The Efficacy of Watch and Wait Strategy or Surgery After Neoadjuvant Immunotherapy for Locally Advanced Colorectal Cancer With dMMR/MSI-H Guided by MRD Dynamic Monitoring(WINDOW): A Single-center, Open-label, Prospective, Phase II Clinical Trial.
Conditions: Clinical Complete Response Rate With Negative MRD
Sponsor: Yunnan Cancer Hospital

The Efficacy of Watch and Wait Strategy or Surgery 
After Neoadjuvant Immunotherapy for Locally 
Advanced Colorectal Cancer with dMMR/MSI-H 
Guided by MRD Dynamic Monitoring(WINDOW): 
A Single-center, Open-label, Prospective, Phase II 
Clinical Trial. 
 
 
 
study protocol 
 
 
 
Protocol version number: V1.0 
Version date: July 02, 2023 
 
 
 
 
 
 
Study director: Associate senior doctor Zhang xuan,  
Chief physician Li Yunfeng 
Research unit: Yunnan Provincial Cancer Hospital 
 
 

Scheme signature page 
I, as the physician / statistical analyst involved in the study, have read the protocol 
for this study. 
I have fully discussed the purpose of the study and the content of the protocol. 
I agree to conduct the study under this protocol, to comply with the ethics 
requirements, and to conduct this clinical study under the guidance of Good Clinical 
Practice (GCP). 
I agree that the contents of this protocol are confidential and will not be disclosed 
to third parties, and the contents of the protocol are only treatment for this study. 
I understand that if the decision to prematurely terminate or suspend it is made 
at any time and for any reason, I will be informed in writing. Similarly, if I decide to 
withdraw from the study, I will immediately notify the study leader and the principal 
investigator. 
 
 
 
Name：________________                              
Signature：________________ 
Date：________________ 
 

Protocol summary 
research topic 
The Efficacy of Watch and Wait Strategy or Surgery After 
Neoadjuvant Immunotherapy for Locally Advanced Colorectal 
Cancer with dMMR/MSI-H Guided by MRD Dynamic 
Monitoring(WINDOW): 
A 
Single-center, 
Open-label, 
Prospective, Phase II Clinical Trial 
version number 
V1.0 
Version date 
July 02, 2023 
by stages 
Il designated time 
Bid unit 
Yunnan Provincial Cancer Hospital 
Principal 
investigator 
Associate senior doctor Zhang xuan, Chief physician Li Yunfeng 
Participating in 
research units 
Single center 
Subject  
investigated 
dMMR/MSI-H Locally advanced colorectal cancer 
research design 
This study is a single arm, single center, phase II, prospective 
clinical study aimed at exploring the effectiveness and safety of 
watch and wait strategy guided by dynamic MRD monitoring to 
achieve 
clinical 
complete 
response 
after 
neoadjuvant 
immunotherapy for locally advanced colorectal cancer with 
dMMR/MSI-H. 
Study duration 
The study is scheduled to begin in July 2023 
Study objective 
/ endpoint 
Main end point  
MRD-negative clinical complete response (cCR). 
Secondary end point 
(1) Optimal number of neoadjuvant immunotherapy cycles. 
(2) Consistency ratio between MRD-negative cCR rate and cCR 
rate assessed by traditional methods. 
(3) 3-year local recurrence/relapse rate. 
(4) 3-year disease-free survival (DFS). 
(5) 3-year overall survival (OS). 

(6) Objective response rate (ORR). 
(7) Tumor downstaging rate (mrTRG). 
(8) Incidence of immune-related adverse events. 
Exploratory endpoints 
(1) Clearance rate of ctDNA in blood MRD-positive patients 2 
weeks after completion of neoadjuvant immunotherapy. 
(2) High-risk period for MRD conversion or relapse in blood 
MRD-negative patients 2 weeks after completion of 
neoadjuvant immunotherapy. 
(3) Dynamic changes and change rates of MRD and their 
correlation with treatment efficacy. 
(4) Detection sensitivity, specificity, positive predictive value, 
and negative predictive value of individualized MRD 
detection protocols (tumor-informed). 
(5) Other multi-omics exploratory analysis results. 
study 
population 
Inclusion criteria: 
1) Disease Characteristics 
• 
Histologically confirmed colorectal adenocarcinoma. 
• 
Immunohistochemically confirmed as dMMR or/and pCR 
or/and NGS as MSI-H. 
• 
Locally advanced colorectal cancer (stage II-III, cT3-4 and/or 
N+) assessed accor detected ding to the UICC/AJCC TNM 
staging system (8th edition, 2017). 
*Clinical staging method: Colon staging by CT, rectal staging 
by pelvic MRI combined with endorectal ultrasonography. 
• 
No signs of intestinal obstruction; or obstruction relieved 
after proximal colostomy. 
• 
No distant metastasis confirmed by comprehensive 
examination (distant organ or/and distant lymph node 
metastasis). 
2) Patient Characteristics 
• 
Age ≥ 18 years and ≤ 75 years at the time of signing the 
informed consent form. 

• 
Eastern Cooperative Oncology Group (ECOG) performance 
status score ≤ 1, with no deterioration within 2 weeks before 
enrollment, and a life expectancy of not less than 12 weeks. 
• 
Hematology: WBC > 4000/mm³; PLT > 100,000/mm³; Hb > 
10g/dL. 
• 
Liver function: SGOT and SGPT less than 1.5 times the 
normal value; bilirubin less than 1.5 mg/dL. 
• 
Renal function: creatinine < 1.8 mg/dL. 
• 
No other malignant diseases (except non-melanoma skin 
cancer or cervical carcinoma in situ) within 5 years or 
concurrently. 
• 
No psychiatric disorders that would prevent informed 
consent. 
• 
No other severe diseases that would shorten survival. 
• 
Female patients of childbearing age should adopt appropriate 
contraceptive measures from screening to 3 months after 
cessation of study treatment and should not breastfeed. 
Pregnancy test should be negative before treatment starts, or 
one of the following criteria should be met to prove no risk 
of pregnancy: 
• 
Postmenopausal defined as age > 50 years and amenorrhea 
for at least 12 months after stopping all exogenous hormone 
replacement therapy. 
• 
Women < 50 years who have stopped all exogenous hormone 
therapy for 12 months or more and have luteinizing hormone 
(LH) and follicle-stimulating hormone (FSH) levels within 
the postmenopausal reference range in the laboratory can also 
be considered postmenopausal. 
• 
Those who have undergone irreversible sterilizing surgery, 
including hysterectomy, bilateral ovariectomy, or bilateral 
salpingectomy, except for bilateral tubal ligation. 
• 
Male subjects should use barrier contraception (i.e., condom) 
from screening to 3 months after cessation of study treatment. 

• 
Patients and their families can understand the research 
protocol and are willing to participate in this study, signing a 
written informed consent form. 
• 
Patients have good compliance and are willing to receive 
follow-up, treatment, laboratory tests, and other research 
steps as planned. 
3) Prior Treatments 
• 
No prior colorectal cancer surgery. 
• 
No prior chemotherapy, immunotherapy, or radiotherapy. 
• 
No prior biological therapy. 
• 
Prior endocrine therapy: no restrictions. 
4) Exclusion Criteria: 
• 
Tumor biopsy specimens showing pMMR or microsatellite 
stability (MSS) by immunohistochemistry or microsatellite 
instability testing. 
• 
Patients with histological types of colorectal cancer other 
than adenocarcinoma (such as neuroendocrine carcinoma, 
sarcoma, lymphoma, squamous cell carcinoma, etc.). 
• 
History of HIV infection or active chronic hepatitis B or C 
(high viral DNA copy). 
• 
Autoimmune diseases. 
• 
Other active clinically significant infections (> NCI-CTC 3.0 
version). 
• 
Patients with clinical stage I disease. 
• 
Preoperative evidence of distant metastasis, including 
isolated, 
distant, 
or 
non-contiguous 
intra-abdominal 
metastases. 
• 
Open surgical procedures ≤ 14 days before enrollment, 
excluding colon surgery. 
• 
Unable to provide surgical tissue for WES testing for 
customized personalized MRD detection panel or patients 
with failed customization of the personalized MRD detection 
panel. 

• 
Unable to provide blood samples for MRD testing at 
treatment and follow-up monitoring points. 
• 
Cachexia, organ dysfunction, or decompensation. 
• 
History of pelvic or abdominal radiation therapy. 
• 
Patients requiring treatment for seizures (e.g., steroid or anti-
epileptic therapy). 
• 
Chronic inflammatory bowel disease, intestinal obstruction. 
• 
Other severe diseases that, in the opinion of the investigator, 
may affect follow-up and short-term survival. 
• 
Patients with a history of blood transfusion within 2 weeks 
before treatment. 
• 
Unable to undergo clinical follow-up using contrast-
enhanced magnetic resonance imaging (MRI) or contrast-
enhanced computed tomography (CT). 
• 
Prior use of anti-tumor traditional Chinese medicine. Patients 
who have used anti-tumor traditional Chinese medicine for 
no more than 7 days and have stopped for 2 weeks or more 
before enrollment can be included. 
• 
Evidence of severe or uncontrollable systemic diseases (e.g., 
severe mental, neurological diseases, epilepsy or dementia, 
unstable or decompensated respiratory, cardiovascular, liver, 
or kidney diseases, left ventricular ejection fraction (LVEF) 
< 50%, uncontrolled hypertension [i.e., hypertension grade 3 
or higher after medication]). Patients with swallowing 
dysfunction, active gastrointestinal diseases, or other diseases 
that significantly affect the absorption, distribution, 
metabolism, and excretion of oral drugs. Patients who have 
undergone major gastrectomy.  
• 
Fever with body temperature above 38°C or clinically 
significant active infection in the past week; active 
pulmonary tuberculosis; active fungal, bacterial, and/or viral 
infection requiring systemic therapy;  

• 
• Active bleeding or newly diagnosed thrombotic disease; 
currently taking anticoagulant medication at therapeutic doses or 
with a tendency to bleed; 
• 
Clinically significant major abnormalities in rhythm, 
conduction, or morphology on resting electrocardiogram 
(ECG), such as complete left bundle branch block, second-
degree or higher atrioventricular block, clinically significant 
ventricular arrhythmia or atrial fibrillation, unstable angina 
pectoris, congestive heart failure, chronic heart failure with 
New York Heart Association (NYHA) class ≥ 2; tendency to 
bleed; 
• 
Myocardial infarction, coronary/peripheral artery bypass, or 
cerebrovascular accident within 3 months; tendency to bleed; 
• 
QT interval (QTc) ≥ 450 ms for males and ≥ 470 ms for 
females on a 12-lead ECG; tendency to bleed; 
• 
Presence of risk factors that cause QT interval prolongation 
or increase the risk of arrhythmia, such as heart failure, ≥ 
CTCAE (version 4.03), second-degree hypokalemia (defined 
as serum potassium < lower limit of normal - 3.0 mmol/L, 
with symptoms requiring treatment), congenital long QT 
syndrome, family history of long QT syndrome; tendency to 
bleed; 
• 
Use of any medication known to prolong the QT interval 
within 2 weeks before enrollment; tendency to bleed; 
• 
Insufficient bone marrow reserve or organ function, reaching 
any of the following laboratory limits (no corrective 
treatment within 1 week before blood sampling for laboratory 
tests): 
a. Absolute neutrophil count < 1.5 × 109 / L; 
b. Platelet count < 90 × 109 / L; 
c. Hemoglobin < 90 g/L (< 9 g/dL); 
d. Alanine aminotransferase > 3 times the upper limit of normal 
(ULN); 

e. Aspartate aminotransferase > 3 × ULN; 
f. Total bilirubin > 1.5 × ULN; 
g. Creatinine > 1.5 × ULN or creatinine clearance  
h. Serum albumin (ALB) < 28 g/L; 
• 
Female subjects who are pregnant, lactating, or plan to 
become pregnant during the study period; 
• 
Subjects with drug abuse and medical, psychological, or 
social conditions that may interfere with their participation in 
the study or have an impact on the evaluation of study results; 
• 
Subjects with known or suspected hypersensitivity to the 
study drugs or any drugs related to this trial; 
• 
Subjects with any unstable condition or situation that may 
jeopardize their safety and compliance; 
• 
Other situations where the investigator believes the subject 
should not participate in this study. 
The planned 
sample size 
Utilizing the non-inferiority test, we set the non-inferiority 
margin with reference to the DYNAMIC II study as -8.5%. Based 
on a power of 0.8, a one-sided alpha of 0.05, a post-conventional 
treatment PD-1 efficacy rate of 30%, and an expected post-
treatment MRD-negative rate of no less than 50%, initial 
calculations using the Non-Inferiority Tests for the Difference 
Between 1 Proportion and 1-Sample function in the PASS (Power 
Analysis and Sample Size) software indicate that a sample size of 
20 cases is required for enrollment.Furthermore, considering 
factors such as the dropout rate during dynamic monitoring, the 
follow-up capabilities of the hospital's execution team, patient 
compliance, and the length of follow-up, the overall dropout rate 
for the 3-year follow-up is expected to be no more than 10%. 
Therefore, the estimated minimum number of subjects required 
for enrollment is 22 cases. 
Treatment and 
monitoring 
programs 
I. Neoadjuvant Immunotherapy Phase:  
1.Immunotherapy Drug: Tislelizumab: Administered at 200mg 
intravenously, every three weeks (Q3W), for a minimum of four 

cycles. The specific number of neoadjuvant immunotherapy 
cycles will be determined based on the results of MRD dynamic 
monitoring. 
2.MRD 
Dynamic 
Monitoring 
Time 
Points: 
Dynamic 
monitoring will be performed at the time of initial diagnosis and 
after the fourth cycle of neoadjuvant immunotherapy. Blood 
sampling 
for 
monitoring 
will 
occur 
1-2 
weeks 
after 
immunotherapy. If consecutive MRD monitoring results are 
negative for two consecutive times, a wait-and-observe strategy 
will be adopted. If MRD remains positive after eight cycles of 
neoadjuvant immunotherapy, surgical intervention will be 
considered. 
(Specific Example: If MRD turns negative after the fourth cycle 
of neoadjuvant immunotherapy, further monitoring of MRD will 
be performed after the fifth cycle. If MRD remains negative, a 
wait-and-observe strategy will be adopted. If MRD is still positive 
after the fourth cycle, immunotherapy will continue for another 
cycle, and MRD monitoring will be performed after the fifth 
cycle. This process will continue accordingly. If MRD remains 
positive after eight cycles, surgical resection will be undertaken.) 
II. "Watch and Wait" Strategy: 
After adopting the MRD dynamic monitoring for the "watch and 
wait" strategy, the time points for recurrence or metastasis 
monitoring are: 1 month, 4 months, 7 months, 13 months, 19 
months, 25 months, and 37 months after the initiation of the 
"watch and wait" period. The follow-up and review strategies, 
along with current routine clinical examination items, include: 
digital rectal examination and tumor markers every 1 to 3 months 
for 3 years, contrast-enhanced CT of the chest, abdomen, and 
pelvis every 3 months, pelvic MRI, transrectal ultrasound, 
colonoscopy with biopsy, and PET-CT if necessary. 

III. Surgery: 
After adopting MRD dynamic monitoring following surgery, the 
time points for recurrence or metastasis monitoring are: 3 to 4 
weeks, 4 months, 7 months, 13 months, 19 months, 25 months, 
and 37 months after radical surgery. The follow-up and review 
strategies, along with current routine clinical examination items, 
include: tumor markers, contrast-enhanced CT of the chest, 
abdomen, and pelvis every 3 months for 3 years, colonoscopy 
every 12 months, and MR and PET-CT if necessary. 
Safety 
Evaluation 
Criteria 
All subjects will be observed for any adverse events during the 
clinical study, including abnormal clinical symptoms and vital 
signs, as well as abnormalities in laboratory tests. The clinical 
manifestations, severity, onset time, duration, treatment methods, 
and prognosis of these adverse events will be recorded. 
Furthermore, the correlation between these adverse events and the 
investigational drug will be determined. The safety of the drug 
will be evaluated using the NCI-CTC AE 5.0 version criteria. 
Evaluation 
Criteria for 
Effectiveness 
During the entire study period, tumor evaluation will be 
conducted primarily through dynamic monitoring of minimal 
residual disease (MRD) to assess the proportion of patients 
achieving clinical complete remission (cCR). The analysis will be 
performed using the Response Evaluation Criteria in Solid 
Tumors (RECIST) standard (version 1.1). Radiological imaging 
scans (CT, MRI) will be performed to measure and calculate the 
cCR, objective response rate (ORR), and major pathological 
tumor regression grade (mrTRG) for all patients. 
Statistical 
Methods 
Statistical analysis will be performed using SPSS23.0 or SAS 
software. The PPS and FAS data will be analyzed for efficacy 
indicators, while the SS data will be analyzed for safety indicators. 
Most of the data in this study are descriptive. For continuous data, 
they will be expressed as mean and standard deviation; categorical 

indicators will be expressed as the number of cases and percentage 
for each category. 
(1) Baseline Demographic and Disease Characteristics: 
Descriptive statistical methods will be used to summarize the 
characteristics of the observed patients, including age, gender, 
clinical stage, ECOG score, etc. 
(2) Primary Endpoint Analysis Method: 
During 
neoadjuvant 
immunotherapy, 
patients 
with 
two 
consecutive negative MRD assessments will be evaluated as 
having clinical complete remission (cCR). 
Secondary Endpoint Analysis Methods: 
A. Imaging results will be evaluated using the RECIST 1.1 
standard to calculate the objective response rate (ORR) and major 
pathological tumor regression grade (mrTRG). After MRD 
becomes negative, two radiologists will jointly assess whether the 
patient has achieved clinically complete remission (cCR) in the 
traditional sense. 
B. Quality of life (QoL) will be assessed using the Chinese version 
of the European Organization for Research and Treatment of 
Cancer Quality of Life Questionnaire (EORTC QLQ-C30) (V3.0). 
C. Safety indicators: Based on the SS set, adverse events and 
serious adverse event incidence rates in this trial will be described 
using the NCI-CTC AE v5.0 standard, along with their clinical 
manifestations, severity, onset time, duration, treatment methods, 
and prognosis. 
D. Tumor downstaging rate, sphincter-preserving rate, surgical 
complication rate, adverse event incidence rate, and serious 
adverse event incidence rate will all be expressed as percentages. 
F. Survival analysis will be performed using the Kaplan-Meier 
method to estimate the 3-year overall survival (OS) and disease-
free survival（DFS）； 

Flow chart of the clinical trial 
Study Step / Visit 1 
Scr
eeni
ng 
peri
od 
Treatment period 3 
W&W/Surgery 
Safety 
follow
-up 4 
Sur
viv
al 
foll
ow-
up 
5 
Neoadjuvant immunotherapy 
One 
mont
h 
after 
W&
W/S
urge
ry  
Onc
e 
ever
y 1-3 
mon
ths 
after 
W&
W/S
urge
ry 
Onc
e 
ever
y 3 
mon
ths 
after 
W&
W/S
urge
ry 
Once 
every 
3 
month
s, once 
every 
6 
month
s after 
half a 
year, 
once 
every 
year 
after 2 
years 
28 
days 
after 
the 
last 
dose 
onc
e 
eve
ry 3 
mo
uth
s 
Visit time 
-
28~
0 
C2
D1 
C3
D1 
C
4
D
1 
C5
D1 
C6
D1 
C7
D1 
C8
D1 
Window time 
0 
±3 
Da
ys 
±3 
Da
ys 
±3 
D
ay
s 
±3 
Da
ys 
±3 
Da
ys 
±3 
Da
ys 
±3 
Da
ys 
±7 
Days 
±7 
Days 
±7 
Days 
±7 
Days 
±7 
Days 
±7 
Da
ys 
Signed 
the 
informed 
consent 
form 2 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic data 
6 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
History of past 
illness 7 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor diagnosis 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissues WES 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height, weight 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
Vital sign 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
Physical 
examination 8 
■ 
 
■ 
 
■ 
 
■ 
 
 
■ 
 
 
■ 
 
ECOG PS Score 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
Enteroscopy+ 
biopsy 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
■ 
 
 
 
A 12-lead ECG 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
MRD dynamic 
monitoring 9 
■ 
 
■ 
 
■ 
 
■ 
 
■ 
 
 
■ 
 
 
CEA 
■ 
 
■ 
 
■ 
 
■ 
 
 
■ 
 
 
■ 
 
Routine blood test 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
Blood biochemical 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
Coagulation 
function 
■ 
 
 
 
 
 
 
 
 
 
 
 
■ 
 
Fecal hidden blood 
■ 
 
 
 
 
 
 
 
 
 
 
 
■ 
 
Routine urine test 
■ 
 
 
 
 
 
 
 
 
 
 
 
■ 
 
Thyroid function 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
■ 
 
HIV、HBV、HCV 
■ 
 
 
 
 
 
 
 
 
 
 
 
■ 
 
Myocardial 
enzymes, 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
 
 

myocardial 
markers 
Peripheral blood 
assay 10 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
 
 
 
 
Genetic testing 11 
■ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Thoracal/ 
abdominal/ pelvic 
enhanced CT 
■ 
 
■ 
 
■ 
 
■ 
 
 
 
■ 
 
■ 
 
Pelvic cavity MRI 
12 
■ 
 
 
 
 
 
 
 
 
 
■ 
 
 
 
Endorectal 
ultrasonography 13 
■ 
 
 
 
 
 
 
 
 
 
■ 
 
 
 
PET-CT 
Conducted according to clinical needs 
Neoadjuvant 
immunotherapy 
 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
 
 
 
 
 
 
Adverse events 14 
 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
 
Simultaneous 
medication 15 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
 
Quality of life 
assessment 16 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
■ 
 
Survival condition 
 
 
 
 
 
 
 
 
 
 
 
 
 
■ 

Flow-chart description of the clinical trial 
1. 
One period in this study was defined as 21 days. The inspection items should be completed within the 
time window listed in the test process. In case of statutory holidays, the reason for the over window can 
be recorded in advance and recorded in the CRF. In addition to the follow-up in the flow chart, the 
investigator may increase the examination items or increase the visit frequency based on the clinical 
condition oft h e subject. 
2. 
The informed consent form for the screening period should be signed within 28 days before treatment. 
Except for the existing tumor imaging examinations and tumor tissue biopsies within the specified 
time limit before the first medication, written informed consent must be obtained prior to performing 
any clinical research procedures. This study allows subjects who have failed previous screenings to 
undergo re-screening, and a new informed consent form must be signed and a new subject number 
must be registered for re-screening. 
3. 
Treatment period: The specific medication is tirelizumab: 200mg, intravenous drip, Q3W, for at least 
4 cycles. The specific number of neoadjuvant immunotherapy cycles will be determined based on the 
MRD dynamic monitoring results. 
4. 
Safety follow-up will be performed 28±7 days after the last medication or before starting a new anti-
tumor treatment, whichever occurs first. All AEs occurring before the safety follow-up visit should be 
recorded until they resolve to grade 0-1 or baseline levels, or until the investigator determines that 
further follow-up is not necessary for reasonable reasons (such as unrecoverable or improved 
condition), whichever occurs first. SAEs occurring within 90 days after the last medication or before 
the subject starts a new anti-cancer treatment (whichever occurs first) should be followed up and 
recorded. If the patient is not due to disease progression, imaging examinations should be performed 
during this follow-up. If there are imaging data within 4 weeks before this follow-up, the investigator 
may assess whether a re-examination is needed. 
Survival follow-up begins after the end of the safety follow-up period, with telephone follow-up every 3 
months to record the subject's survival status until the last subject's follow-up is completed or the trial 
ends. 
5. 
Demographic data includes: age, gender, etc. 
6. 
Past medical history includes: tumor location, disease stage, histological stage, history of perforation 
or obstruction. 
7. 
Physical examination: During the treatment period, rectal examination by digital palpation is 
mandatory for rectal cancer patients. Other examination items may be specified based on clinical 
symptoms or clinical needs. 
8. 
MRD monitoring: Dynamic monitoring will be performed at the time of initial diagnosis and after the 
4th cycle of neoadjuvant immunotherapy. Blood sampling for MRD monitoring should be performed 
1-2 weeks after immunotherapy. If two consecutive MRD monitoring results are negative, a wait-and-
observe strategy will be adopted. If MRD remains positive after 8 cycles of neoadjuvant 
immunotherapy, surgical treatment will be considered. 

9. 
Tumor peripheral blood: This includes CD3+, CD4+, and CD8+ T cell expression levels. 
10. Genetic testing: This includes routine testing for colorectal cancer, such as MSI status (or 
immunohistochemical assessment of MMR status), POLE/POLD1 mutation status, PTEN mutation 
status, TMB status, RAS and BRAF mutation status, etc. 
11. Pelvic MRI: This will only be performed for rectal cancer patients. 
12. Rectal endosonography: This will only be performed for rectal cancer patients. 
13. Adverse event collection begins at the start of treatment. Once a subject terminates the trial treatment, 
only new or unresolved adverse events related to the trial treatment should be recorded. 
14. Concomitant medication records should include any medication taken within 28 days of the screening 
period and during the trial period. Once a subject interrupts the trial treatment, only concomitant 
medications and treatments used for new or unresolved adverse events related to the trial treatment 
should be recorded. Concomitant medications for other diseases do not need to be recorded. 
15. Quality of life assessment will be performed using the Wexner scoring scale, EORTC QLQ-C30 
scale, and EORTC-QLQ-CR29 scale. 
 
 
 

1. research background 
Colorectal cancer (CRC) is one of the most common malignant tumors of the 
digestive tract. According to the global cancer statistics in 2018, CRC ranks third in 
incidence (10.2%) and second in mortality (9.2%) [1]. Locally advanced colorectal cancer 
(LACRC) is defined as CRC in stage II (cT3-4, N0) or stage III (cT1-4, N+). If these 
patients undergo direct surgical resection, it will lead to a high local recurrence rate and a 
low overall survival rate. Therefore, for locally advanced rectal cancer (LARC), the 
globally recognized standard treatment is the "neoadjuvant chemoradiotherapy (NCRT) + 
total mesorectal excision (TME) + postoperative adjuvant chemotherapy" recommended 
by the National Comprehensive Cancer Network (NCCN) guidelines. For locally 
advanced colon cancer (LACC), the NCCN guidelines also recommend considering 
neoadjuvant chemotherapy with FOLFOX or CAPEOX for patients with bulky lymph 
nodes or clinical T4 colon cancer [2]. 
In clinical practice, among LARC patients who undergo radical surgery after NCRT, 
15% to 20% of specimens show no residual tumor cells, achieving pathological complete 
response (ypCR). The 5-year overall survival rate for ypCR patients is approximately 
90%, indicating excellent prognosis [3]. Based on this, some scholars have proposed the 
idea of non-surgical treatment for patients predicted to achieve pCR after neoadjuvant 
therapy. Subsequent studies, including our own, have confirmed that patients with LARC 
who achieve clinical complete response (cCR) after NCRT and undergo a watch-and-wait 
(W&W) approach have no statistical difference in overall survival compared to those who 
undergo surgery and achieve pCR [4]. Additionally, patients on the W&W protocol avoid 
unnecessary surgical trauma and risks, significantly improving their quality of life. For 
patients who achieve near-clinical complete response (near-cCR), over half have the 
opportunity to adopt W&W for organ preservation. The recurrence-free survival (RFS) 
and metastasis-free survival (MFS) in the near-cCR group are not significantly different 
from those in the cCR group [5]. In summary, NCRT has been proven to significantly 
improve tumor downstaging rates, radical resection rates, and even allow some patients to 
avoid surgery altogether. However, the incidence of perioperative complications is 
relatively high in LARC patients undergoing radical surgery after NCRT, including 
anastomotic leakage, impaired bowel function, sexual function, and reproductive function 
caused by radiotherapy and chemotherapy. Patients undergoing low anterior resection also 

face the possibility of temporary stoma failure and long-term anterior resection syndrome 
(ARS). Similarly, LACC patients undergoing neoadjuvant chemotherapy (NCT) may 
experience adverse reactions such as nausea, vomiting, and diarrhea during treatment, and 
some may even discontinue treatment due to intolerance to these side effects. 
Certainly, are there alternative neoadjuvant treatment modalities, beyond 
neoadjuvant (chemo)radiotherapy, that are compatible with the Watch & Wait strategy 
while circumventing the associated acute and chronic toxic side effects, as well as the 
decline in quality of life? Amidst the advent of precision oncology and the burgeoning 
field of genetic testing, researchers have uncovered a distinct molecular subset of 
malignancies characterized by deficient mismatch repair (dMMR) or high microsatellite 
instability (MSI-H). This subgroup comprises approximately 15% of all colorectal cancer 
patients, exhibiting robust immunogenicity, profound lymphocytic infiltration within the 
tumor microenvironment, and favorable prognosis. However, these patients often display 
insensitivity to traditional neoadjuvant (chemo)radiotherapy [6, 7]. Mismatch repair 
(MMR) is a pivotal DNA repair mechanism, encompassing four essential proteins: 
MLH1, MSH2, MSH6, and PMS2. This intricate system precisely identifies and rectifies 
base mismatches, as well as small-scale base deletions or insertions that arise during DNA 
replication or recombination, thereby safeguarding genomic stability. dMMR represents 
mutations in MMR-related genes, leading to dysfunctional repair capabilities and 
diminished or absent mismatch repair activity. MSI-H, on the other hand, refers to 
alterations in the length of microsatellite sequences, often triggered by insertions or 
deletions during DNA replication, frequently stemming from MMR defects associated 
with dMMR [8]. 
Tumor cells in MSI-H/dMMR colorectal cancer patients exhibit significantly 
upregulated expression of programmed death-ligand 1 (PD-L1) on their surface. This PD-
L1 can bind to the programmed death receptor 1 (PD-1) on effector T cells, inhibiting 
their immune killing effect against tumor cells. However, immune checkpoint inhibitors 
can block the binding of PD-L1 to PD-1 receptors, thereby enhancing the body's immune 
system's ability to kill tumor cells [9]. In 2021, the NCCN guidelines formally 
recommended pembrolizumab as a 1A-level evidence-based treatment for first-line 
therapy in patients with dMMR/MSI-H metastatic colorectal cancer (mCRC) [10]. 
However, dMMR/MSI-H mCRC accounts for less than 5% of all CRC patients, and most 

dMMR/MSI-H CRC cases are still locally advanced. Therefore, neoadjuvant 
immunotherapy (NIT) for dMMR/MSI-H locally advanced colorectal cancer (LACRC) 
has gradually become a current research hotspot. 
Numerous studies on neoadjuvant immunotherapy conducted by our research team 
have been reported as follows: In 2018, the NICHE study [11], as the world's first 
neoadjuvant immunotherapy trial for non-metastatic colon cancer, has garnered 
significant attention since its inception. The study included two patient populations: those 
with dMMR colon cancer and those with proficient mismatch repair (pMMR) colon 
cancer. Among the 32 patients with dMMR colon cancer, 100% achieved pathological 
remission after neoadjuvant immunotherapy, with 69% achieving pathological complete 
response (pCR). However, due to the small sample size of only 32 dMMR patients in the 
NICHE study, larger-scale studies are needed to further validate the findings. Against this 
backdrop, researchers embarked on the NICHE-2 study [12], targeting a larger patient 
population.In 2022, the NICHE-2 study enrolled 112 patients with non-metastatic dMMR 
colon cancer. They received one dose of ipilimumab (1mg/kg) and two doses of 
nivolumab (3mg/kg) within 6 weeks before surgery. The primary endpoint was safety and 
3-year disease-free survival (DFS), while secondary endpoints included major 
pathological response (MPR) and pCR. The results showed that in terms of safety, the 
incidence of adverse events (AEs) of any grade was 61%, with 4 patients (4%) 
experiencing grade 3-4 AEs. Only 2 patients had surgery delayed for more than 2 weeks 
due to immune-related AEs. All patients underwent R0 surgical resection, and 
pathological remission was observed in 99% of patients, including 95% MPR and 67% 
pCR rates. After a median follow-up of 13.1 months, no patient experienced disease 
recurrence. The NICHE-2 study further demonstrated that neoadjuvant immunotherapy 
for non-metastatic dMMR colon cancer achieves significant efficacy with good 
tolerability. Based on the NICHE/NICHE-2 studies, our research team believes that 
neoadjuvant immunotherapy regimens contribute to the preservation of organ function in 
patients with dMMR/MSI-H colon cancer. In 2021, the PICC study conducted by 
Professor Deng Yanhong's team at the Sixth Affiliated Hospital of Sun Yat-sen University 
[13] also confirmed the favorable efficacy of neoadjuvant immunotherapy. The study 
enrolled 34 patients with dMMR/MSI-H CRC and randomly divided them into two 
groups: the Toripalimab monotherapy group and the Toripalimab combined with 

Celecoxib (COX-2 inhibitor) group. The results showed that the pCR rate in the 17 
patients receiving PD-1 monotherapy was 65%, while the pCR rate in the 17 patients 
receiving Toripalimab combined with Celecoxib was as high as 88%.In 2023, a 
nationwide multicenter retrospective study conducted by Professor Ding Peirong from the 
Sun Yat-sen University Cancer Center, along with Professor Li Yunfeng from our 
hospital, analyzed the efficacy of PD-1 inhibitor-based neoadjuvant therapy in terms of 
tumor remission, surgical resection rate, long-term survival, and recurrence rate [14]. A 
total of 73 patients were enrolled in this study, most of whom had LACRC. 79.5% of the 
patients received PD-1 inhibitor monotherapy, and ultimately, 62 patients achieved 
objective remission in imaging assessment, with 17 achieving complete response (CR) 
and opting for the watch-and-wait (W&W) approach instead of surgery. 45 patients 
achieved partial remission, with a median response time of 9.6 weeks. There was no 
difference in remission rates between cT4a/4b patients and cT2-3 patients (84.0% vs. 
85.4%; P=0.999). With a median follow-up time of 17.2 months, among patients who 
underwent surgery or achieved CR, the 2-year tumor-specific disease-free survival (DFS) 
rate and overall survival (OS) rate were both 100%. This is the largest study to date in the 
field of CRC NIT treatment worldwide and the first report on the long-term efficacy of 
NIT in this area.Moreover, in 2022, our research team also published a retrospective 
analysis exploring the efficacy and safety of neoadjuvant immunotherapy monotherapy 
for patients with dMMR/MSI-H LACRC [15]. A total of 32 patients were included, with a 
median NIT duration of 6 cycles. The objective response rate (ORR) was 100%. 
Ultimately, 3 patients achieved clinical complete response (cCR) and adopted the W&W 
strategy, while 29 patients underwent radical surgery. The pathological remission rate was 
100%, with a major pathological response (MPR) of 86.2% and a pCR rate of 75.9%. In 
summary, it is evident that patients with dMMR/MSI-H CRC can achieve a high pCR rate 
with NIT, and preoperative immunotherapy alone can achieve efficient and safe results. 
So, can these patients who achieve pCR avoid surgery through preoperative 
assessment as cCR? Based on this idea, more and more researchers are exploring the 
direction of organ function preservation for patients with dMMR/MSI-H CRC. In June 
2022, MSKCC published a significant study in the New England Journal of Medicine 
[16]. This study enrolled 12 patients with stage II/III rectal cancer with dMMR/MSI-H, 
who were treated with 500mg Dostarlimab q3w for 6 months and followed up for at least 

6 months. Ultimately, all 12 patients achieved cCR and adopted the W&W approach. No 
cases of progression or recurrence were reported during the follow-up period, and no 
adverse events of grade 3 or higher occurred.In April 2023, a study led by Professor Xu 
Ruihua, Professor Chen Gong, and their gastrointestinal oncology team from the Sun Yat-
sen University Cancer Center [17] also found that dMMR or MSI-H LARC patients, 
without the need for "sandwich therapy" (neoadjuvant chemoradiotherapy → surgery → 
adjuvant chemotherapy), could achieve cCR after PD-1 antibody treatment alone, 
avoiding the damage caused by chemoradiotherapy and surgical treatment. This study 
enrolled 17 newly diagnosed patients with dMMR/MSI-H LARC from October 2019 to 
June 2022, who received 4 cycles of neoadjuvant treatment with Sintilimab and had the 
option of choosing the following subsequent treatment options based on their treatment 
response. Among the 16 patients evaluable for efficacy, 15 patients showed tumor 
shrinkage after treatment. Six patients underwent radical surgery, with 3 achieving pCR. 
Another 3 patients had residual tumor cells after surgery, but they achieved radical 
treatment after surgical resection. Nine patients achieved cCR and chose the W&W 
approach. Follow-up of this group until November 2022 showed no tumor recurrence. 
This study once again demonstrates that neoadjuvant PD-1 monoclonal antibody 
immunotherapy can give patients the opportunity to achieve cCR, thus avoiding surgery 
and preserving organ function, fundamentally changing the treatment approach for this 
disease. Similarly, in 2022, another domestic multi-center real-world retrospective study 
led by Professor Ding Peirong's team from the Cancer Center of Sun Yat-sen University 
and involving our hospital [18] included 19 patients with dMMR/MSI-H rectal cancer 
who achieved cCR after PD-1 inhibitor treatment. Among them, 16 patients received PD-
1 inhibitor as first-line treatment, and 11 patients received PD-1 inhibitor monotherapy. 
The median time from the start of treatment to cCR was 3.8 months. After achieving cCR, 
the median follow-up duration was 17.1 months, during which no local or distant 
recurrence was observed. The 2-year local RFS rate, 2-year distant metastasis-free 
survival rate, 2-year DFS rate, and 2-year OS rate were all 100%. This is the largest 
sample size and longest follow-up study on the exemption of surgery and radiotherapy 
after immunotherapy for dMMR/MSI-H rectal cancer in the international arena to date. A 
recently accepted retrospective study by this research team also found that the W&W 
strategy may become a new treatment model after NIT for dMMR/MSI-H LARC 

patients. We included a total of 20 patients with dMMR/MSI-H LARC who received NIT. 
Among them, 90% of patients achieved CR after a median of 7 cycles, including 11 
patients with postoperative pCR and 7 patients who chose the W&W strategy after being 
assessed as cCR or near-cCR. The median follow-up duration for both groups was 25 
months, with 2-year DFS and OS both at 100%. There was no statistical difference in the 
incidence of immune-related adverse events (irAEs) between the two groups. However, in 
the pCR group, 2 patients underwent permanent colostomy, and 2 patients experienced 
surgical-related adverse events (srAEs). The above studies suggest that the W&W 
strategy may be a safe and reliable option for patients with dMMR/MSI-H rectal cancer, 
and even colon cancer, after achieving cCR through neoadjuvant immunotherapy. 
However, in previous clinical practice, we have observed that although most patients 
exhibited significant radiographic regression after neoadjuvant immunotherapy (NIT), the 
rate of complete clinical response (cCR) was relatively low. Paradoxically, a high rate of 
pathological complete response (pCR) was confirmed in postoperative pathological 
analysis. This inconsistency in radiographic and pathological CR rates has made it 
difficult for clinicians to accurately assess the true efficacy of preoperative immune 
neoadjuvant therapy, resulting in many patients with deficient mismatch repair 
(dMMR)/microsatellite instability-high (MSI-H) colorectal cancer (CRC) undergoing 
potentially avoidable surgeries and incurring associated risks and complications. 
Therefore, clinical medicine experts are further exploring whether there exist more 
sensitive and precise detection technologies that can overcome the limitations of 
radiographic assessment of NIT efficacy, thereby guiding and optimizing wait-and-watch 
(WW) strategies. 
The emergence of minimal disease residual (MRD) has brought us a ray of hope. 
Studies have found that MRD is a significant factor for recurrence and metastasis after 
radical tumor treatment, yet it represents tumor remnants at the cellular level that are 
undetectable by traditional imaging or laboratory methods. The next-generation 
sequencing (NGS) technology based on circulating tumor DNA (ctDNA) in blood serves 
as a favorable means for detecting MRD. Initial research on MRD detection in colorectal 
cancer tends to guide the selection of adjuvant therapy for patients in stages II and III. 
Adjuvant chemotherapy can improve overall survival (OS) in patients with stage III colon 
cancer. However, 40% of patients may achieve cure even without adjuvant therapy, while 

30% may experience recurrence despite adjuvant treatment [19]. Therefore, especially in 
stage II and III colon cancer, we have the opportunity to intensify treatment for patients 
who are likely to benefit, thereby increasing the cure rate, and reduce treatment for those 
who are unlikely to benefit, thus lowering treatment-related toxicity. 
The DYNAMIC study [20] is a prospective study specifically targeting patients with 
stage II colon cancer. This study aimed to assess whether ctDNA-guided therapy could 
reduce the need for adjuvant treatment without increasing the risk of recurrence compared 
to standard treatment methods. Additionally, it further explored the prognosis of patients 
with positive ctDNA who received adjuvant chemotherapy and those with negative 
ctDNA who did not receive adjuvant therapy. From August 10, 2015, to August 2, 2019, a 
total of 455 patients with stage II colon cancer were enrolled. Blood samples were 
collected from all patients at weeks 4 and 7, and they were randomly assigned to a 2:1 
ratio into a ctDNA-Guided group (ctDNA-positive - adjuvant chemotherapy; ctDNA-
negative - observation) and a standard treatment group. Ultimately, 289 patients entered 
the ctDNA-Guided group, and 147 entered the standard treatment group. With a median 
follow-up time of 37 months, the results showed that the ctDNA-guided management 
group had a 46% lower acceptance rate of adjuvant chemotherapy compared to the 
standard treatment group, significantly sparing some patients from unnecessary 
chemotherapy. The 2-year recurrence-free survival (RFS) rates were 93.5% vs 92.4% for 
the ctDNA-guided management group and the standard management group, respectively, 
with an HR of 0.96, indicating no significant difference and achieving the predefined 
non-inferiority endpoint. Among the ctDNA-guided management group, the 3-year RFS 
rate was better for ctDNA-negative patients than for ctDNA-positive patients, suggesting 
that ctDNA-positive patients could benefit from adjuvant therapy. The approach of using 
ctDNA-guided therapy for stage II colon cancer reduced the use of adjuvant 
chemotherapy without affecting recurrence-free survival. This study's findings may 
potentially change the guidelines' recommendation level for ctDNA testing, and ctDNA 
testing may even become one of the clinical standard detection methods. A prospective 
study led by Professor Chen Gong's team from the Cancer Center of Sun Yat-sen 
University [21] utilized patient-specific tumor-informed ctDNA analysis to detect MRD 
in patients with stage I to IV colorectal cancer. The study enrolled 117 patients with 
surgically resected stage I to IV colorectal cancer. An individualized tumor-informed 

technology called brPROPHET was used to detect MRD at baseline (n=117), 7 days after 
surgery (n=117), and 30 days after surgery (n=93). With a median follow-up time of 213 
days, the overall positive rate of baseline ctDNA was 97%. Among patients with stage I, 
II, III, and IV colorectal cancer, the positive rates of ctDNA were 88%, 98%, 98%, and 
100%, respectively. The median ctDNA level was higher in patients with advanced 
colorectal cancer and was significantly correlated with tumor volume. In two patients 
who relapsed, ctDNA detection identified the risk of recurrence one and two months 
earlier than imaging methods. In the subgroup analysis, two other MRD detection 
technologies - a fixed panel (covering 168 genes, 273kb) tumor-informed method (fixed 
panel with informed calling [FI method]) and a tumor-agnostic method (fixed panel with 
agnostic calling [FA method]) - were compared head-to-head with brPROPHET. Among 
the 74 patients included in the parallel comparison of the three MRD detection methods, 
the preoperative ctDNA positive rates were 97.3%, 75.7%, and 68.9% when detected by 
brPROPHET, FI method, and FA method, respectively. Only 15 patients could be detected 
with ctDNA by the brPROPHET method at baseline. The ctDNA levels in these patients 
were below the detectable level of the FI and FA fixed panel methods. A total of 135 
postoperative blood samples were tested using the three methods, and the positive rates of 
brPROPHET, FI, and FA fixed panel methods were 14.8%, 8.1%, and 6.7%, respectively. 
This study demonstrated the clinical performance of patient-specific brPROPHET in 
detecting ctDNA in patients with colorectal cancer, showing higher sensitivity in 
detecting preoperative and postoperative ctDNA compared to fixed panel detection 
methods. 
ctDNA detection, as a non-invasive method, boasts high accessibility, safety, and 
convenience, allowing for continuous dynamic monitoring. Moreover, its short half-life 
enables real-time reflection of tumor status. Currently, there are two major technical 
approaches for monitoring MRD using ctDNA in patients with early or locally advanced 
colorectal cancer: the tumor-informed approach based on tissue sequencing and the 
tumor-naive approach solely relying on plasma analysis [22]. The tumor-informed 
approach involves sequencing tumor tissue to obtain mutation information, selecting 
specific mutations for personalized customization, and then detecting ctDNA through 
blood sampling and high-depth sequencing to track the selected mutations in tumor tissue. 
In contrast, the tumor-naive technique solely relies on plasma ctDNA detection using a 

fixed-pattern approach, where the detection range is the same for everyone. If a patient 
does not have these mutations, they cannot be detected, resulting in a higher false-
negative rate and lower accuracy. The Signatera technology, based on the tumor-informed 
approach, can reliably detect tumor-specific mutations with a variant allele frequency 
(VAF) of 0.01%. By filtering out clonal hematopoiesis and germline-derived variations of 
unknown significance from the analysis, it significantly reduces the false-positive rate. In 
contrast, tumor-naive detection has lower sensitivity with a reliable detection limit of 
0.1% to 1% VAF. The GALAXY study [23] reported a preoperative ctDNA positive rate 
of over 90% in stage II-III colorectal cancer, suggesting that tumor-informed 
individualized customization may have better application prospects in MRD monitoring. 
The role of dynamic monitoring of ctDNA in advanced colorectal cancer and the 
post-surgical adjuvant phase has been increasingly recognized, but its role in the 
neoadjuvant therapy stage before surgery for colorectal cancer remains largely 
unexplored. Currently, there is only one study [24] on dynamic monitoring of ctDNA 
after radical treatment for anal squamous cell carcinoma (ASCC) that included 31 patients 
receiving definitive chemoradiation (dCRT). Among them, 65% had stage III tumors, 
with a median radiation dose of 54 Gy/27 fractions. 84% of patients received fluorouracil 
(5-FU or capecitabine) combined with mitomycin chemotherapy, while 16% received 
capecitabine monotherapy. The median follow-up time was 32 weeks. The final results 
showed that 27 patients successfully underwent baseline ctDNA testing, of which 23 
patients had detectable baseline ctDNA, with a positive detection rate of 85%. Except for 
one patient, all patients with negative ctDNA achieved clinical complete response (cCR) 
in subsequent clinical assessments. Patients who did not achieve cCR were assessed as 
having residual tumor despite negative ctDNA, and the researchers are conducting 
follow-up monitoring to further evaluate this patient's condition. Survival analysis 
showed that the 1-year disease-free survival (DFS) rate for all patients was 96%, and the 
1-year overall survival (OS) rate was 100%. Notably, the time to ctDNA remission was 
significantly shorter than the time to cCR. 
Similarly, there is a lack of research on the use of MRD dynamic monitoring to 
guide the wait-and-watch (W&W) strategy after achieving complete response (CR) with 
neoadjuvant immunotherapy for dMMR/MSI-H locally advanced colorectal cancer 
(LACRC). Therefore, this study aims to customize individualized protocols based on the 

tumor-informed approach, dynamically monitoring MRD status after neoadjuvant 
immunotherapy for dMMR/MSI-H stage II/III CRC patients using ctDNA technology. 
This will provide support for patients considering the W&W strategy or selecting surgical 
treatment. 
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2. research contents 
 
2.1 fundamental purpose 
MRD-negative Clinical Complete Response Rate (cCR) 
2.2 Secondary purpose 
1) Optimal Number of Neoadjuvant Immunotherapy Cycles 
2) Inconsistency Rate between MRD-negative cCR Rate and cCR Rate Using Traditional 
Assessment Methods 
3) 3-Year Local Recurrence/Regrowth Rate 
4) 3-Year Disease-Free Survival (DFS) 
5) 3-Year Overall Survival (OS) 
6) Objective Response Rate (ORR) 

7) Tumor Downstaging Rate (mrTRG) 
8) Incidence Rate of Immune-Related Adverse Events. 
2.3 Exploratory research purpose 
1). Clearance rate of ctDNA in blood MRD-positive patients after 1-2 weeks of completion of 
neoadjuvant immunotherapy.;  
2)  High-risk period for MRD conversion or recurrence in blood MRD-negative patients after 
1-2 weeks of completion of neoadjuvant immunotherapy； 
3)  Dynamic changes and rates of change in MRD and their correlation with treatment 
efficacy； 
4)  Detection sensitivity, specificity, positive predictive value, and negative predictive value 
of the tumor-informed, individually customized MRD detection scheme;  
5)  Results of other exploratory multi-omics analyses. 
 
3. study protocol 
2.3 Treatment plan： 
2.3.1 Neoadjuvant Immunotherapy Protocol 
1. Immunotherapy Drug: Tirelizumab: 200mg, intravenous drip, Q3W (every 3 weeks), for at 
least 4 cycles. The specific number of neoadjuvant immunotherapy cycles will be determined 
based on the results of dynamic MRD (Minimal Residual Disease) monitoring. 
2. Time Points for Dynamic MRD Monitoring: The first monitoring will be conducted at the 
initial diagnosis, and subsequent monitoring will be performed after the 4th cycle of 
neoadjuvant immunotherapy. Blood sampling for monitoring will be done 1-2 weeks after 
immunotherapy. If consecutive monitoring of MRD reveals negative results twice, a wait-
and-watch strategy will be adopted. If MRD remains positive after 8 cycles of neoadjuvant 
immunotherapy, surgical intervention will be considered. 
(Specific Example: If MRD turns negative after the 4th cycle of neoadjuvant immunotherapy, 
continued monitoring of MRD after the 5th cycle will be performed. If MRD remains 
negative, the wait-and-watch strategy will be maintained. If MRD is still positive after the 4th 
cycle, an additional cycle of immunotherapy will be administered, and MRD will be 
monitored after the 5th cycle. This process will continue accordingly. If MRD remains 
positive after 8 cycles, surgical resection will be performed.) 

2.3.2 Wait-and-Watch" Strategy 
After four cycles of neoadjuvant immunotherapy, if consecutive monitoring of MRD reveals 
negative results twice, a wait-and-watch strategy will be adopted. Following the wait-and-
watch period, the time of recurrence or metastasis will be observed through dynamic MRD 
monitoring, with MRD testing conducted at 1 month, 4 months, 7 months, 13 months, 19 
months, 25 months, and 37 months after the observation period. The follow-up and review 
strategy, along with current routine clinical examination items, include: digital rectal 
examination and tumor markers every 1 to 3 months for 3 years, chest, abdomen, and pelvic 
contrast-enhanced CT, pelvic MRI, transrectal ultrasound, colonoscopy with biopsy every 3 
months, and PET-CT if necessary. 
2.3.3 Surgical Resection 
If MRD remains positive after eight cycles of neoadjuvant immunotherapy, surgical resection 
will be performed. After surgery, the time of recurrence or metastasis will be observed 
through dynamic MRD monitoring, with MRD testing conducted at 3-4 weeks, 4 months, 7 
months, 13 months, 19 months, 25 months, and 37 months after the radical surgery. The 
follow-up and review strategy, along with current routine clinical examination items, include: 
tumor markers, chest, abdomen, and pelvic contrast-enhanced CT every 3 months for 3 years, 
colonoscopy every 12 months, and MR and PET-CT if necessary. 
 
 
The research process is as follows： 
 
MRD: Minimal Residual Disease; +: Positive; -: Negative; dMMR: Deficient Mismatch 
Repair; MSI-H: Microsatellite Instability-High; pMMR/MSS: Proficient Mismatch 
Repair/Microsatellite Stable; W&W: Wait-and-Watch Strategy; WES: Whole Exome 

Sequencing; PD: Progression of Disease; MRD Status Assessment: Based on the gene 
mutation results of WES detection in tumor tissue from each patient, variant sites are 
screened to develop an individualized MRD detection panel for the patient. 
2.4 MRD Monitoring Technology and Sequencing Platform 
2.4.1 MRD Detection Technique 
Through Whole Exome Sequencing (WES) of tumor tissue and control blood samples, 
variations unrelated to tumor progression and of unknown significance are filtered out to 
obtain the gene mutation spectrum of tumor lesions for each subject. The Signatera 
proprietary algorithm is utilized to select 16 primary clonal somatic mutations, thereby 
customizing an individualized MRD monitoring panel. Based on this customized MRD 
monitoring panel, ultra-high-depth ctDNA-NGS sequencing is performed on the subject's 
blood samples to evaluate the status and content of ctDNA/MRD. By continuously 
monitoring changes in blood ctDNA, the disease progression of the subject can be indicated. 
1.2.2 MRD Sequencing Platform 
1. Whole Exome Sequencing Platform: MGISEQ-2000. 
Sequencing Strategy: PE100; 
Sequencing Depth: Tumor Sample > 500X, Control Blood > 200X. 
2.ctDNA Sequencing Platform: MGISEQ-2000. 
Sequencing Strategy: PE50; 
Sequencing Depth: > 100,000X. 
3. Tumor tissue specimens and blood samples from enrolled patients, i.e., the subjects 
undergoing testing, will be sent to Tianjin BGI Medical Testing Lab, which possesses a 
medical institution practice license and testing qualifications, for free monitoring. 
2.5 Treatment Evaluation  
1.Effectiveness: Tumor evaluation will be conducted throughout the study period, primarily 
through dynamic MRD monitoring to assess the proportion of patients achieving clinical 
complete response (cCR). The Response Evaluation Criteria in Solid Tumors (RECIST) 
standard (version 1.1) will be adopted for analysis. Radiological imaging (CT, MRI) scans 
will be performed to measure and calculate cCR, objective response rate (ORR), and 
modified tumor regression grade (mrTRG) for all patients. 

2.Safety: Adverse events will be observed and graded according to the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 5.0. Tumor markers, blood 
routine tests, blood biochemistry, liver and kidney function, thyroid function, etc., will be 
checked every 2 cycles. During the treatment process, adverse reactions such as edema, 
gastrointestinal reactions, leukopenia, anemia, hand-foot syndrome, rash, muscle spasms, 
diarrhea, etc., will be closely monitored. The time, severity, management methods, and 
outcomes of these adverse reactions will be recorded. 
2.6 Dose adjustment and adverse reaction treatment 
Hematological toxicity dose adjustment 
All dose changes should be documented and treated with the most adequate supportive 
therapy. If the symptoms resolve immediately after supportive care and continued study 
treatment is considered appropriate and the investigator believes the treatment is beneficial to 
the patient, consider continuing the same dose of study treatment plus appropriate continued 
supportive care. If the reduction is medically required, the reduction of chemotherapy is 
allowed. Adjust this cycle dose according to the lowest blood count after last dose (refer to 
the table below):：  
low 
The next cycle dose 
Neutrophil number（×109/L）  Platelet count（×109/L）  
≥0.5  
Platelet count≥50  
invariant  
＜0.5  
Or＜50  
The subsequent dose of 
chemotherapy will bereduced 
by 20% at the discretion of the 
investigator 
 
After two reductions, another 4 degrees of neutropenia or III degree grain deficiency 
with fever (> 38.5°C) or above 3 degrees occurred, without the third reduction, the 
investigator will discuss whetherto continue the trial according to the specific situation. 
 
hepatotoxicity  
• 
Bilirubin: Ifthe bilirubin levels are abnormal in the study, the next cycle should be 
delayed. If 4 weeks, the trial;； 

• 
Liver enzymes: AST and / or ALT and / or alkaline phosphatase levels abnormal in the 
absence o fdisease progression, liver protection treatment, within 2 weeks, not returning 
to normal, dose can be adjusted according to the table below. 
If the liver fuction recovers in the next cycle, the dose should be increased to the previous 
level. 
AST/ALT price 
Alkaline phosphatase values 
dose titration 
＜1.5×ULN 
＜5×ULN 
No dose adjustment is required 
＞1.5×ULN 至＜2.5×ULN 
＜2.5×ULN 
No dose adjustment is required 
2.5×ULN 至＜5×ULN 
＜2.5×ULN 
Subsequent dose reduction of 
chemotherapy was 20-25% as 
determined by the investigator 
＞1.5×ULN 至＜5×ULN 
＞2.5×ULN＜5×ULN 
＞5×ULN and /or＞5×ULN 
Delay by up to two weeks. if 
still not 
Recovery, at the discretion of 
the investigator. Ill Whether the 
person withdrew 
from the study. 
 
Immune-related adverse effects 
Based on the mechanism of action, patients receiving its treatment may develop immune-
related adverse reactions, including severe and fatal cases. Most immune-related adverse 
effects are reversible and can be managed by suspension or cessation of the treatment and 
administering corticosteroid therapy and / or supportive therapy. Depending on individual 
patient safety and tolerability, dose suspension or permanent discontinuation may be 
required. Increase or dose reduction is not recommended. See the table below for 
recommended Tirelizumab. 
As recommended for Tirelizumab 
Immune-related adverse effects 
order of severity ‡ 
Treatment adjustment 
protocol 
pneumonia 
Level 2 
Suspension 
until 
adverse 
effects return to grade 0-1 
Grade 3 or 4 or recurrent 
grade 2 
Permanent 
withdrawal 
of 
drugs 

Diarrhea and colitis 
Level 2 or 3 
Suspension 
until 
adverse 
effects return to grade 0-1 
Level 4 
Permanent 
withdrawal 
of 
drugs 
hepatitis 
Grade 
2, 
aspartate 
aminotransferase (AST) or 
alanine 
aminotransferase  
(ALT)> 3 to 5 times the upper 
limit of normal (ULN); and / 
or total bilirubin (TBIL)> 1.5 
to 3 times ULN 
Suspension 
until 
adverse 
effects return to grade 0-1 
and prednisone 10mg / day or 
equivalent dose 
Grade ,3 AST or ALT> 5x 
ULN, and / or TBIL> 3 x 
ULN 
Permanent 
withdrawal 
of 
drugs 
Nephritis and renal dysfunction 
Grade 2, creatinine> 2 – 3 
times ULN 
Suspension 
until 
adverse 
effects return to grade 0-1 
Grade 3, creatinine>3 times 
ULN or>4.0 mg/dL, with 
indication for hospitalization; 
life- threatening, indication 
for dialysis treatment 
Permanent 
withdrawal 
of 
drugs 
endocrine disease 
≥ Grade 2 hyperthyroidism 
Suspension until symptom 
improvement 
or 
adverse 
response remission to grade 
0-1 
≥ Grade 2 hypothyroidism 
Continue the medication, by 
standard therapy, by hormone 
replacement therapy Control 
≥ Grade 2 hyperglycaemia or 
type 1 diabetes mellitus 
Suspension 
until 
adverse 
effects return to grade 0-1. 
Start 
insulin 
replacement 
therapy 
as 
clinically 
indicated, 
and 
use 
hypoglycemic 
drugs 
for 
hyperglycemia 
Grade 2 hypophysitis 
Suspension until the subject 
is clinically stable 

Grade 3 or 4 hypophysitis 
Suspension until hormone 
replacement therapy To when 
the subject was clinically 
stable 
≥ Grade 2 hypadrenia 
Suspension until the subject 
is clinically stable 
Skin adverse effects 
Grade 
,3 
or 
suspected 
Stevens-Johnson 
syndrome 
(Stevens Johnson syndrome, 
SJS) or toxic epidermal 
necrolysis (Toxic Epidermal 
Necrolysis, TEN) 
Suspension 
until 
adverse 
effects return to grade 0-1 
Level 4, or to confirm the SJS 
or TEN 
Permanent 
withdrawal 
of 
drugs 
carditis 
Level 1 
Suspension of dosing 
≥ level 2 
Permanent 
withdrawal 
of 
drugs 
thrombocytopenia 
Level 3 
Suspension 
until 
adverse 
effects return to grade 0-1 
Level 4 
Permanent 
withdrawal 
of 
drugs 
Other immune-related adverse 
effects 
Grade 1 encephalitis 
Grade 2 or 3 pancreatitis 
Grade 2 myasthenia gravis 
Grade 3 or 4 with elevated 
blood amylase or elevated 
lipase 
Other 
immune-related 
adverse reactions that first 
occurred in grade 2 or 3 
include dermatitis, etc 
The administration should be 
suspended until the adverse 
reaction returns to grade 0-1, 
and the encephalitis should 
lipase decide whether to 
continue 
the 
medication 
based on clinical judgment 

≥grade 2 encephalitis 
Grade 4 pancreatitis 
Grade 3 or 4 myasthenia 
gravis  
Guillain 
Barre 
Svndrome
（Guillain-Barrésyndrome，
GBS） 
Other 
adverse 
immune-
related 
reactions 
first 
occurring in grade 4 include 
dermatitis 
Permanent 
withdrawal 
of 
drugs 
Recurrent or persistent adverse 
effects 
Recurrent Grade 3 or Grade 4 
(except 
for 
endocrine 
disorders)  
Within 12 weeks after the last 
dose: Grade 2 or 3 adverse 
effects did not improve to 
Grade 0-1 (except endocrine 
disease) or the corticosteroid 
dose was not reduced to 
prednisone 10mg daily, or 
equivalent dose 
Permanent 
withdrawal 
of 
drugs 
Iniection site reaction 
Level 2 
continue 
Level 3-4 
Permanent 
withdrawal 
of 
drugs 
hypersensitivity 
Level 2 
Stop the drug administration 
immediately. Depending on 
the observed intensity of 
response, 
antihistamines 
should be given in advance in 
the next cycle of treatment 
and 
the 
subcutaneous 
injection is slowed. 
Level 3-4 
Immediately 
continue 
immediately with subsequent 
permanent withdrawal 
Toxicity grading was determined using theNational Cancer Institute Common Terminology Assessment Criteria forAdverse 
Events version 4.03 (NCICTCAE v4.03). 
›For advice on hormone replacement therapy, see the envafolimab insert [Notes]. 
 
Treatment of adverse reactions 

The treatment of other adverse reactions can refer to the drug instructions or the routine 
treatment principles of the test center. 
2.7 Follow-up 
1）Before treatment 
All patients participating in this trial should sign an informed consent form and receive a 
copy of the informed consent form. If the patient agrees to participate in the trial, the 
participation form should be completed and signed immediately, and thereafter the patient 
can be enrolled. Within 28 days prior to initiation oftreatment, the investigator should 
evaluate the following clinical and laboratory indicators. 
1 Past history, including: age, sex, tumor location, disease stage, histological stage, 
history of perforation or obstruction, duration of surgery, and the procedure 
2 Physical strength status score (see Appendix 
3 Clinical examination, height (pre-treatment measurement only), body weight 
4 Electrocardiogram 
5 Collection of clinical laboratory data:： 
Routine blood indicators: hemoglobin, white blood cells, platelets and neutrophils; 
Blood biochemical indexes: creatinine, alkaline phosphatase, total bilirubin, SGOT, 
SGPT; 
Coagulation function: prothrombin time (PT), activated partial thromboplastin time 
(APTT) and international standardized ratio (INR); 
Thyroid function: thyroid-stimulating hormone (ISH), free I3, free T4; 
Urine routine: including urine specific gravity, pH value, white blood cells, red 
blood cells, protein, glucose, ketone body, and tube type. Two consecutive routine urine 
testing of urine protein + +, it is recommended to test 24 hours of urine protein quantification 
as soon as possible (such as within 72 hours; 
fecal occult blood testing； 
HIV check; 
HBV infection: including hepatitis B five and HBV DNA;  
HCV-infected persons: including HCV RNA.。 
6 Detectionof tumor markers: CEA 
7  
8 pelvic cavity M R I Ctoluminal ultrasound 

9 Genetic testing: MSI status (or MMR status assessed by immunohistochemistry), 
POLE / POLD 1, PTEN mutation status, TMB status, RAS and BRAF gene mutation 
testing 
10 13 
11 Peripheral tumor blood: CD3 +, CD4 +, and CD8 + I cell 
12 14 
13 Concomitant medication: collect medication within the 3 months before treatment 15 
14 Quality of life assessment 
2）During the treatment process 
During the treatment process, researchers can add follow-up content or increase the 
frequency of follow-up according to needs. The follow-up items during the treatment period 
need to be completed after the 2nd, 4th, 6th, and 8th cycles of treatment. 
The follow-up content of the trial plan is as follows: 
1．Body weight: Measurements will be taken before neoadjuvant immunotherapy, after 
2 cycles, 4 cycles, 6 cycles, and 8 cycles of neoadjuvant immunotherapy. 
2．ECOG PS (Eastern Cooperative Oncology Group Performance Status) Score: 
Assessment will be conducted before neoadjuvant immunotherapy, after 2 cycles, 4 cycles, 6 
cycles, and 8 cycles of neoadjuvant immunotherapy. 
3．12-lead electrocardiogram (ECG): This test will be performed before neoadjuvant 
immunotherapy, after 2 cycles, 4 cycles, 6 cycles, and 8 cycles of neoadjuvant 
immunotherapy. 
4．Complete blood count (CBC): Blood samples will be collected for CBC before 
neoadjuvant immunotherapy, after 2 cycles, 4 cycles, 6 cycles, and 8 cycles of neoadjuvant 
immunotherapy. 
5．Blood biochemistry: Biochemical analysis will be done before neoadjuvant 
immunotherapy, after 2 cycles, 4 cycles, 6 cycles, and 8 cycles of neoadjuvant 
immunotherapy. 
6．MRD (Minimal Residual Disease) detection: MRD will be assessed before 
neoadjuvant immunotherapy, after 2 cycles, 4 cycles, 6 cycles, and 8 cycles of neoadjuvant 
immunotherapy. If MRD becomes negative after neoadjuvant immunotherapy for the first 
time, a repeat MRD test will be performed after the next cycle of immunotherapy. 
7．Tumor Marker Detection: To be performed before neoadjuvant immunotherapy, and 
after 2, 4, 6, and 8 cycles of neoadjuvant immunotherapy. 

8．Thyroid Function Test: To be performed before neoadjuvant immunotherapy, and 
after 2, 4, 6, and 8 cycles of neoadjuvant immunotherapy. 
9．Peripheral Blood Testing (Th1/Th2, Lymphocyte Immune Panel): To be performed 
before neoadjuvant immunotherapy, and after 2, 4, 6, and 8 cycles of neoadjuvant 
immunotherapy. 
10．Colonoscopy with Biopsy: To be performed before neoadjuvant immunotherapy, 
and after 2, 4, 6, and 8 cycles of neoadjuvant immunotherapy. 
11．Thoracic, Abdominal, and Pelvic CT Scan with Contrast: To be performed before 
neoadjuvant immunotherapy, and after 2, 4, 6, and 8 cycles of neoadjuvant immunotherapy. 
12．Pelvic MRI (for rectum only): To be performed before neoadjuvant immunotherapy, 
and after 2, 4, 6, and 8 cycles of neoadjuvant immunotherapy. 
13．Adverse Events: Collection of adverse events after the start of treatment. 
14．Concomitant Medications: Continuous collection. 
15．Quality of Life Assessment: To be collected every cycle. 
3）After the completion of the treatment 
After completion of treatment, patients should be reviewed at the items and time in the 
table below; until tumor recurrence or at least 3 years. Review beyond the time of the 
following table can be conducted according to the habits of different centers, with PET / CT 
if necessary. 
  
proiect 
After the first moon 
1 
/ 
3 
/ 
6 
/ 
9 
/ 
12 
/ 
15 
/ 
18 
/ 
21 
/ 
24 
/ 
symptom 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
check-up 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
routine blood test 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Blood biochemical 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
MRD detection 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
CEA 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Peripheral blood 
detection 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Chest/abdominal/pelvic 
CT 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
colonoscope* 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 

Assessment of quality 
of life 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Observe the efficacy 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
* If the lesion is found by colonoscopy, the colonoscopy period can be shortened according to the specific situation. 
 
4）Safety follow-up 
Safety follow-up was performed 28 days after the last dose and the trial planned follow-up is 
as follows: 
1  weight 
2 check-up 
3 ECOG PS Score 
4 And a 12-lead ECG 
5 routine blood test 
6 Blood biochemical 
7 routine urine test 
8 coagulation function 
9 thyroid function 
10 Enhanced thoracic, abdominal, and pelvic CT scans: Patients who are discontinued 
due to disease progression do not need further follow-up enhancement 
11 Adverse events: Continuous follow-up until adverse event recovery or further 
observation is not required by the investigator. 
12 Concomitant medication: Continuous collection until treatment for the adverse event 
is completed or the investigator. 
13 Quality of life assessment 
 
5）Survival follow-up 
After disease progression, patient survival status was collected every 3 months 
allowing telephone follow-up. 
6）Patient compliance observation 
The investigator's responsibility to ensure patient compliance with the trial will be 
examined by itinerant representatives. 

2.8 data collection 
All data shall be timely, truthfully and detailed in the medical record register (Case 
Report Form, CRF) or CRF software. The medical record form should be filled in by special 
personnel and signed by the project leader. The medical records (CRFs) shall be checked and 
collected regularly, and recorded according to the time node required by the clinical trial 
management center of the hospital. 
After the clinical trial, the unit shall write the clinical summary report according to the 
standard requirements.。 
2.9 Clinical safety assessment 
1）Adverse events 
Definition o fthe adverse events 
Adverse event (or adverse experience, AE: any adverse medical event occurring in a 
subject or clinical subject, not necessarily causally related to treatment. 
The AE can therefore be any bad or non-intended signs (e. g., including abnormal 
laboratory results), symptoms, or transient drug-related disease, which should be considered 
for medication involvement. 
Adverse events occurring both before and after treatment were considered as adverse 
events according on management needs. Therefore, safety monitoring (reporting adverse 
events or serious adverse events) should be performed from subject enrollment to the end of 
the study. Therefore, adverse events occurring during signing the informed consent and 
initiation of study treatment were also considered as AEs. 
Adverse drug reactions (ADRs): Al toxicities and non-intended reactions to the drug 
related to any dose should be considered as adverse drug reactions (ADRs). 
Response to the drug means that there is at least a reasonable possibility of a causal 
relationship between the drug and the AE, meaning that this relationship cannot be excluded. 
Serious Adverse Event (SAE: means all adverse medical events occurring at any drug 
dose: 
• 
Lead to death 
• 
life-threatening 
Note: "serious" and "life threatening" are defined as an adverse event when the 
subject has the adverse event; rather than an adverse event that the more serious may 
lead to death.。 

• 
Patients require hospitalization or extension of existing hospitalization  
• 
Leading in persistent or significant incapacity / disability 
• 
Congenital malformations or birth defects 
Important medical events: important medical events, do medical and scientific appraisal 
to decide whether to report is appropriate, these important medical events may not 
immediately life threat or cause death or hospitalization, but may harm subjects or may need 
interference to prevent the occurrence of the other results, usually should also be considered 
serious. For example, some adverse events require severe treatment in the emergency room, 
or management of allergic bronchial asthma at home; malignant fluid or convulsions without 
hospitalization; drug dependence and abuse, or malignant tumors histologically different from 
the primary tumor. 
Other events that should be addressed as SAEs: Drug exposure during pregnancy / 
lactation: In principle, pregnancy and lactation are under the exclusion criteria. If a pregnancy 
occurs during the study, the patient should immediately withdraw from the study and inform 
the investigator immediately and follow up the patient throughout the pregnancy and 
postpartum. Even if the mother and child are completely normal without any adverse events, 
the consequences should be recorded. Even if the pregnancy is not a SAE, use the SAE report 
form.  
Events that shouldb e handled as SAEs: disease progression. 
2）Record and evaluation methods ofadverse events 
All A s should be recorded in the appropriate part ofthe Medical Record Report Form 
(CRF). SAE report forms (including initiation or follow-up reports) should also be 
completed. 
The following aspects of each event should be recorded in the CRF:： 
• 
AEs in medical terms, not as a subject; 
• 
Date of occurrence (start date); 
• 
Octime (start time); 
• 
Recovery date (end date); 
• 
Recovery time (end time). 
The grading was assessed by the investigator as defined by NCI-CTC version 5.0. 
• 
Level 1= mild; 
• 
Level 2= moderate; 
• 
Level 3= Heavy; 

• 
Grade 4= life-threatening or disabling;； 
• 
Level 5 = death. 
The investigator should assess the causal relationship between the adverse events and 
the study drug; the decisive factor assessed in the record is the temporal correlation 
between the AEs and the study drug. The causal relationship between the adverse event and 
the study drug or study protocol is judged as follows: 
• 
Uncorrelation = no temporal relationship with the study drug (too early, too late or not 
medication), or a reasonable causal relationship between the A and another drug, 
associated disease or environment; 
• 
Impossible = a temporal relationship with study drug, but no reasonable causal 
relationship between AE and study drug; 
• 
Probably = a reasonable causal relationship between A and study drug. Lack of 
withdrawal information (withdrawal information) or unclear; 
• 
It is likely that there is a reasonable causal relationship between AE and study drug. 
Discontinuation (withdrawal from the drug study) had an effect on the response. No 
redose to prove; 
• 
Clear / definite = a reasonable causal relationship between A and study drug. 
Discontinuation has an effect on response and can occur if readministered when 
clinically feasible. 
Measures taken for this study treatment (none, discontinuation, dose reduction, delayed 
treatment, slowing of intravenous infusion) and other measures (no, concomitant medication, 
need for hospitalization, or prolonged hospitalization, surgery, delayed chemotherapy, 
discontinuation of chemotherapy, chemotherapy reduction) were defined as follows: 
• 
Recovery with sequelae 
• 
Healing without sequelae 
• 
Not healed, but no treatment is needed  
• 
Not healed and need treatment 
• 
die 
Toxicity grade / severity was changed 
• 
Serious: Yes or No; 
• 
If the patient has the same AE several times, each must be recorded and re-evaluated. 
3) Reporting procedures for serious adverse events3 
Any serious or medically significant clinical adverse event or laboratory abnormality 

occurring during the study obligates the investigator to report every serious adverse 
event to the ADR by telephone or email within 24 hours. 
After the telephone report, the written information should be sent by fax. The report 
shall provide the information of the reporter and recipient including name, address, telephone 
and fax number, and indicate that the report is "preliminary" report or "follow-up" report. If 
necessary, the report form shall be accompanied by the relevant medical record report form. 
The investigator shall guarantee to the public ethics committee or competent authority 
any additional information regarding the death of the subject. 
All forms must be dated and signed by the responsible person, or signed by an 
authorized colleague ofthe responsible person. 
4）Monitoring of the adverse events of the subjects 
Any adverse events occurring during the study were monitored and followed up until the 
end of the study. In addition, the SAEs must be reported through the SAE table. 
5）Laboratory Indicator Assessment 
All laboratories involved in clinical evaluations in the hospital should comply with the 
basic principles of Good Laboratory Practice (GLP) and the requirements of their respective 
hospitals. 
6）Clinical Safety Assessment 
NCI-CTCAE version 5.0 will be used to evaluate the clinical safety of the treatment in 
the study. The occurrence of adverse events in the subjects must be assessed during each 
clinical visit. 
2.10 
statistical analysis 
tatistical analysis technique 
Using computer (SPSS or R) software package, count data adoption rate, mean and 95% 
credible interval for measurement data: t-test for measurement data when comparing two 
groups, chi-square test for count data, Kaplan-Meier and Log Rank methods for survival 
analysis, and Cox proportional risk model for prognosis analysis. The probability P-value was 
used at any stage, and the statistical significant difference bound was used at 0.05. 
Safety analysis: The toxicity evaluation criteria were evaluated according to the NCI- 
CTC 5.0 criteria. Specific statistical methods are the same as above. 
Type of analysis 
Prior to database lock, the following three datasets were identified. 

The primary efficacy analysis will be performed in all enrolled populations (the intent- 
to-treat population or the ITT population) and the per-protocol population (PP). Secondary 
efficacy analysis and exploratory analysis will be performed in the intention-to-treat 
population. Non-inferiority analysis will be performed in the per-protocol population. 
1.Intention-to-treat population 
The intention-to-treat population consists of all patients randomized to any of the two 
treatment arms. Analysis will be performed according to the treatment groups 
randomassigned by the patients. 
2.Population conforming to the protocol 
Patients who have not received any dose of chemotherapy or chemoradiotherapy, or 
those who have suffered a serious violation of the inclusion or exclusion criteria. Analysis 
will be performed according to the treatment groups randomassigned by the patients. 
3.Safety population 
The safety population includes all patients who have received at least one dose of 
chemotherapy or radiotherapy. The Safety population was used to analyze all safety 
parameters. Patients were assigned to the treatment group based on the medication they 
actually received. 
4.The data that should be excluded during the analysis 
There were no excluded data available. 
Medium-term analysis 
An interim analysis of the efficacy endpoints is not performed in this study. An 
independent data security monitoring team will monitor the safety data throughout the study. 
All safety data descriptive analysis will be performed after study treatment for all patients 
enrolled in this study. This analysis will be strictly limited to the safety data and will not 
include any efficacy endpoints. 
3 research contents 
3.2 Sample size of the study 
Using a non-inferiority test, we have set the non-inferiority margin based on the 
DYNAMIC II study as -8.5%. With a power of 0.8, one-sided alpha of 0.05, an effective rate 
of PD-L1 after conventional treatment of 30%, and an expected MRD-negative rate after 
treatment of no less than 50%, an initial calculation using the Non-Inferiority Tests for the 
Difference Between 1 Proportion and 1-Sample function in the PASS (Power Analysis and 

Sample Size) software indicates that a sample size of 20 patients is required for enrollment. 
The dropout rate for dynamic monitoring is related to the follow-up capabilities of the 
hospital's execution team, patient compliance, and the length of follow-up. The overall 
dropout rate for 3-year follow-up is expected to be no more than 10%. Therefore, the 
estimated minimum number of patients required for enrollment is 22. 
 
3.3 subject investigated 
The research subjects of this project originate from 22 newly diagnosed patients with 
dMMR/MSI-H locally advanced colorectal cancer who were admitted to Yunnan Cancer 
Hospital (The Third Affiliated Hospital of Kunming Medical University) after the project's 
launch. 
 
3.4 Enrollment criteria 
1）1) 
Disease characteristics 
• 
Histological confirmation of rectal adenocarcinoma: 
• 
Immunohistochemistry confirmed pMMR or / and pCR or / and NGS as MSS: 
• 
The tumor location is within 12cm from the anal margin: 
• 
Local advanced rectal cancer (stage II-III, cT 3-4 and / or N +): 
* Preoperative staging method: pelvic MRI / transrectal ultrasound. 
• 
No signs of intestinal obstruction: or obstruction relieved after proximal colostomy surgery: 
• 
Preoperative thoracic, abdominal, and pelvic CT excluded distant metastases.； 
2）Patient characteristics 
• 
Age: from 18 years old to 75 years old: 
• 
Activity status score: ECOG 0-1: 
• 
Life expectancy: greater than 2 years: 
• 
Hematology: WBC> 3500106/L ; PLT>100000a 106/L ; Hb>10g/dL: 
• 
Liver function: SGOT and SGPT were less than 1.5 times the normal value: bilirubin was 
less than 1.5 mg/dL: 
• 
Renal function: creatinine was ml.8 mg/dL: 

• 
Other: non-pregnant or lactating women: no other malignant disease (except non- 
melanoma or carcinoma of the cervix) within 5 years or concurrent: no mental illness that 
prevents informed consent: no other serious disease that leads to shortened survival. 
• 
Patients or family members can understand the study protocol and are willing to participate 
in the study, and sign a written informed consent form: 
• 
Patients had good compliance and volunteered for scheduled follow-up, treatment, 
laboratory tests, and other study procedures.。 
3）Previous treatment 
• 
No previous rectal cancer surgery: 
• 
No prior chemotherapy or radiotherapy: 
• 
No previous biological treatment: 
• 
Previous endocrine therapy: no restriction. 
4.4 Exclusion criteria 
 
• Tumor biopsy specimens with immunohistochemical indications of pMMR or 
microsatellite stability (MSS). 
 
Patients with histological types of colorectal cancer other than adenocarcinoma (such 
as neuroendocrine carcinoma, sarcoma, lymphoma, squamous cell carcinoma, etc.). 
 
History of HIV infection or active chronic hepatitis B or C (high viral DNA copy). 
 
Autoimmune diseases. 
 
Other active clinically severe infections (> NCI-CTC v3.0). 
 
Patients in clinical stage I. 
 
Preoperative evidence of distant metastasis, including isolated, distant, or non-
contiguous intra-abdominal metastases. 
 
Patients who have undergone open surgery on sites other than the colon within 14 days 
prior to enrollment. 
 
Patients unable to provide surgical tissue for WES testing for customized personalized 
MRD detection panel or those with failed customization of the personalized MRD 
detection panel. 
 
Patients unable to provide blood samples for MRD testing during the treatment and 
follow-up periods. 
 
Cachexia or organ dysfunction. 
 
History of pelvic or abdominal radiation therapy. 
 
Patients requiring treatment for seizures (such as steroid or anti-epileptic therapy). 

 
Chronic inflammatory bowel disease or intestinal obstruction. 
 
Patients with other severe diseases that may affect follow-up and short-term survival, 
as determined by the investigator. 
 
Patients with a history of blood transfusion within 2 weeks before treatment starts. 
 
• Patients unable to undergo clinically required contrast-enhanced magnetic resonance 
imaging (MRI) or contrast-enhanced computed tomography (CT) for follow-up. 
 
Prior use of traditional Chinese medicine (TCM) with anti-tumor effects. However, if 
the duration of use was less than 7 days and the patient has discontinued the medication 
for 2 weeks or more before enrollment in this study, they may be eligible for inclusion. 
 
Evidence of severe or uncontrollable systemic diseases, such as severe psychiatric, 
neurological disorders, epilepsy, dementia, unstable or decompensated respiratory, 
cardiovascular, liver, or kidney diseases, left ventricular ejection fraction (LVEF) < 
50%, uncontrolled hypertension (defined as greater than or equal to CTCAE grade 3 
hypertension despite medication), dysphagia, active gastrointestinal diseases, or other 
conditions significantly affecting the absorption, distribution, metabolism, and 
excretion of oral medications. Patients who have undergone subtotal gastrectomy in the 
past are also excluded. 
 
Fever with a temperature of 38°C or higher in the past week, clinically significant active 
infections, or active pulmonary tuberculosis. Active fungal, bacterial, and/or viral 
infections requiring systemic treatment. 
 
Active bleeding, new-onset thrombotic disease requiring therapeutic anticoagulation, 
or patients with a bleeding tendency. 
 
Clinically significant major abnormalities in rhythm, conduction, or morphology on 
resting electrocardiogram (ECG), such as complete left bundle branch block, second-
degree or higher heart block, clinically significant ventricular arrhythmias or atrial 
fibrillation, unstable angina, congestive heart failure, or chronic heart failure with New 
York Heart Association (NYHA) class ≥ 2. Patients with a bleeding tendency are also 
excluded. 
 
• History 
of 
myocardial 
infarction, 
coronary/peripheral 
artery 
bypass, 
or 
cerebrovascular accident within the last 3 months. 
 
Patients with a bleeding tendency. 
 
12-lead electrocardiogram (ECG) QTc interval ≥ 450 ms for males and ≥ 470 ms for 
females. 

 
Presence of risk factors for QT interval prolongation or increased risk of arrhythmia, 
such as heart failure, grade 2 hypokalemia (defined as serum potassium < lower limit 
of normal - 3.0 mmol/L, with symptoms requiring treatment), congenital long QT 
syndrome, or a family history of long QT syndrome. 
 
Use of any medication known to prolong the QT interval within 2 weeks before 
enrollment. 
 
Insufficient bone marrow reserve or organ function, meeting any of the following 
laboratory limits (with no corrective treatment within 1 week before blood sampling): 
o Absolute neutrophil count < 1.5 × 109 / L 
o Platelet count < 90 × 109 / L 
o Hemoglobin < 90 g/L (< 9 g/dL) 
o Alanine aminotransferase > 3 times the upper limit of normal (ULN) 
o Aspartate aminotransferase > 3 × ULN 
o Total bilirubin > 1.5 × ULN 
o Creatinine > 1.5 × ULN or creatinine clearance < 45 mL/min (calculated using 
the Cockcroft-Gault formula) 
o Serum albumin (ALB) < 28 g/L 
 
Female subjects who are pregnant, breastfeeding, or plan to become pregnant during 
the study. 
 
Substance abuse and medical, psychological, or social conditions that may interfere 
with patient participation in the study or impact the assessment of study results. 
 
Known or suspected hypersensitivity to the study drugs or any drugs related to this trial. 
 
Any unstable condition or situation that may compromise patient safety and compliance. 
 
Any other situation deemed inappropriate for participation in the study by the 
investigator. 
 
4.5Exit standard 
• 
The subject withdrew his informed consent and requested his withdrawal: 
• 
Poor compliance, not administered according to the study protocol: 
• 
 After enrollment from the study, the patient requests withdrawal from 
the investigator or fails to complete the study schedule for any 
reason: 
• 
Other circumstances deemed necessary to conclude the study. 

4.6 Termination of treatment 
• 
Serious adverse events or serious adverse reactions (SAEs) during the study: 
• 
Delay treatment for more than 4 weeks for any reason (meaning all 
drugs in the delayed regimen). 
4.7 Evaluation of the study findings 
1）Efficacy evaluation o f chemoradiotherapy 
— 
CR: Pelvic MR: complete regression of primary tumor or only fibrotic scar, 
MR no mesangial lymph node enlargement: ultrasound colonoscopy: 
complete regression of primary tumor or only erythematous tiny ulcer or scar, 
no cancer remaining on biopsy: digital rectal tumor disappeared and no 
stiffness or nodules of intestinal wall. PR: tumor volume reduced by more 
than 30%, no new lesions: 
— 
PD: tumor volume increased more than 20%, or new lesions. 
— 
SD: The tumor volume did not change significantly, the reduction degree did 
not reach PR, and the increase degree did not reach PD: 
2）Evaluation of the degree of radical operation 
— 
R0 microscopic pathology examination cut margin has no 
residual tumor cells: The R1 microscopic pathological 
examination of the resection margin showed residual tumor 
cells: 
— 
Residual tumor cells were visible at the macroscopic lower margins of R2. 
3）Tumor response assessment 
It is recommended to evaluate postoperative pathological specimens according 
to the tumor pathological withdrawal grade (tumor regression grading, TRG) criteria 
(see Annex 2): if not, a complete pathological response must be reported. 
 
4） Evaluation of local recurrence or metastasis 
When clinical symptoms occur (anal pain, hematochezia, lower limb edema, etc.), 
progressive elevation of CEA, or suspicious signs in chest or abdominal imaging, further 
examination is needed to find local recurrence or disease metastasis progression. Regional 
recurrence mainly refers to tumor recurrence in the local area or near the adjacent organs: 
distant metastasis refers to tumor recurrence outside the above areas. Disease-free survival is 

the absence of tumor recurrence or the occurrence of new colorectal cancer by systematic 
evaluation. The diagnosis of clinical recurrence and metastasis must meet at least one of the 
following criteria: 
1.Imaging suggested recurrence (ultrasound, CT, MRI, PET-CT) 
2.Positive cytology biopsy (ascites, anastomotic recurrence, suspicious imaging 
findings) 
The reported date of recurrence is the date when recurrence was detected using the 
diagnostic method described above. In case of recurrence, the investigator should specify the 
site of recurrence and the diagnostic method used. When definitive imaging evidence cannot 
be obtained, a positive result of cytology or biopsy should be obtained. Elevated CEA alone 
could not be used as evidence of local recurrence or metastasis of rectal cancer. 
5）Calculation of the survival period 
Local-regional recurrence-free survival was defined as the time interval from the onset 
of randomization to the appearance of a local recurrence event. Tumor evaluation (CT / MRI 
of the abdomen and pelvic cavity or ultrasound and chest CT) and CEA testing must be 
performed every 6 or 12 months after randomization, or when the patient shows signs of 
progression (i. e., clinically indicated). 
Disease-free survival (DFS) is defined as the time interval from the start of 
randomization and the occurrence of the event in the next segment. Tumor evaluation (CT / 
MRI of the abdomen and pelvic cavity or ultrasound and chest CT) and CEA testing must be 
performed every 6 or 12 months after randomization, or when the patient shows signs of 
progression (i. e., clinically indicated). The suspected lesion detected by ultrasound must be 
confirmed by CT / MRI. All re-operations or further anticancer therapy should also be 
documented. 
Based on the purpose of this study, events that identified patients were no longer in the 
disease-free condition were defined as follows: 
• 
The patient showed signs of the original tumor recurrence: 
• 
The patient showed signs of a new colorectal cancer, 
• 
Death from any cause. 
Note: recurrence of any original tumor or occurrence of new colorectal cancer should be 
determined by cytological or histological methods Confirm. No supporting evidence of other 
specific test findings (such as radiography, histology / cytology) cannot be based on isolated 
events such as increased CEA or unexplained clinical deterioration of the disease. The date of 

relapse was defined as the date of final confirmation of the targeted findings. Patients will 
thereafter be followed up for survival as planned. 
If a recurrence of a confirmed colorectal cancer or the occurrence of a new colorectal 
cancer occurs during the study treatment period, patients will be withdrawn from the study 
treatment for survival follow-up. If the recurrence of colorectal cancer or the occurrence of a new 
colorectal cancer occurs during the study treatment period, the patient may be treated further 
according to the guidance of the investigator. 
All tested patients will be followed for at least 2 years. For a biopsy, a biopsy report should 
be provided. Overall survival was the time from randomization to death. When the patient 
survived, the time to the last follow-up was taken as the overall survival period. 
 
6）Evaluation of the toxic and side effects of adjuvant therapy 
Toxicity evaluation according to the CTC criteria (version 5.0) 
Follow-up of patient safety should include during treatment and 30 days after the end of 
the last cycle. The reason for the delay or interruption should be recorded in the CRF table 
7）ECOG (Appendix) 
3.5 Ethical and informed consent aspects 
3.5.1 Ethical requirements 
The investigator ensured that the study was conducted in accordance with the Declaration 
of Helsinki principles for maximum protection of the individual. This study was reviewed and 
approved by the ethics committee of Yunnan Provincial Cancer Hospital. 
3.5.2 informed consent 
The investigator or a person assigned by the investigator is responsible (as permitted by local 
regulations) to obtain written informed consent for the purpose, methods, prospective benefits, 
and potential hazards of the study. Subjects who fail or fail to sign a legal consent must sign an 
informed consent by their legal representative. If the subject and his legal representative will not 
read, a notary should be present during the informed consent discussion. After the subject and 
his representative verbally agree to participate in the trial, the witness should sign the informed 
consent form to prove that the contents of the agreement are accurately interpreted and 
understood. The Investigator or designee should also state that the subject may refuse to 
participate in the study or withdraw from the study at any time for any reason. The CRF of this 
study includes the section documenting the subject's informed consent, which should be 
completed accurately. If new safety information leads to significant changes in the hazard / 

benefit evaluation, the informed consent should be re-reviewed or updated as needed. All 
subjects (including those who have started treatment) should be informed of the new 
information, given an updated informed consent form, and obtained their consent to continue the 
study. 
3.5.3 confidentiality 
During the study, your name, gender and other personal data will be replaced by code 
names or numbers, and kept strictly confidential. Only the relevant doctor knows your 
information, and your privacy will be well protected. The principal investigator of this project will 
publish the final research results in a journal after the end of the project for academic 
communication and promoting medical progress, but will not disclose any of your personal 
information. 
 
 

4 Project implementation years and annual plan 
The total length of the project 
implementation 
3 years 
General schedule: June 2023 – June 2026 
Phase time schedule 
Main contents and results of the stage objectives 
June 2023 - December 2023 
（6 Mouths） 
Study Enrollment Phase: 10 patients were enrolled 
for the initial assessment of efficacy, safety, and 
other relevant factors. 
December 2023- June 2024 
（6 Mouths） 
Completion of Enrollment with Follow-up Phase: 
Upon completion of enrolling 22 patients, follow-
up assessments were conducted to evaluate the 
cCR rate of MRD, safety, feasibility, toxic side 
effects of neoadjuvant immunotherapy, tumor 
regression, and other factors. 
June 2024- December 2024 
（6 Mouths） 
Six-Month Follow-up Report after Enrollment of 
All Cases: Following the enrollment of all cases, a 
six-month follow-up report was issued to assess 
the cCR rate of MRD, safety, local recurrence rate, 
distant metastasis rate, and the incidence of 
surgical complications. 
December 2024- June 2025  
（6 Mouths） 
One-Year Follow-up Report after Enrollment of 
All Cases: After the enrollment of all cases, a one-
year follow-up report was issued to analyze the 
local recurrence rate, distant metastasis rate, and 
other relevant factors. 
June 2025- June 2026 
（12 Mouths） 
Two to Three-Year Follow-up Report after 
Enrollment of All Cases: After the enrollment of 
all cases, a two to three-year follow-up report was 
issued to analyze the 3-year local recurrence rate, 

3-year distant metastasis rate, 3-year DFS 
(Disease-Free Survival), and 3-year OS (Overall 
Survival). 
Conclusion and Reporting: The study evaluated the 
safety and effectiveness of utilizing MRD dynamic 
monitoring results to guide the wait-and-watch 
strategy after neoadjuvant immunotherapy for 
dMMR/MSI-H colorectal cancer. 
 
 
 

6. appendix 
6.1Attachment 1: ECOG PS Scoring criteria 
classify 
Behavioral state 
0 
Ability to perform all daily activities without restriction 
1 
Severe physical activity is limited, but he can walk and can perform light 
physical work. 
2 
Can walk, live can take care of themselves, but can not engage in any 
work, awake, more than half of the time can get out of bed and walk 
3 
Can only have limited self-care, more than half of the waking time to 
bed or chair. 
4 
Unable to move: unable to take care of themselves: bed or chair. 
 
6.2 Attachment 2: Efficacy Evaluation Criteria for Solid Tumor Version 
(Response Evaluation Criteriain Solid Tumors RECIST Version1.1) 
1Measureability of tumors at baseline levels 
1.1Definition 
At baseline, tumor lesions / nodes will be measurable and not measurable as defined 
below： 
1.1.1Measureable lesions 
Tumor lesion: at least one diameter (which can be recorded as maximum) with the 
following minimum length: 
 R CT scan 10mm (CT scan layer thickness not greater than 5mm) 
 0 Clinical routine examination instrument 10mm (tumor lesion cannot be 
accurately measured with diameter instrument should be recorded as 
unmeasurable) 
 0 Chest X-ray at 20mm 
 0 Malignant lymph nodes: Pathologically enlarged and measurable, short CT 
scan diameter 15mm (CT scan thickness recommended not more than 5mm). 
At baseline and follow-up, only short paths were measured and followed up. 
1.1.2Non-measurable lesions 
All other lesions, including small lesions ( 10mm or 10mm to 15mm) and 
unmeasurable lesions. Unmeasured lesions include meningeal disease, ascites, pleural or 

pericardial effusion, inflammatory breast cancer, cancerous lymphangitis of the skin / lung, 
imaging of unconfirmed and follow-up abdominal mass, and cystic lesions. 
1.1.3 Special considerations regarding lesion measurement 
Bone lesions, cystic lesions, and previously locally treated lesions should be specifically 
noted: 
Bone lesions: 
 
Bone scan, PET scan or plain film are not suitable for measuring bone lesions, but can be 
used to confirm the presence or disappearance of bone lesions: 
 
If an osteolytic lesion or a mixed osteogenic lesion has a defined soft tissue component 
that meets the above measurable definition, these lesions can be considered as 
measurable lesions if they can be evaluated by tomographic imaging techniques such as 
CT or MRI: 
 
Osteogenic lesions are non-measurable lesions. 
Cystic lesions： 
 
Lesions that meet the criteria of the definition of pure cyst by radioimaging should not be 
considered malignant because they are a simple cyst by defmition, neither measurable 
lesions nor unmeasurable lesions: 
 
R If it is a cystic metastatic lesion and meets the above measurable definition, it may be a 
measurable lesion. However, if non-cystic lesions exist in the same patient, non-cystic 
lesions should be preferred as the target lesion. 
Topically treated lesions: 
 
Lesions located at sites previously irradiated or treated with other regional areas are 
generally considered as non-measurable lesions, unless the lesion has clearly progressed. 
The study protocol should describe in detail the conditions under which these lesions are 
measurable lesions.。 
1.2 Description of the measurement method 
1.2.1 Lesion measurement 
For clinical evaluation, all tumor measurements were recorded in the metric rice system. All 
baseline assessments of tumor lesion size should be completed as close as possible prior to 
treatment initiation and must be completed within 28 days (4 weeks) before treatment initiation. 
1.2.2 Evaluation methot 

The same technique and methods should be used for the baseline assessment and 
subsequent measurements of the lesions. All lesions must be evaluated using imaging, except 
for those that cannot be evaluated by imaging but only by clinical examination. 
Clinical lesions: Clinical lesions can only be considered as measurable lesions (such as 
skin nodules) when they are superficial and measured at 10mm in diameter. For patients with 
skin lesions, color photographs containing a scale to measure the size of the lesion, are 
recommended for archiving. When the lesions are evaluated using both imaging and clinical 
examination, imaging evaluation should be used as far as possible because the imaging is 
more objective and reproducible at the end of the study. 
Chest X X: When tumor progression is an important study endpoint, chest CT should be 
preferred because CT is more sensitive than X-ray, especially for new lesions. Chest X-ray 
detection is applicable only if the measured lesions are well defined and the lungs are well 
ventilated. 
CT, MRI: CT is currently the best available and reproducible method for efficacy 
evaluation. The definition of measurable ability is based on the 5mm thickness of the CT scan 
layer. If the CT layer thickness is greater than 5mm, the minimum measurable lesion should be 2 
times the layer thickness. MRI is also acceptable in some cases (e. g. whole body scan). 
Ultrasound: Ultrasound should not be used as a measurement method to measure the 
lesion size. Because of its operation dependence, the ultrasound examination is not 
reproducible after the end of the measurement, which cannot guarantee the identity of the 
technology and measurement between different measurements. If new lesions are identified 
using ultrasound during testing, they should be confirmed using CT or MRI. If radiation 
exposure to CT is considered, MRI can be used instead. 
Endoscopy, laparoscopy: These techniques are not recommended for objective tumor 
evaluation, but they can be used to confirm CR in biopsy specimens obtained or in trials of 
recurrence after the study endpoint of CR or surgical resection. 
Tumor markers: Tumor markers cannot be used alone to evaluate objective tumor 
response. However, if the marker level exceeds the upper normal limit at baseline, it must 
return to normal when used to evaluate complete remission. Because tumor markers vary by 
disease, this factor needs to be taken into account when writing the measurement criteria into 
the protocol. Specific criteria for CA-125 remission (recurrent ovarian cancer) and PSA 
(recurrent prostate cancer) remission have been published. In addition, the International 
Gynecological Cancer Organization has formulated the CA-125 progression criteria, which 

will be added to the tumor objective evaluation criteria for the first-line treatment of ovarian 
cancer. 
Cytology / histology techniques: in the specific circumstances specified in the protocol, 
these techniques can be used to identify PR and CR (such as residual benign tumor tissue 
often present in lesions of germ cell tumors). When exudation may be a potential side 
reaction of a therapy (e. g., treatment with taxane compounds or angiogenesis inhibitors), and 
a measurable tumor meets the criteria for remission or disease stabilization, tumor-related 
exudation occurrence or aggravation during treatment can be confirmed by cytology to 
distinguish between remission (or disease stabilization) and disease progression. 
2 2 It was used for tumor remission assessment 
2.1Evaluation of all tumors and measurable lesions 
To evaluate the objective response or possible future progression, a baseline assessment 
of the total tumor burden in all tumor lesions is necessary for the subsequent measurements. 
In a clinical regimen with objective remission as the primary treatment endpoint, only 
patients with measurable lesions at baseline will be enrolled. A measurable lesion was defined 
as the presence of at least one measurable lesion. For trials with disease progression (time to 
progression or degree of fixed date progression) as the primary treatment endpoint, the 
protocol inclusion criteria must be limited to patients with measurable lesions or no 
measurable lesions can be included. 
2.22.2 Baseline recordings of target lesions and non-target lesions 
When there are more than one measurable lesions during the baseline assessment, all 
lesions should be recorded and measured, a total of not more than 5 (no more than 2 each 
organ), as the target lesions represent all involved organs (that is, patients with only one or 
two cumulative organs up to choose two or four target lesions as the baseline measurement 
lesions). 
The target lesion must be selected based on size (longest diameter), representative of all 
organs involved, and the measurements must be well reproducible. Sometimes when the 
largest lesion cannot be repeatedly measured, the largest lesion can be reselected. 
Lymph nodes need special attention because they are normal tissue and can be detected 
by imaging even in the absence of tumor metastasis. Pathological lymph nodes defined as 
measurable nodules or even target lesions must meet the following criteria: CT measurement 
of a short diameter of 15mm. Baseline only detects the short diameter. Radiologists usually 
use the short diameter of the nodule to determine whether the nodule has a metastatic tumor. 
The cle size is generally expressed by two-dimensional data from imaging detection (CT 

axial plane and MRI selects a plane from the axial, sagittal or coronal plane). The minimum 
value is the short diameter. For example, a 20mm 30mm abdominal nodule with a short 
diameter of 20mm can be regarded as a malignant, measurable nodule. In this example, 
20mm is the measurement of the nodule. Nodules with 10mm diameter but 
15mm should 
not be considered as target lesions. However, nodules 
10mm do not belong to the category 
of pathological nodules and do not need to be recorded and further observed. 
The sum of the total diameter calculated together (including the longest diameter of the 
non-nodular lesion and the short diameter of the nodular lesion) will be reported as the sum 
of the baseline diameters. If containing the lymph node diameter, as mentioned above, only 
the short diameter is included. The sum of the baseline diameters will serve as the reference 
value for the disease baseline level. 
All remaining lesions including pathological lymph nodes may be considered non- 
target lesions, but should be recorded at baseline assessment. If recorded as "present", 
"missing" or in rare cases "clear progress". Widespread target lesions can be recorded with 
target organs (e. g., massive expanded pelvic lymph nodes or large liver metastases). 
2.3 Mitigation criteria 
2.3.1 Evaluation of the target lesions 
Complete response (CR): All target lesions disappear, and the short diameter of all 
pathological lymph nodes (including target nodules and non-target nodules) must be reduced 
to 10mm. 
Partial response (PR): The sum of target lesion diameter decreased by at least 30% from 
baseline. 
Disease progression (PD): Using the minimum value of the sum of all measured target 
lesions throughout the experimental study, the diameter and relative increase is at least 20% 
(baseline value if the minimum): otherwise, the absolute increase of at least 5mm must be 
met (occurrence of one or more new lesions is also considered as disease progression). 
Disease stability (SD): the degree of target lesion reduction does not reach the PR, the 
degree of increase does not reach the PD level, in between the two, the minimum value of the 
sum of the diameter can be used as a reference. 
2.3.2 2.3.2 Precautions for target lesion evaluation 
Lymph node: Even if the lymph nodes identified to be the target lesion decrease to less 
than 10mm, the actual short diameter value corresponding to the baseline should be recorded 
at each measurement (consistent with the anatomical plane at the baseline measurement). 

This means that if the lymph node belongs to the target lesion, even if the criteria for 
complete remission are achieved, it cannot be said that the lesion has all disappeared, because 
the short diameter of the normal lymph node is defmed as 10mm. The target lymph short 
lesion needs to be recorded at a specific location in the CRF table or other recording methods: 
for CR, all lymph short diameters must be 10mm: for PR, SD, and PD, the actual 
measurements of the target lymph short diameter will be included in the sum of the target 
lesion diameter. 
Small to unmeasurable target lesions: In clinical studies, all baseline recorded lesions 
(nodules or non-nodules) should be recorded again in the later evaluation, even if the lesions 
are very small (e. g. 2mm). But sometimes it may be too small to cause the CT scan to be 
blurred, and the radiologist is struggle to define the exact value, potentially it as "too small to 
measure."In this case, it is important to record a value on the CRF table. If the radiologist 
believes that the lesion may have disappeared, it should also be recorded as Omm. If the 
lesion is indeed present but is vague and a precise measurement cannot be given, the default 
can be 5mm.(Note: lymph nodes are unlikely to do this because they normally have a 
measurable size or are often enclosed by adipose tissue as in the retroperitoneal cavity: but if 
this measurement cannot be given, the default is 5mm). The default value of 5mm stems from 
the cut thickness of the CT scan (this value is not changed by the different cut thickness values 
of the CT scan). Since the same measurement is not repeated, providing this default will reduce 
the risk of misassessment. But it should be reiterated that if the radiologist can give the exact 
value of the lesion size, the actual value must be recorded even if the lesion is less than 5mm in 
diameter. 
Isolated or combined lesion: When the non-nodular lesion divides into fragments, the 
longest diameter of the separated part together calculates the total diameter of the lesion. 
Similarly, for the binding lesions, they can be distinguished by the plane between the bound parts, 
and then the respective maximum diameter is calculated. However, if the combination is 
inseparable, the longest diameter should be taken as the longest of the fusion lesion. 
2.3.3Assessment of non-target lesions 
This section defines the remission criteria for non-target lesion tumors. Although some 
non-target lesions are actually measurable, no measurement requires only qualitative 
assessment at the time points specified in the protocol. 
Complete response (CR): All non-target lesions disappeared and tumor markers returned 
to normal levels. All lymph nodes were of non-pathological dimensions (short diameter
 
10mm). 

Non-complete response / non-disease progression: presence of one or more non-target 
lesions and / or persistence of tumor markers at levels greater than normal. 
Disease progression: a definite progression of preexisting non-target lesions. Note: The 
presence of one or more new lesions is also considered as disease progression. 
2.3.42.3.4 
Special considerations for the progression assessment of non-target lesions 
The supplementary explanation of the definition of non-target lesion progression is as 
follows: When a patient has a measurable non-target focus, even if the target lesion is assessed 
as stable or partial remission, to make a clear definition of progression based on the non-target 
focus, the overall deterioration of the non-target lesion has reached the point that the treatment 
must be terminated. However, the general increase in the size of one or more non-target lesions is 
often not enough to meet the progression criteria. Therefore, the overall tumor progression is 
almost rare in the target lesion when the target lesion is stable or partially relieved. 
This occurs when none of the non-target lesions are measurable: in some phase trials, 
when measurable lesions are not specified in the inclusion criteria. The overall assessment 
was based on the above criteria, but because there was no measurable data for the lesion in 
this case. The deterioration of the target lesions is not easy to assess (according to the 
definition: must all non-target lesions are not measured), so when the target lesions change to 
increase the overall disease burden is the target of the disease progression, according to the 
definition of the target focus progress, need to establish an effective detection method to 
evaluate. As described, an increase in tumor burden is equivalent to an additional 73% 
increase in volume (equivalent to a 20% increase in measurable lesion diameter). Or 
peritoneal exudation from "trace" to "massive": lymphangiopathy from "local" to 
"widespread spread": or described in the protocol as "sufficient to change treatment". 
Examples include pleural exudate from trace to mass, spread of lymphatic involvement 
from the primary site to the distance, or may be described as "necessary therapeutic changes" 
in the protocol. If definite progression is found, the patient should be treated as disease 
progression at that time point. It is best to have objective criteria that can be applied to the 
assessment of unmeasurable lesions, noting that the increased criteria must be reliable 
2.3.5New lesions 
The appearance of new malignant lesions indicates the progression of the disease: 
therefore some evaluation of new lesions is very important. There are no specific criteria for 
imaging detection of lesions, however the discovery of a new lesion should be clear. For 
example, progression cannot be attributed to differences in imaging techniques, changes in 
imaging morphology, or other lesions other than tumors (for example, some so-called new 

bone lesions are merely the cure of the original lesion, or the recurrence of the original 
lesion). This is important when a patient's baseline lesion is partially or completely reactive, 
for example, a necrosis of a liver lesion may be identified as a new cystic lesion on the CT 
report, but not. 
Lesions detected at follow-up but not detected at baseline will be considered as new and 
estive of disease progression. For example, if a patient with a visceral lesion on baseline 
examination has metastases on CT or MRI, his intracranial metastases will be considered as 
the basis for disease progression, even if he did not have a cranial examination at baseline 
examination. 
If a new lesion is unclear, for as due to its small morphology, further treatment and 
follow-up evaluation are needed to confirm whether it is a new lesion. If repeated 
examinations confirms it is a new lesion, the time of disease progression should be counted 
from the time of its initial discovery. 
FDG-PET assessment of lesions generally requires additional testing for complementary 
confirmation, and the combination of FDG-PET and supplementary CT test results is 
reasonable to evaluate progression (especially for new suspected diseases). New lesions may 
be identified by FDG-PET according to the following procedures: 
Baseline FDG-PET was negative and the following up FDG-PET was positive, 
indicating progression of the disease. 
No baseline FDG-PET test was performed, and the subsequent FDG-PET test result was 
positive: 
If the follow-up FDG-PET positive test results matched the CT test results, the disease 
progression was proved. 
If the new lesion found by the positive test result of the follow-up FDG-PET is not 
confirmed by the CT test result, the CT test should be confirmed again (if confirmed, the time 
of disease progression starts from the previous FDG-PET abnormality). 
If the follow-up FDG-PET results with a preexisting lesion by CT and the lesion does 
not progressive on imaging tests, no disease progression. 
2.42.4 Best of the overall efficacy evaluation 
The best overall efficacy evaluation is the best efficacy record from the beginning of the 
trial to the end of the trial, with any necessary conditions taken into consideration for 
confirmation. Sometimes the efficacy response occurs after the end of treatment, so the 
regimen should specify whether the efficacy evaluation after the end of treatment should be 
considered in the best overall efficacy evaluation. The protocol must clarify how any new 

treatment affects optimal efficacy response. The best response response depends on the 
outcome of the target and non-target lesions and the performance of the new lesions. In 
addition, it relies on the nature of the trial, protocol requirements, and outcome measures. 
Specifically, the efficacy response profile is the primary goal in non-randomized trials, and 
the confirmation of efficacy by PR or CR is mandatory to confirm which is the best overall 
efficacy response. 
2.4.1Time-point reaction 
It is assumed that an efficacy response will occur at the specific time point of each 
regimen. Table 6 will provide a summary of the overall efficacy response of a patient 
population with disease measurable at baseline level at each time point. 
If the patient has no measured lesions (no target lesion), the evaluation is presented in 
Table 7. 
2.4.2 Missing evaluation and non-evaluable description 
If lesion imaging or measurement cannot be performed at a particular point in time, the 
patient will not be evaluated at that time point. If only some of the lesions can be evaluated in 
an evaluation, it is usually considered impossible to evaluate at that time point, unless there is 
evidence that the missing lesion does not affect the efficacy response evaluation at the 
specified time point. This situation is very likely to occur in the case of disease progression. 
For example, a patient with three lesions with a total of 50mm at baseline, but then only 
two lesions, with a total of 80mm, will be evaluated as disease progression, regardless of the 
impact of the missing lesion 
2.4.3 Best total response: all time points 
Once all the patient data are available, the best total response can be determined. 
Assessment of best total response when the study does not require confirmation of a 
complete or partial efficacy response: best efficacy response in the trial was best response at 
all time points (e. g., a patient was evaluated as SD in the first cycle, PR in the second cycle, 
PD in the last cycle, but the best total response as PR. When the best total response is 
evaluated as an SD, it must meet the minimum time from the baseline level specified in the 
protocol. If the standard of the shortest time is not met, even if the best overall response 
evaluation as SD is not approved, the best overall response of this patient will depend on the 
subsequent evaluation. For example, a patient was evaluated as SD in the first cycle and the 
second cycle as PD, but it did not meet the shortest time requirement of SD, and its best 
overall response was evaluated as PD. The same patient lost to follow-up after the first cycle 
evaluation of SD will be considered as nonevaluable. 

Assessment of the best total response when the study requires confirmation of complete 
or partial response: a complete or partial response is confirmed only if each subject meets the 
partial or complete response criteria specified in the trial and is specifically mentioned in the 
protocol at subsequent time points (usually four weeks later). In this case, the best total 
response is as described in Table 7. 
2.4.4Special tips for efficacy assessment 
When nodular lesions are included in the total target lesion assessment and the nodule 
size decreases to a "normal" size ( 10mm), they will still have a lesion size scan report. To 
avoid excessive assessment based on the increased nodule size, the measurements will be 
recorded even if the nodule is normal. As already mentioned, this means that subjects with 
complete response will not be recorded as 0 on the CRF table. 
During the trial. The analytical plan of the trial must state that these missing data / 
assessments can be explained when determining efficacy. For example, in most trials, the 
response of a subject PR-NE-PR can be confirmed as the efficacy. 
It should be reported as symptomatic progression when the subject experienced an 
overall deterioration of his or her health, but with no objective evidence. Objective 
progression should be assessed even after treatment termination. Symptomatic deterioration 
is not an objective assessment of assessment: it is the reason for discontinuing treatment. The 
objective response of such subjects will be assessed by the target and non-target lesion 
conditions shown in Table 6 to 8. 
Defined as early progression, early death and nonevaluable conditions are study 
exceptions and should be clearly described in each protocol (depending on the treatment 
interval and treatment cycle). 
In some cases, it is difficult to identify local lesions from normal tissue. When 
evaluation of complete response is based on such a definition, we recommend biopsy before 
efficacy evaluation of local focal complete response. When some subjects with abnormal 
local lesion imaging findings are considered to represent lesion fibrosis or scar formation, 
FDG-PET is used as a similar assessment criterion to biopsy to confirm the efficacy of 
complete response. In such cases, the application of FDG-PET should be prospectively 
described in the protocol, while supported by reports of the specialist medical literature for 
this situation. However, it must be realized that the limitation of FDG-PET and biopsy itself 
(including their resolution and sensitivity) will lead to false positive results in complete 
remission evaluation. 

Table 6 Time point response: Subjects with target lesions (including or excluding non- 
target lesions) 
Target focus 
Non-target lesions 
New lesions 
Total relief 
CR 
CR 
mistake 
CR 
CR 
Non-CR / non-PD 
mistake 
PR 
CR 
Can't evaluate 
mistake 
PR 
PR 
Non-progressive or 
could not be fully 
assessed 
mistake 
PR 
SD 
Non-progressive or 
could not be fully 
assessed 
mistake 
SD 
Not fully evaluated 
PD 
Non-progression 
Any situation 
mistake 
Yes or no 
 
PD 
Any situation 
PD 
Yes or no 
PD 
Any situation 
Any situation 
Yes 
PD 
CR= complete 
remission 
PR= partial 
remission 
SD= stable 
disease 
PD= progression 
disease 
NE= Not 
assessable 
 
Table 7 Time point responses-Only subjects with non-target lesions 
Non-target lesions 
New lesions 
Total relief 
CR 
mistake 
CR 
Non-CR or non-PD 
mistake 
Non-CR or non-PD 
Not fully evaluated 
mistake 
Can't evaluate 
Can not be clearly defined 
Yes or no 
PD 
for the PD 
 
 
Note: For non-target lesions, "non-CR/non-PD" refers to the therapeutic effect that is better than SD. Since SD is 
increasingly being used as an endpoint for evaluating therapeutic effects, a therapeutic effect for non-CR/non-PD has been 
defined to target situations where no lesions are measurable. 
 
Treatment with ambiguous progressive findings (e. g. very small uncertain new lesions: 
preexisting cystic or neurotic lesions) can be continued until the next evaluation. If disease 

progression is confirmed in the next assessment, the date of progression should be the date of 
prior suspected progression。 
Table 78 Optimal overall response for CR and PR efficacy 
Total 
remission at 
the first time 
point 
Total 
remission at 
the subsequent 
time point 
Best total relief 
CR 
CR 
CR 
CR 
PR 
SD，PD or PRa 
CR 
SD 
SD if SD lasts sufficient duration, otherwise PD 
CR 
PD 
SD if SD lasts sufficient duration, otherwise PD 
CR 
NE 
SD if SD lasts sufficient duration, otherwise NE 
PR 
CR 
PR 
PR 
PR 
PR 
PR 
SD 
SD 
PR 
PD 
SD if SD lasts sufficient duration, otherwise PD 
PR 
NE 
SD if SD lasts sufficient duration, otherwise NE 
NE 
NE 
NE 
Note: CR is complete response, PR is response, SD for stable disease, PD is progressive disease, and NE is not evaluable. 
Superscript "a": If CR truly occurs at the first time point and any disease appears at subsequent time points, even if the 
subject meets the PR criteria relative to baseline, the therapeutic response evaluation at subsequent time points will still be 
PD (because disease recurrence after CR). The best response depends on whether SD occurs within the shortest treatment 
interval. However, sometimes when the first evaluation is CR, but subsequent scans suggest that small lesions still appear, 
the subject's actual response at the first time point should be PR rather than CR. In this case, the initial CR judgment should 
be revised to PR, and the best response is PR.。 
 
2.5. Frequency of tumor reevaluation 
The frequency of tumor reevaluation during treatment is determined by the treatment 
regimen and should be consistent with the type and schedule of treatment. However, in phase 
trials where the benefit of treatment is unclear, follow-up every 6 to 8 weeks (designed at the 
end point of a cycle) is reasonable, and the length of the interval can be adjusted under 
special protocol or circumstances. The protocol should specify which tissue sites require 
baseline evaluation (usually those most likely to be closely associated to the metastatic lesion 
of the tumor type studied) and the frequency of evaluation repeats. Normally, target lesions 
and non-target lesions should be evaluated at each evaluation. In some optional cases, some 

non-target lesions can be evaluated less frequently. For example, the bone scan should be 
repeated when the efficacy evaluation of the target disease is confirmed as CR or bone lesion 
progression is suspected. 
After completion of treatment, the tumor re-evaluation depends on taking the response 
rate or the time to an event (progression / death) as the clinical trial endpoint. Time for an 
event (e. g. TTP / DFS / PFS) requires the routine repeat evaluation specified in the protocol. 
In particular, in randomized comparative trials, scheduled evaluations should be included in 
the schedule (e. g., 6 to 8 weeks during treatment, or 3 to 4 months after treatment) and 
should not be affected by other factors, such as treatment delay, dosing interval, and any other 
events that may lead to unbalanced treatment arm in the choice of disease evaluation time. 
2.6. Efficacy assessment / confirmation of remission period 
2.6.1. Confirmation 
For non-randomized clinical studies with efficacy as the primary study endpoint, the 
efficacy of PR and CR must be confirmed to ensure that efficacy is not the result of 
misevaluation. This also allows for a reasonable interpretation of the results where historical 
data are available, but the efficacy in the historical data of these trials should also have been 
confirmed. However, in all other cases, such as randomized trials (or periods) or studies with 
disease stability or disease progression as the primary endpoint, efficacy confirmation is no 
longer required because this is of no value in the interpretation of trial results. Removing the 
requirement for efficacy confirmation, however, makes the central review even more 
important, especially in unblinded experimental studies. 
In the case of SD, at least one measurement during the shortest time interval after the 
start of the trial (generally not less than 6 to 8 weeks) will meet the SD criteria specified in 
the protocol. 
2.6.2 Overall remission period 
The total remission period was from the time of measuring the first CR or PR (which was 
measured first) to the time of the first true record of disease recurrence or progression (using the 
minimum measurement recorded in the trial as a reference for disease progression). The time to 
total complete response was from time to first meeting CR criteria to time of first true recording 
of disease relapse or progression. 
2.6.3. Stable disease period 
Is the time from the start of treatment to disease progression (in the randomized trial, from 
the time of randomization), with the smallest sum in the trial as a reference (if the sum of 
baseline is the minimum, as the reference for PD calculation). The clinical relevance of 

disease stabilization varies between studies and different diseases. If the proportion of the 
patients maintaining the shortest time stability period is used as the study endpoint, the 
protocol should specifically state the shortest time interval between the two measurements in 
the SD definition. 
Note: Response, stabilization, and PFS were affected by the frequency of follow-up after 
baseline evaluation. Defining standard follow-up frequency is not within this guideline. The 
frequency of follow-up should consider many factors, such as disease type and stage, 
treatment cycle and standard norms. However, if inter-trial comparisons are required, the 
endpoint accuracy limitations of these measurements should be considered. 
2.7.PFS/TTP 
2.7.1. Ⅱ Phase of the clinical trial 
This guideline focuses on the use of objective remission as a research endpoint in phase 
clinical trials. In some cases, remission rates may not be optimal for evaluating the potential 
anticancer activity of new drugs / novel regimens. In these cases, PFS / PPF at the 
demarcation time points can be considered a suitable surrogate indicator for the original 
signal that provides the biological activity of new drugs. But it is clear that in a noncontrolled 
trial, these evaluations will be questioned, because seemingly valuable observations may be 
related to biological factors such as patient screening, rather than the role of pharmacological 
interventions. Therefore, phase clinical trials with these as study endpoints are best designed as 
randomized controls. But the clinical manifestations of some tumors is consistent (usually 
always poor) and non-randomized trials are reasonable. However, in these cases, the evidence of 
efficacy should be carefully documented when assessing the expected PFS or PPF due to the 
lack of positive controls. 

6.3 Attachment 4: The EORTC QLQ-C30 scale and the EORTC-QLQ-
CR29 scale 
1）EORTC Quality of Life Measurement Scale QLQ-C30 (V3.0) 
We would like to know something about you and your health, please answer all the 
questions for yourself. There is no "right" or "wrong" here, but only to draw circles on the 
number that best reflects your situation. The information you provide will be kept strictly 
confidential. 
 
 
Not 
have 
some match extraordinary 
1 
Do you have difficulty engaging in some laborious 
activities, Like carrying a very heavy shopping bag 
or a suitcase? 
1 
2 
3 
4 
2 
Is long-distance walking so difficult for you? 
1 
2 
3 
4 
3 
Is it difficult for you to walk over a short distance 
outdoors？ 
1 
2 
3 
4 
4 
Do you need to stay in bed or in a chair during the 
day？ 
1 
2 
3 
4 
5 
Do you need help while eating, dressing, bathing, or 
going to the bathroom？ 
1 
2 
3 
4 
 
 
 
 
 
 
 
In the past one week： 
Not 
have 
some match extraordinary 
6 
Are you restricted in your work and daily 
activities？ 
1 
2 
3 
4 
7 
Are you restricted in engaging in your hobbies or 
leisure activities? 
1 
2 
3 
4 

8 
Do you have the gas to promote？ 
1 
2 
3 
4 
9 
Do you have any pain？ 
1 
2 
3 
4 
10 Do you need a break？ 
1 
2 
3 
4 
11 Do you have trouble sleeping？ 
1 
2 
3 
4 
12 Do you feel weak？ 
1 
2 
3 
4 
13 Do you lose your appetite (have no appetite)？ 
1 
2 
3 
4 
14 Do you feel sick？ 
1 
2 
3 
4 
15 Do you vomit？ 
1 
2 
3 
4 
16 Do you have constipation？ 
1 
2 
3 
4 
 
 
 
 
 
 
 
In the past one week： 
Not 
have 
some match extraordinary 
17 Do you have any diarrhea？ 
1 
2 
3 
4 
18 Do you feel tired？ 
1 
2 
3 
4 
19 Does the pain affect your daily activities？ 
1 
2 
3 
4 
20 
Do you have difficulty concentrating on doing 
things, such as reading a newspaper or watching 
TV？ 
1 
2 
3 
4 
21 Do you feel nervous？ 
1 
2 
3 
4 
22 Do you feel worried？ 
1 
2 
3 
4 
23 Do you feel hot-tempered？ 
1 
2 
3 
4 

24 Do you feel depressed (depressed)？ 
1 
2 
3 
4 
25 Do you feel difficulty remembering？ 
1 
2 
3 
4 
26 
Does your physical condition or treatment affect 
your family life？ 
1 
2 
3 
4 
27 
Does your physical condition or treatment affect 
your family activities？ 
1 
2 
3 
4 
28 
Does your physical condition or treatment get you 
into financial difficulties？ 
1 
2 
3 
4 
 
 
 
 
 
 
 
For the following questions, select a number best for you between 1 and 7 and circle. 
29 How do you evaluate your overall health in the past week？ 
 
1 
2 
3 
4 
5 
6 
7 
 
very bad 
 
 
 
 
 
beyond compare 
30 How do you evaluate your total quality of your life over the past week？ 
 
1 
2 
3 
4 
5 
6 
7 
 
very bad 
 
 
 
 
 
beyond compare 
 
The EORTCQLQ-C30 scale, which the European Organization for Research on Cancer 
Treatment (EORTC: The European Organization for Research and Treatment of Cancer), 
launched the cross-cultural and cross-national QOL-C30 (Quality of Life Questionnare- 
Core 30) in 1993, Evaluation QOL from a multidimensional perspective, Can better reflect 
the QOL connotation, Has been applied to QOL measurements in cancer patients in 
multiple European countries and regions 
1. Calculation of the entry score 

The EORTC QLQ- -C30 (V3.0) is a core scale for all cancer patients with a total of 30 
entries. Among them, items 29 and 30 are divided into seven levels, ranging from 1 to 7 
points according to their answer options: other items are divided into four levels: from 
none, one point, more to many, directly rated from 1 to 4 points. 
dimension 
Number 
of 
indicators 
content 
Physical function 
5 
Physical activity: carrying objects, walking, staying in bed 
for days and basic self-care conditions 
Role function 
2 
Whether daily activities, work and hobbies are restricted 
emotional function 
4 
Tension, worry, emotional control ability 
cognitive function 
2 
Whether it is difficult to remember things, and whether you 
can concentrate on doing things 
social function 
2 
Family life and social activities are hindered 
Overall health status / quality of life 
2 
Self-rated overall health status and overall quality of life 
physical symptom 
12 
Fatigue, nausea and vomiting, pain, dysphagia, sleep, 
disturbance, and decreased appetite 
Health-related economic 
1
situation 
1 
The impact of the disease and the treatment on the patient's 
economy 
 
2. Calculation of the domain (dimension) score (coarse score) 
For the convenience of statistical analysis and application, the scales are often divided 
into certain fields (domain). The domain is an aspect of the quality of life component, also 
known as dimensionality (dimension), when analyzed as an independent variable. 
EORTC QLQ- -The 30 items of C30 (V3.0) can be divided into 15 areas, with 5 
functional domains (body, role, cognitive, emotional, and social function), 3 symptom 
domains (fatigue, pain, nausea and vomiting), 1 general health / quality of life area, and 6 
single items (each as one area). The score of the field is obtained by adding up and dividing 
it by the number of items included in the field (coarse RS, Raw Score), namely RS=
（Q1+Q2+Q？）／n。 
QLQ-C30（V3.0）Scoring method for each fields (coarse RS） 

Field (dimension) 
proper 
Number of 
entries 
Score full 
distance（R） 
scoring method 
somatic function 
functional 
form 
5 
3 
(Q1+Q2+Q3+Q4+Q5)/5 
Role function 
functional 
form 
2 
3 
(Q6+Q7)/2 
emotional function 
functional 
form 
4 
3 
(Q21+Q22+Q23+Q24)/4 
cognitive function 
functional 
form 
2 
3 
(Q20+Q25)/2 
social function 
functional 
form 
2 
3 
(Q26+Q27)/2 
Total health status 
 
2 
6 
(Q29+Q30)/2 
tired 
Symptomatic 
type 
3 
3 
(Q10+Q12+Q18)/3 
nausea and 
vomiting 
Symptomatic 
type 
2 
3 
(Q14+Q15)/2 
pain 
Symptomatic 
type 
2 
3 
(Q9+Q19)/2 
anhelation 
Symptomatic 
type 
1 
3 
Q8 
lose sleep 
Symptomatic 
type 
1 
3 
Q11 
Loss of appetite 
Symptomatic 
type 
1 
3 
Q13 
astriction 
Symptomatic 
type 
1 
3 
Q16 
diarrhea 
Symptomatic 
type 
1 
3 
Q17 
economic hardship 
Symptomatic 
type 
1 
3 
Q28 
 
3. Calculation of standard and chemical scores 

In order to make the scores of each field compare with each other, the extreme 
differential method is further used for linear transformation to convert the coarse score into 
a standardized score (standard score, SS) within 0 100. In addition, the purpose is to change 
the direction of the score. Because QLQ-C30 scale, except item 29,30 are reverse entry (the 
greater the value, the worse the quality of life), and in the scoring rules: the 
functional and overall health field score higher that functional condition and life 
quality is better, the higher the score for symptom areas indicates that the more symptoms 
or problems (worse quality of life). Therefore, the calibration time of the computing 
function field has to change the direction. Specifically, the following formula is calculated 
respectively (where R is the full score distance of each field or item). 
Functional area: SS= [l- (RS-1) / R] 100 
Symptom areas and general health status areas: SS=[（RS-1)／R]×100 
 
2）The EORTC-QLQ-CR29 scale 
We would like to know something about you and your health, please answer all the 
questions for yourself. There is no "right" or "wrong" here, but only to draw circles on the 
number that best reflects your situation. The information you provide will be kept strictly 
confidential. 
 
In the past wee 
Not 
have 
some match extraordinary 
31 
Have you urinate  frequently during the day in the 
past week？ 
1 
2 
3 
4 
32 
Have you urinate  frequently during the day in the 
past week？ 
1 
2 
3 
4 
33 Have you involuntarily urinate or leaked in the 
past week？ 
1 
2 
3 
4 
34 Have you had any pain in urinating in the past 
week？ 
1 
2 
3 
4 

35 Have you had any abdominal pain in the past 
week？ 
1 
2 
3 
4 
36 Have you had pain in the hip, anal area or rectum 
in the past week？ 
1 
2 
3 
4 
37 Have you filled your abdomen in the past week？ 
1 
2 
3 
4 
38 Have you had blood stools in the past week？ 
1 
2 
3 
4 
39 Did you have mucus in your stool in the past 
week？ 
1 
2 
3 
4 
40 What have you done in the past week？ 
1 
2 
3 
4 
41 Have you treated your hair loss in the past week？ 
1 
2 
3 
4 
42 Have you had any problems with your taste 
patterns in the past week？ 
1 
2 
3 
4 
43 Have you been worried about your future health 
in the past week？   
1 
2 
3 
4 
44 Have you been worried about your weight in the 
past week？ 
1 
2 
3 
4 
45 Have you experienced less illness or treatment in 
the past week？ 
1 
2 
3 
4 
46 Have you felt that illness or treatment has reduced 
your feminine / masculine taste in the past week？ 
1 
2 
3 
4 
47 Have you been dissatisfied with your body in the 
past week？ 
1 
2 
3 
4 
 
 
 
 
 
 

48 Do you have a stoma pocket（colostomy or 
ileostomy）？ (select Yes/No) 
Yes 
No 
 
 
 
Question 48 for patients with ”yes”, please 
answer the following questions：  
Not 
have 
some match extraordinary 
49 In the past week, have you had an involuntary 
pocket exhaust or flatulence？ 
1 
2 
3 
4 
50 Have you leaked in your pocket in the past 
week？ 
1 
2 
3 
4 
51 Have you had pain in the skin around the fistula in 
the past week？ 
1 
2 
3 
4 
52 Have you changed your pockets frequently during 
the day in the past week？ 
1 
2 
3 
4 
53 Have you changed your pocket frequently at night 
in the past week？ 
1 
2 
3 
4 
54 Have you been embarrassed in the past week？  
1 
2 
3 
4 
55 Have you had difficulty nursing the fistula in the 
past week？ 
1 
2 
3 
4 
 
 
 
 
 
 
 
Question 48 for patients with ”No”, please 
answer the following questions：：  
Not 
have 
some match extraordinary 
49 In the past week, have you had an involuntary 
pocket exhaust or flatulence？ 
1 
2 
3 
4 
50 Have you leaked in your pocket in the past 
week？  
1 
2 
3 
4 

51 Have you had pain in the skin around the fistula in 
the past week？ 
1 
2 
3 
4 
52 Have you changed your pockets frequently during 
the day in the past week？  
1 
2 
3 
4 
53 Have you changed your pocket frequently at night 
in the past week？ 
1 
2 
3 
4 
54 Have you been embarrassed in the past week？  
1 
2 
3 
4 
 
 
 
 
 
 
 
In the past 4 weeks, men should answer 
questions 56、57 
Not 
have 
some match 
extraordinary 
56 How interested have you been in sex in the last 4 
weeks？ 
1 
2 
3 
4 
57 Have you had difficulty achieving or maintaining 
an erection in the last 4 weeks？ 
1 
2 
3 
4 
 
 
 
 
 
 
 
In the past 4 weeks, women should answer 
questions 58、59 
Not 
have 
some match 
extraordinary 
58 How interested have you been in sex in the last 4 
weeks？ 
1 
2 
3 
4 
59 Have you had any pain or discomfort during sexual 
intercourse in the last 4 weeks？ 
1 
2 
3 
4 

The Colorectal Cancer Quality of Life Questionnaire (QLQ-CR29), a module of the 
Quality of Life Questionnaire-Core 30 (QLQ-C30), developed by the European 
Organization for Research and Treatment of Cancer to assess quality of life for specific 
aspects of colorectal cancer in cancer patients. 

6.4Attachment 5: Anal Incontinence Wexner Scale Scale 
一，Scale introduction 
1. Evaluation method: conducted by doctors or test-experienced personnel. 
2. Scale function: The Wexner incontinence scale was prepared by Wexner, Jorge 
and other researchers to assess the severity of patient defecation. 
3. Applicable population: patients with fecal incontinence caused by various 
reasons. 
二，operating guide 
1. Content of the scale: The scale mainly includes four aspects of stool morphology, 
gastrointestinal incontinence, wearing pads and lifestyle, lasting for more than 4 
weeks. There are 5 items, and each score is calculated according to Table 1. 
2. Results analysis: the total score is the sum of all scores, the score range is 0-20 
points, 0 points = normal, 20 points = complete incontinence. 
Table 1 The Wexnei’ scoring scale of anal incontinence 
variable 
never 
once in 
a while 
sometimes 
often 
always 
score 
exhaust 
0 
1 
2 
3 
4 
 
Loose stools 
0 
1 
2 
3 
4 
 
formed stool 
0 
1 
2 
3 
4 
 
Sanitary belt 
0 
1 
2 
3 
4 
 
Lifestyle change 
0 
1 
2 
3 
4 
 
total points 
（      ）scores 
 
三，Results and interpretation 
Total score：（）scores。 
Explanation: Wexner score scale introduces the analysis index is the total score, the 
score range is 0-20 points, 0 points = normal, 20 points = complete incontinence.