
criteria/Withdrawal
criteria during the study
See the plan
Expected research
duration
2025-2 to 2025-12
Statistical analysis plan
See the plan
Research background
(1) Research significance
Esophageal cancer is one of the common malignant digestive tract tumors in China.
According to the data from the World Health Organization, the incidence and mortality
rates of esophageal cancer in China in 2022 were 224,000 and 187,000 cases,

respectively, ranking 7th and 5th among all cancers [1]. The esophageal cancer patients
in China are mainly esophageal squamous cell carcinoma (ESCC) [2], and the
recurrence rate is high when treated with radical surgery alone. Therefore,
comprehensive treatment strategies are usually used in the treatment of ESCC to
prolong the recurrence-free survival and overall survival of patients.
Neoadjuvant therapy refers to the comprehensive treatment before tumor resection,
mainly including neoadjuvant radiotherapy, chemotherapy, and immunotherapy. For
most resectable esophageal cancers, neoadjuvant therapy can reduce tumor stage,
increase the rate of R0 resection, and prolong patient survival and improve quality of
life without increasing mortality. Neoadjuvant chemoradiotherapy has been proven to
significantly prolong patient survival and is widely used nationwide. However, the
response to neoadjuvant therapy varies greatly among different patients, and about 40%
of patients experience recurrence [3]. Therefore, how to improve neoadjuvant therapy
and predict the clinical response of patients to individual chemotherapy have become
urgent clinical problems. In recent years, tumor immunotherapy, which activates
immune responses, has developed rapidly, and immunotherapy such as immune
checkpoint inhibitors has been approved as first-line treatment for various solid tumors
(such as melanoma and non-small cell lung cancer) [4]. However, the response to
neoadjuvant immunotherapy varies significantly among different ESCC patients, and
only tumors with rich lymphocytes and high PD-L1 expression can benefit significantly
from the treatment, accounting for only about 30% of all ESCC patients [5]. Currently,
the main method to determine whether patients can benefit from neoadjuvant therapy is
gene sequencing or immunohistochemistry, which has relatively low predictive
accuracy. To further improve the benefit rate of neoadjuvant therapy, we urgently need
to explore more efficient efficacy prediction models.
Organoids are 3D stem cell-derived tissue cultures that can self-organize into
isolated "mini-organs." Human tumor organoid models (patient derived organoids,
PDO) are biological models obtained by culturing patient tumor samples, which can
better retain the original tumor characteristics and interpatient variability, with structural
and genetic integrity, and can be long-cultured and form biological sample banks [6].
Studies have shown that PDOs greatly exceed traditional cancer cell lines in accurately
reproducing 3D tumor structure, heterogeneity, and drug response [7,8]. Based on this
property, in the future era of precision medicine, PDOs will have broad application
prospects, such as individualized prediction of drug efficacy, investigation of drug
resistance mechanisms, and potential translational research.
(2) Current research status at home and abroad
PDO models and human tumor xenograft models (patient derived xenografts, PDX)
are currently the most cutting-edge tools for predicting the efficacy of tumor drugs.
PDX largely maintain the original histological and genetic characteristics of patient
tumor cells, offering potential for detecting biomarkers and predicting drug efficacy.
However, PDX also has certain disadvantages; on one hand, its tumors are mixed with
some mouse-derived cells, leading to differences between PDX and the primary tumor
in terms of genomics and gene expression profiles; on the other hand, PDX modeling is
time-consuming, has a low success rate, and is costly, which limits its application in
clinical efficacy prediction [9]. In contrast, PDO not only retains the characteristics of
human tumor cells but can also be co-cultured with the patient's autologous immune

cells, thereby simulating the interaction between immune cells and tumor cells in vivo
[6], more accurately predicting the clinical efficacy of drugs.
Currently, there are two main types of immune co-culture models supported by
technology. The first is to culture tumors together with the in situ immune cells within
the tumor. The advantage of this method is that it retains all immune cells, including
exhausted T cells. However, since the culture medium required for immune cells and
PDO is different, immune cells cannot maintain their activity for long periods, so this
model is more suitable for investigating treatment strategies that take effect in the short
term [10]. The second method involves culturing PDO and peripheral blood immune
cells separately. The advantage of this method is that it can maintain the activity of
immune cells for longer periods and can continuously amplify and add activated
autologous immune cells outside the PDO [10]. Evidence suggests that this model can
obtain tumor-reactive CD8+ T cells after co-culture for 14 days [11].
Currently, there are multiple studies that have successfully established such models
[12–14], and some research has already validated that the PDO and peripheral blood
immune cell co-culture model can well predict the efficacy of neoadjuvant immune
combined with chemotherapy regimens in diseases such as gastric cancer [15],
colorectal cancer [16], and skin cancer [17]. However, there are currently no similar
studies in esophageal cancer, which may be due to the relatively greater difficulty in
culturing squamous epithelial cells, and this is one of the innovations of this study.
The main purpose of this study is to establish PDO models of tumors from ESCC
patients and explore the consistency between the PDO model and the clinical efficacy of
patient's neoadjuvant personalized therapy, as well as to use the PDO model to explore
possible drug mechanisms and resistance mechanisms, providing precise guidance for
efficacy prediction and personalized medication in future neoadjuvant immunotherapy
and chemotherapy.
Research Objective
1. Main research objectives
Establish human-derived tumor organoids (PDO) models for ESCC patients,
comparing the pathological and genetic features of PDOs with corresponding tumor
tissues for consistency.
2. Secondary research objectives
Construct an immune co-culture model for esophageal squamous cell carcinoma
PDOs, by comparing the drug sensitivity results of different chemotherapy immune
combination regimens and their consistency with clinical efficacy, to evaluate the
feasibility of using the esophageal squamous cell carcinoma organoid immune co-
culture model to guide the selection of clinical chemotherapy immune combination
regimens.
Exploratory research objectives
Investigating the possible resistance mechanisms of esophageal cancer to
neoadjuvant personalized therapy using patient PDO and immune co-culture models.
Research overview
This study is a single-center, prospective, observational study that uses patient-
derived human tumor organoid models (PDO) for drug sensitivity tests and calculates

consistency by comparing actual efficacy, thereby evaluating its clinical application
value.
Enrolled patients should be confirmed by endoscopic biopsy histology and
evaluated for tumor resectability by CT or PET/CT scans. Enrolled patients should have
an ECOG performance score ≤1 and no significant organ dysfunction.
The tumor biopsy samples collected by endoscopy will be used for PDO modeling,
and blood samples will be used to construct an immune co-culture model and perform
drug sensitivity experiments. Patients will be grouped for data analysis based on drug
sensitivity test results and compared with the final disease remission status of the
patients.
Enrolled patients will receive neoadjuvant therapy, and the specific treatment plan
will be determined by the treating physician. This study does not interfere with clinical
treatment plans.
Patients will receive the following follow-up:
(1) Safety follow-up: Adverse reactions related to the treatment drug within 90 days
after the last dose of medication, followed up every 30 days, including survival status,
adverse reactions, concomitant medications, and concomitant treatment information.
(2) Survival follow-up: Every 3 months, collect survival information and data on
antitumor treatment and disease progression time after the study ends, until the
participant dies, is lost to follow-up, or the study is terminated.
Design roadmap
Research population
1. Data source
All patients enrolled in this study were from Zhongshan Hospital, histologically
confirmed esophageal squamous cell carcinoma patients who were reviewed and
considered resectable by two experienced thoracic surgeons.
2. Inclusion/Exclusion/Withdrawal criteria
Inclusion criteria

Patients with squamous cell carcinoma confirmed by gastroscopic biopsy pathology, and
who have gastric endoscopy tissue and blood samples collected at this center before
treatment;
The primary tumor is located in the chest, and the primary site of esophageal cancer is
determined by the center of the mass in the esophagus (upper esophageal segment: from
the thoracic inlet to the lower margin of the aortic arch level, endoscopic examination
distance from the incisor is 20 cm to <25 cm; middle esophageal segment: from the
lower margin of the aortic arch to the level of the inferior pulmonary vein, endoscopic
examination distance from the incisor is 25 cm to <30 cm; lower esophageal segment:
from the level of the inferior pulmonary vein to the stomach, endoscopic examination
distance from the incisor is 30 cm to <40 cm)
Based on the above examinations, preoperative clinical assessment can be performed for
esophageal cancer patients who can undergo surgical resection (Whether the tumor had obvious
external invasion, mediastinal lymph node enlargement, and distant organ metastasis were
evaluated. If the primary tumor is suspected to be T4b, multiple mediastinal lymph node
metastases, or distant metastases, whole-body PET-CT and endoscopic ultrasonography (EUS) ,
or other optional examinations to further confirm the clinical stage, should be performed.
)；
Age ≥18 years, ≤75 years, ECOG performance status 0-1 (see Appendix 1), expected
survival ≥12 months;
Subjects must not have major organ dysfunction, and basic normality in complete blood
count, lung, liver, kidney function, and cardiac function. Laboratory test values must
meet the following requirements: Blood: White blood cells > 4.0×10^9/L, absolute
neutrophil count (ANC) ≥2.0×10^9/L, platelet count > 100×10^9/L, hemoglobin >
90g/L; Lung function: FEV1 ≥1.2L, FEV1% ≥50% and DLCO ≥50%. Note: FEV1
(L): Forced vital capacity (FVC) measured value (liters). FEV1%: FVC measured
value/estimated value (%). DLCO%: Carbon monoxide single-breath diffusion capacity
measured value/estimated value (%); Liver function: Serum bilirubin less than 1.5 times
the maximum normal value; ALT and AST less than 1.5 times the maximum normal
value. Kidney function: Serum creatinine (SCr) ≤120µmol/L, creatinine clearance
(CCr) ≥60ml/min
Have legal capacity, able to understand the situation of this study and sign an informed
consent form.
2.2 Exclusion criteria
Exclusion criteria related to cancer:
Patients with clinical stage T4b who are not resectable (determined by senior thoracic
surgeons), have multiple lymph node enlargements (estimated lymph node metastasis ≥
6), multiple lymph node stations (estimated number of lymph node metastasis stations ≥
3), or distant metastasis (M1) as determined by imaging studies such as contrast-
enhanced CT of the chest and abdomen, neck lymph node ultrasound, whole-body PET-
CT (optional), or EBUS (optional) (AJCC/UICC 8th Edition);
Patients who have already received or are currently receiving other chemotherapy,
radiotherapy, or targeted therapy;
Patients with non-squamous cell carcinoma on endoscopic examination pathology;

A history of other tumors (previous cervical in situ carcinoma or local basal cell
carcinoma of the skin that has been cured is excluded);
Other exclusion criteria:
History of autoimmune diseases;
Use of hormones or immunosuppressants recently or currently;
Has previously received immunotherapy;
Has a history of severe hypersensitivity to antibody drugs;
Has a history of or is currently suffering from chronic or recurrent autoimmune diseases;
Interstitial lung disease, pulmonary fibrosis, diverticulitis, or systemic ulcerative
gastrointestinal inflammation;
A confirmed history of congestive heart failure; angina with poor medication control;
transmural myocardial infarction confirmed by electrocardiogram (ECG); poorly
controlled hypertension; clinically significant valvular heart disease; or uncontrolled
high-risk arrhythmia;
Severe uncontrolled systemic intermittent diseases, such as active infection or poorly
controlled diabetes; coagulation disorders, with a tendency to bleed or receiving
thrombolytic or anticoagulant therapy;
Women who are serum pregnancy test positive or breastfeeding, as well as fertile men
and women who are unwilling to use adequate contraception during the study of drug
treatment;
Known active infection with human immunodeficiency virus (HIV), hepatitis B virus
(HBV), or hepatitis C virus (HCV), or known HIV seropositivity, including positive
surface antigen for HBV or HCV (RNA);
Known allergy to any research drug;
History of organ transplantation (including autologous bone marrow transplantation and
peripheral stem cell transplantation);
Presence of peripheral neuropathy or history of significant psychiatric or central nervous
system disorders;
Concomitant use of antineoplastic drugs outside the research protocol;
Based on the researcher's judgment, there are other factors that may lead to the forced
termination of the study, such as having other serious illnesses (including mental illness)
requiring combined treatment, alcoholism, drug abuse, family or social factors;
2.3 Withdrawal criteria (if applicable)
Participants can withdraw informed consent at any time for any reason. If an
adverse event occurs, the researcher can decide to terminate the study treatment for the
participant. Additionally, if the participant does not meet the inclusion and exclusion
criteria, violates the trial plan, or due to management and/or other safety reasons, the
researcher can terminate the treatment and disqualify the participant.

Terminating study treatment does not mean the patient has withdrawn from this trial,
as long as the participant has not withdrawn informed consent to refuse further follow-up,
the participant still needs to continue to complete the remaining study visits according to
the protocol requirements after termination of treatment. The main reasons for the
participant's termination of treatment are recorded in the CRF.
If any of the following situations occur, the subject must terminate treatment but
continue follow-up:
The subject withdraws informed consent or refuses to continue receiving the original
drug regimen treatment;
The investigator judges that the subject's clinical symptoms worsen or physical
condition declines;
Imaging disease progression/recurrence or metastasis, unless the trial group subject
meets the criteria for continued treatment after disease progression and agrees to
continue treatment;
Occurrence of intolerable adverse events, or other events affecting the subject's safety,
including but not limited to the occurrence of any clinical AE, laboratory abnormalities,
or other medical conditions;
Significant protocol deviation;
Other situations deemed necessary to terminate research drug treatment by the
investigator;
Subject's request;
Pregnancy;
Initiation of other systemic antitumor therapy;
Subjects will withdraw from the study if any of the following occur:
Subject withdraws informed consent, or refuses further follow-up;
Lost to follow-up;
Death;
Study termination;
Other reasons considered by the investigator as necessary to withdraw from the study;
When an eligible subject is found to be 不合格after enrollment, the investigator
must discuss with the principal investigator (sponsor) whether the subject should
continue or withdraw from the study.
Study Grouping
This study is observational, and no patient grouping or intervention will be
implemented during the treatment phase. Grouping will be conducted during the data
analysis phase, primarily based on the drug sensitivity test results of PDO for data
grouping and statistical analysis.
Study Endpoints

Primary research endpoint
Culture formation indicators and pathological identification indicators of esophageal
squamous cell carcinoma; interpretation of drug sensitivity results: the maximum
inhibition rate of the measured drug.
Secondary research endpoint
Clinical efficacy evaluation: (to be performed according to the RECIST 1.1 version
for solid tumor response evaluation criteria)
Efficacy evaluation
Efficacy evaluation
Clinically effective
CR, PR, SD
Clinically Ineffective
PD
Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 version:
Complete Response (CR): All target lesions disappeared;
Partial Response (PR): Sum of the longest diameters of baseline target lesions decreased
by ≥30%;
PD: Sum of baseline lesion longest diameters increased by ≥20% or new lesions
appeared
SD: Sum of baseline lesion longest diameters decreased but not reaching PR or
increased but not reaching PD
Safety endpoint
Adverse events: Any adverse medical event experienced by a patient or clinical
trial subject after receiving a drug, which may not necessarily have a causal relationship
with the treatment. The incidence and severity (including serious adverse events and
immune-related adverse events) are determined according to the National Cancer
Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) 5.0 standard
(see Appendix 2).
Research Steps
1. Screening Phase:
After the patient is admitted, medical history collection, physical examination, and
relevant ancillary examinations (see inclusion/exclusion criteria) must be completed
within 2 weeks to determine if the patient meets the inclusion criteria. For patients who
meet the inclusion criteria, the doctor must introduce the details of the trial, and after the
patient consents, they must sign the "Informed Consent Form". After completing all
screening items, the researcher must review the results/data, and only after passing the
review can the subject be enrolled.
1) Record the patient's basic information, current medical history, past medical history,
physical examination, vital signs, and BMI index;
2) Record pre-treatment complete blood count, liver function, renal function, blood
biochemistry, tumor markers (CEA, CA19-9, CA125, CYFRA21-1, and SCC, the same
below), cardiopulmonary status, enhanced CT of the chest and abdomen, ultrasound of
cervical lymph nodes, PET-CT, EUS, etc.;

3) Collect pre-treatment gastroscopic biopsy samples, and possibly 1 blood sample
(10ml) depending on the organoid culture conditions and the patient's condition, for
PDO culture and other exploratory research.
4) Collect post-treatment pathological tissue samples for exploratory research.
2. Organoid modeling:
PDGF culture method
Collect surgical tissue, digest, wash, and resuspend in organoid culture medium;
Add matrix gel, mix until non-segregated, then inoculate into culture dishes and place in
a 37℃constant temperature incubator for fixation;
After fixation, add organoid culture medium and place in a 37℃constant temperature
incubator.
Change the culture medium every 2-3 days until ESCC organoids are grown.
Tumor organoid model identification
Collect appropriate organoid samples and perform consistency identification with the
source tissue;
Screen appropriate organoids for paraffin embedding, sectioning, HE staining, and
immunohistochemical staining, and compare with the pathology and
immunohistochemistry of the source tissue.
2) Culture of Tumor-Invading Lymphocytes (TIL)
Collect tumor tissue, take about 0.1g, wash and then cut the tissue into small pieces of
about 1mm³;
The small tissue pieces are transferred separately to different wells of a 24-well plate
and immune cell induction medium is added;
After observing the amplification of immune cells, filter or sort using a sieve, enrich the
immune cells, and continue the culture;
After increasing to a sufficient number of immune cells, freeze for the next experiment.
3) Culture of peripheral blood lymphocytes (PBMC)
Collect blood samples, use EasySepTM Direct Human PBMC Isolation Kit to isolate
PBMCs;
First perform organoid culture;
The organoids after cultivation are digested into small clusters using TrypLE, and the
organoids are mixed with PBMCs at a certain ratio for co-culture.
4) Drug sensitivity test
According to the experimental design, co-culture of immune cells and the addition of
immune combination chemotherapy drugs are performed.
Through microscopy and immunohistochemical staining, the sensitivity of the co-
culture model to immune combination chemotherapy drugs is detected.

In the organoid drug sensitivity test, one or more of the following drugs are planned to
be used:
Paclitaxel Injection
Albumin-bound Paclitaxel Injection
Cisplatin Injection
Cisplatin Injection
Nivolumab
Pembrolizumab
Trastuzumab
Tislelizumab
Camrelizumab
The specific drug and its combination to be used should refer to the clinical
treatment plan that the patient may adopt. The final plan will be decided jointly by
the principal investigator and the responsible physician.
3. Follow-up period:
Safety Follow-up: Adverse reactions related to the treatment drug within 90 days
after the last dose of medication, followed up every 30 days, including survival status,
adverse reactions, concomitant medications, and concomitant treatment information.
Survival Follow-up: The first follow-up 30 days after surgery, and then every 3
months, collecting survival information and data on antitumor treatment and disease
progression time after the study ends, until the participant dies, is lost to follow-up, or
the study is terminated.
Data Governance/Data Management Plan
All cases must complete the case report form (CRF) or EDC.
1. Filling and Handing Over of Case Report Forms (CRFs):
All relevant information for the cases is recorded by the treating physician in the
CRF according to the trial protocol, ensuring that the data is recorded promptly,
complete, accurate, and truthful. The CRF should generally not be altered; if there is a
definite need for correction, the person making the change must sign and date it.
Completed CRFs must be reviewed and signed by the clinical researcher at each center,
and then reviewed by the monitor dispatched by the sponsor. If there are any questions,
the researcher must decide whether to make changes, and once confirmed, no further
modifications can be made on the CRF. The CRF is in triplicate; after the trial ends, one
copy is retained by the trial hospital, one is given to the clinical lead hospital, and one is
given to the data administrator for data entry management. After data management and
statistical analysis are completed, it is returned to the sponsor. All processes must be
documented.
2. Data Entry and Modification:

The designated data administrator is specifically responsible for this process. A
dedicated database system for this trial is established. After training the data entry
personnel, data entry is performed using the double-entry method, with two individuals
completing it independently.
3. The emergence and resolution of questions:
When the data manager conducts a comprehensive check, data entry, or data review
of the CRF, and has questions, they must fill out a question form and return it to the
monitor. The investigator is required to provide written answers to the questions in the
question form and sign it, before returning it to the data manager to correct the data. The
data manager is not authorized to modify the original data. The question form should be
properly stored.
4. Data review:
The data manager should report on the data management process and general
situation, the enrollment and completion of cases (including a list of dropouts), the items
involved when determining the statistical analysis population, and the issues to be
discussed and resolved (such as inclusion/exclusion criteria review, completeness check,
logical consistency check, outlier data check, time window check, concomitant
medication check, adverse event check, etc.), and it should be confirmed by the
principal investigator, sponsor, and statistician.
5. Database Locking:
After the data has been reviewed and confirmed to be correct, the database will be
locked by the principal investigator, sponsor, and statistician. Once the database is
locked, it should not be modified in principle. If there is indeed a problem, corrections
can only be made in the statistical analysis program after confirmation by the leading
unit. All modifications must be documented and explained. Data security must be
ensured, and unauthorized personnel should not have access to or modify the data. The
data must be backed up.
6. Privacy Concerns:
To ensure strict protection of patient privacy, all research procedures will exclusively
utilize unique research identifiers (Research ID) to replace any personally identifiable
information. Upon screening and eligibility confirmation, each participant will be
assigned a sequential, alphanumeric Research ID (e.g., ESCC-PDO-001), which will
serve as the sole identifier throughout the study and be recorded on all documents,
biological sample containers, and imaging records, with no patient names or contact
information linked to data unless required by regulatory authorities. All clinical data,
including tumor stage, treatment response, and adverse events, will be documented
under the Research ID, while biological samples like tissue biopsies and blood samples
will be stored in containers marked with the ID, with sample tracking systems using the
ID to link samples to clinical data without referencing identities. The informed consent
form will include the patient’s name for legal purposes but will be cross-referenced
with the Research ID in a secure database, stored separately from study data.
For analysis and reporting, all statistical analyses will use the Research ID to aggregate
results, with group comparisons based on ID-linked data to prevent individual
identification, and research findings will be reported in aggregate form without

disclosing case-specific data that could reveal identities. Privacy controls will include
access restrictions for authorized personnel with confidentiality agreements, physical
and digital data storage security measures, and the conversion of data shared with
external collaborators to anonymous datasets. The research team will also conduct
annual audits to ensure compliance with privacy regulations, verifying no personal
information is included in study materials. This approach ensures all research activities
remain anonymous, with the Research ID serving as a non-identifying link between
data points to uphold patient confidentiality while maintaining scientific rigor.
Consideration of Bias
1. Selection Bias: The potential correlation between modeling success rate and patient
response to treatment may cause selection bias. Some studies have shown that patients
with poor treatment response have tumors with relatively stronger stress resistance,
making it more likely that PDO modeling will be successful [18]. This means that the
patients whose PDO cultures ultimately succeed and are included in the analysis have
been screened, which may introduce selection bias. We will statistically analyze the
correlation between modeling success rate and tumor responsiveness and discuss its
impact in the results.
2. Confounding Bias: Uneven distribution of characteristics such as age, gender, and
tumor stage among included patients may lead to confounding bias. We will conduct
statistical tests for confounding factors on the sensitive and insensitive groups predicted
by the PDO drug sensitivity test, aiming to minimize the impact of confounding factors.
Statistical analysis plan
1. Sample Size Estimation

The main objective of this study is to explore the predictive accuracy of the PDO
model for drug sensitivity experiments on PD-1 antibody immunotherapy, specifically
investigating whether the model has statistical significance.
This research project is primarily aimed at subsequent modeling and scientific
exploration, therefore the sample needs to meet the requirements for modeling and
testing. Based on the budget support and the duration of the research, this study plans to
successfully culture 20 patient-derived organoids for subsequent consistency analysis.
Assuming the PDO predicts sensitivity and specificity to be both 80%, according to the
research, the clinical response rate of ESCC patients to PD-1 antibody combined with
chemotherapy is approximately 58.3% [19. Based on the above data, calculate the
sample size. Perform a four-fold table exact Fisher test:
Clinical Efficacy
Total
Effective
Ineffectiv
e
PDO
Predictio
n Group
Sensitive
10
2
12
Insensitiv
e
2
6
8
Total
12
8
20
Fisher's exact test results: bilateral P=0.0194, which can validate the statistical
significance of the PDO prediction results.
Since the modeling success rate for esophageal squamous cell carcinoma is
approximately 71.4%18, a preliminary plan is to include 30 patients. If the modeling
success rate of PDO, the drug sensitivity test success rate, and the expected results do

not match during the subsequent research process, the researcher needs to appropriately
adjust the expected number of patients to be enrolled.
2. Dataset Definition
The safety dataset includes all enrolled patients, including those who completed the
experiment and those who withdrew due to reaching safety endpoints or 中途退出, and
did not complete the experiment. The effectiveness dataset only includes patients whose
PDO culture was successful and who successfully underwent drug sensitivity testing.
3. Handling of Missing Data
For patients with missing data, assess whether the missing data include the primary
endpoint. If the missing data do not include the primary endpoint or the missing data do
not significantly affect the main analysis, the patient should not be excluded. If the
missing data significantly affect the main analysis, the patient should be excluded.
4. Descriptive Analysis
Descriptive analysis primarily focuses on the "sensitive" and "insensitive" groups
of the PDO drug sensitivity test, statistically analyzing confounding factors such as age,
gender, stage, etc., in both groups, and performing statistical tests; it also statistically
analyzes and tests the primary and secondary endpoints between the two groups.
Primary Analysis
Statistical analysis of drug sensitivity results of organoids and clinical gold
standard in the form of a contingency table, calculate clinical sensitivity, clinical
specificity.
Clinical Efficacy Imaging
Interpretation (Gold Standard)
Total
Valid Invalid
Organoid drug
sensitivity result
interpretation
Sensitive
a b
a+b (R1)
Insensitiv
e
c d
c plus d (R2)
Total
a plus c (C1) b plus d (C2)
a plus b plus c
plus d (N)
Clinical sensitivity (%) = [ a / (a + c) ] × 100%
Clinical specificity (%) = [ d / (b + d) ] × 100%
Consistency (%) = [ (a + d) / ( a + b + c + d) ] × 100%
Kappa value analysis: kappa =
N( a+d)
−(RC+RC)
N−(RC+RC)
All statistical tests are two-tailed. A P value less than 0.05 will be considered statistically
significant. The confidence interval (CI) is 95%.
Quality Control
1. Laboratory quality control measures: The laboratory establishes standard operating
procedures and quality control programs. Special examination items must be handled by

dedicated personnel;
2. Researchers participating in clinical trials must have professional expertise,
qualifications, and capabilities in clinical trials, undergo qualification review, and
require relatively fixed personnel;
3. Trial participants receive training on the trial protocol before the trial starts, enabling
trial personnel to have a full understanding and recognition of the clinical trial protocol
and its specific connotations of each indicator.
Research-related ethics
1. Ethics committee review
This protocol and written informed consent forms, as well as materials directly
related to the subjects, must be submitted to the ethics committee and may only be
formally initiated upon written approval from the ethics committee. The researcher must
submit a continuing review report one month before the expiration of the ethics approval
letter to apply for an extension of the approval. Upon termination and/or completion of
the study, the researcher must notify the ethics committee in writing; the researcher must
promptly report all changes that occur in the research work to the ethics committee
(such as revisions to the protocol and/or informed consent forms) and may not
implement these changes until they receive approval from the ethics committee, except
for changes made to eliminate obvious and direct risks to the subjects. In such cases, the
ethics committee will be notified.
2. Informed consent and the process of obtaining informed consent
Researchers must provide an easily understandable informed consent form
approved by the ethics committee to the participant or their legal representative, and
give the participant or their legal representative sufficient time to consider this research.
No participant may be enrolled before obtaining a signed written informed consent form
from the participant or their legal representative. During the participant's involvement,
all updated versions of the informed consent form and written information will be
provided to the participant. The informed consent form should be kept as an important
document of the clinical trial for reference.
Confidentiality Measures
The results of this project research may be published in medical journals, but we
will keep the personal information of patients confidential in accordance with legal and
regulatory requirements, unless required by relevant laws, the personal information of
patients will not be disclosed. When necessary, government administrative departments,
hospital ethics committees, and relevant personnel may review patient information in
accordance with regulations.
Expected Schedule and Completion Date of the Research Project
2025/02 – 2025/09 Completion of patient enrollment and sample collection, model
construction and testing
2025/09 – 2025/12 Completion of data analysis and reporting
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