Neoadjuvant Chemoradiotherapy Combined With Tislelizumab With or Without Probio-M9 in pMMR/MSS Locally Advanced Middle and Low Rectal Cancer: A Single-Center, Prospective, Randomized Controlled Trial (NCRIT-PM9 Trial)
Phase: PHASE2
Conditions: Rectal Cancer, Adenocarcinoma, Neoadjuvant Therapy, Immunotherapy, Probio-M9, Tislelizumab, Randomized
Sponsor: Seventh Medical Center of PLA General Hospital

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Neoadjuvant chemoradiotherapy combined with PD-1 monoclonal 
antibody (tislelizumab) and Probio-M9 for the treatment of 
pMMR/MSS in locally advanced mid-low rectal cancer: A single-
center, prospective, randomized controlled study 
Research Plan 
I. Research Background 
Rectal cancer is one of the most common digestive tract tumors in 
China, with 253,000 new cases in China each year, accounting for about 
18.6% of the world's total. With changes in people's lifestyles and the 
improvement of living standards, the incidence of rectal cancer is still 
gradually increasing [1] . According to the 2017 edition of the Chinese 
Expert Consensus on the Diagnosis and Treatment of Locally Advanced 
Rectal Cancer, locally advanced rectal cancer (LARC) is currently 
defined as: rectal cancer in which the primary tumor, as discovered by 
pathological examination or imaging, invades the muscular layer of the 
intestinal wall and extends to the surrounding tissues (c/p T3-4) or lymph 
node metastasis occurs in the mesentery and true pelvis (c/p N1-2) 
without distant metastasis (M0) [2] . Rectal cancer has an insidious onset, 
and early clinical symptoms are not obvious. Moreover, most people lack 
knowledge about the diagnosis and treatment of rectal cancer. Therefore, 
about 75% of the clinically diagnosed rectal cancer cases in China are 
already in the middle and late stages, and some patients have even lost the 
opportunity for surgery when they seek medical attention [3] . Among 

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them, mid-to-low rectal cancer (the distance between the tumor and the 
anus is ≤10cm) accounts for the majority of rectal cancer patients 
diagnosed in my country. Due to its proximity to the anus, the rate of anal 
preservation is low, and patients have a poor quality of life after surgery. 
For patients with locally advanced rectal cancer, the current standard 
treatment regimen is neoadjuvant chemoradiotherapy (nCRT) combined 
with 
total 
mesenteric 
resection 
(TME), 
including 
long-course 
chemoradiotherapy (LCRT, 50 Gy/25 Fx, concurrent 5-FU or 
capecitabine) and short-course radiotherapy (SCRT, 25 Gy/5 Fx). 
However, the pathologic complete response ( pCR) rate for patients 
receiving neoadjuvant chemoradiotherapy before surgery is currently 
about 11%-15%, which still needs to be improved [4-5] . Meanwhile, some 
literature reports that the overall survival (OS) of neoadjuvant 
chemoradiotherapy before surgery is not significantly improved 
compared to postoperative adjuvant chemoradiotherapy [6-7] . In recent 
years, immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway 
(PD-1/PD-L1 monoclonal antibodies) have provided a new treatment 
option for malignant tumors. The Keynote-177 trial showed that PD-1 
monoclonal antibodies have good efficacy and safety in patients with 
advanced colorectal cancer (CRC) with high microsatellite instability 
(MSI-H) or loss of mismatch repair (dMMR) [8] . The NICHE trial was 
the first study to evaluate the efficacy and safety of immunotherapy in 

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neoadjuvant therapy for colorectal cancer patients. The results showed 
that neoadjuvant immunotherapy was safe and showed good tumor 
response [9] . However, dMMR/MSI-H patients account for only 5%-20%, 
and the vast majority of CRC patients are patients with no loss of 
mismatch repair (pMMR) or microsatellite stable (MSS). Improving the 
sensitivity of pMMR/MSS patients to immunotherapy remains a 
challenge. 
A neoadjuvant study initiated by Zhang Zhongtao's team in China (a 
single-arm, multicenter, phase II clinical trial of neoadjuvant 
chemoradiotherapy combined with tislelizumab for locally advanced mid-
to-low rectal cancer, ClinicalTrials.cn registration number: NCT04911517) 
enrolled patients with initially resectable, solitary, mid-to-low rectal 
adenocarcinoma. Patients received neoadjuvant chemoradiotherapy 
combined with tislelizumab. From April 2021 to June 2022, 46 patients 
with pMMR/MSS locally advanced rectal cancer underwent surgical 
treatment. The median age was 63 years, and the median distance of the 
tumor from the anal verge was 5.1 cm. Efficacy analysis showed that 20 
patients 
achieved 
pathological 
complete 
response 
(pCR) 
after 
neoadjuvant chemoradiotherapy with tislelizumab (pCR rate 43.5%). The 
objective response rate was 76.1%, and the radical resection rate was 
100%. The incidence of grade 3 or higher treatment-related adverse 
events (irAEs) after treatment was 4.3% (2/46), indicating that this 

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treatment strategy has good safety [10-11] . 
Professor Zhang Heping of China isolated a probiotic strain, 
Lactobacillus rhamnosus Probio-M9, from human colostrum [12] and 
verified its role in tumor prevention and synergistic therapy [13-14] . The 
team demonstrated that intestinal microbiota dysbiosis caused by 
inflammation or antibiotics can be effectively restored after treatment 
with Probio-M9, thereby improving the anti-tumor efficacy of colorectal 
cancer treatment. Further studies have shown that Probio-M9 promotes 
the growth of beneficial intestinal microorganisms (such as lactobacillus 
and bifidobacterium animalis), produces beneficial metabolites including 
butyrate in the intestine, and accumulates α-ketoglutarate, N-acetyl-L-
glutamate and pyridoxine from the blood, thereby promoting the 
infiltration and activation of cytotoxic T lymphocytes (CTLs) and 
inhibiting the function of regulatory T cells (Tregs) in the tumor 
microenvironment (TME), thereby enhancing the immunotherapy 
response [15] . 
In recent years, the gut microbiota, as an important immune 
regulatory hub, has been recognized as a key factor influencing the tumor 
immune microenvironment and the efficacy of immunotherapy, and has 
gradually become an important research direction for overcoming the 
bottlenecks in immunotherapy. Numerous basic and translational studies 
have shown that the gut microbiota can influence the local immune status 

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of tumors through its metabolites and immunomodulatory effects, thereby 
regulating the intensity and persistence of anti-tumor immune responses. 
Probio-M9 (Lactobacillus rhamnosus), a marketed food-grade 
probiotic strain with clear immunomodulatory potential ( Production 
License No.: SC10633070200513, Standard: Q/JYH0006S) , has been 
shown in previous studies to participate in regulating the tumor 
microenvironment 
through 
its 
metabolites, 
improving 
immunosuppression, and to some extent enhancing the anti-tumor effect 
of immunotherapy . However, its clinical application value in patients 
with pMMR/MSS locally advanced rectal cancer, especially its 
synergistic effect in the context of neoadjuvant therapy, still lacks a 
systematic review of prospective randomized controlled trials. 
The necessity of this study lies in exploring whether Probio-M9 can 
synergistically modulate the tumor immune microenvironment through 
the introduction of gut microbiota intervention, thereby promoting the 
transformation from immunosuppressive "cold tumors" to immune-
activated "hot tumors," and ultimately improving the pathological 
complete response rate (pCR) and sphincter-preserving rate in 
pMMR/MSS rectal cancer patients. This research not only holds promise 
for providing a new supplementary strategy to existing neoadjuvant 
therapy modalities but also offers new insights and clinical evidence for 
the exploration of precision treatment for refractory MSS rectal cancer. 

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References: 
1.W. Chen, R. Zheng, PD Baade, S, et al. Cancer statistics in China, 2015, 
CA Cancer J Clin 66(2) (2016) 115-32. 
2. Peng Junjie, Zhu Ji, Liu Fangqi, Chen Zhiyu, Tong Tong, Huang Dan, 
Li Wenhua, Yang Lifeng. Expert consensus on the diagnosis and 
treatment of locally advanced rectal cancer in China [J]. Chinese Journal 
of Cancer, 2017, 27(01):41-80. 
3. Zhu J , Liu A , Sun X , et al. Multicenter, Randomized, Phase III Trial 
of Neoadjuvant Chemoradiation With Capecitabine and Irinotecan 
Guided by UGT1A1 Status in Patients With Locally Advanced Rectal 
Cancer. Journal of clinical oncology 2020; 38(36):4231-4239. 
4. Gérard J, Conroy T, Bonnetain F, et al. Preoperative radiotherapy with 
or without concurrent fluorouracil and leucovorin in t3-4 rectal cancers: 
results of ffcd 9203. J Clin Oncol. 2006;24(28):4620-5. 
5. Bosset J, Calais G, Mineur L, et al. Enhanced tumorocidal effect of 
chemotherapy with preoperative radiotherapy for rectal cancer: 
preliminary results-EORTC 22921. J Clin Oncol. 2005;23(24):5620-7. 
6. Bosset JF, Collette L, Calais G, et al. Chemotherapy with preoperative 
radiotherapy in rectal cancer. N Engl J Med. 2006;355(11):1114-23. 
7. Wu F, Zhou C, Wu B, et al. Adding Adjuvants to Fluoropyrimidine-
based Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal 

number : V1.1    Version Date: January 26, 2026 
 7/ 39 
 
Cancer: An Option Worthy of Serious Consideration. J Cancer. 2021; 
12(2):417-427. 
8. Andre T, Shiu KK, Kim TW, et al. Pembrolizumab in microsatellite-
instability-high 
advanced 
colorectal 
cancer. 
N 
Engl 
J 
Med. 
2020;383(23):2207–18. 
9. Verschoor YL, van den Berg J, Beets G, et al: Neoadjuvant nivolumab, 
ipilimumab, and celecoxib in MMR-proficient and MMR-deficient colon 
cancers: Final clinical analysis of the NICHE Study. Am Soc Clin Oncol 
2022. 
10. Yao H, Yang Z, Gao J, et al. Safety and efficacy evaluation of long 
course neoadjuvant chemoradiotherapy plus tislelizumab followed by 
total mesorectal excision for locally advanced rectal cancer: Short-term 
results of a multicenter, phase II study. J Clin Oncol, 2022 (suppl 16): 
e15599. 
11. Yang Z, Gao J, Zhang 
12. Liu W, Chen M, Duo L, Wang J, Guo S, Sun H, Menghe B, Zhang H. 
Characterization of potentially probiotic lactic acid bacteria and 
bifidobacteria isolated from human colostrum. J Dairy Sci. 2020 
May;103(5):4013-4025. 
2021 Mar 30;13(4):1143. 
14. Gao G, Ma T, Zhang T, Jin H, Li Y, Kwok LY, Zhang H, Sun Z. 
Adjunctive Probiotic Lactobacillus rhamnosus Probio-M9 Administration 

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Enhances the Effect of Anti-PD-1 Antitumor Therapy via Restoring 
Antibiotic-Disrupted Gut Microbiota. Front Immunol. 2021 Dec 
14;12:772532. 
15. Gao G, Shen S, Zhang T, Zhang J, Huang S, Sun Z, Zhang H. 
Lacticaseibacillus rhamnosus Probio-M9 enhanced the antitumor 
response to anti-PD-1 therapy by modulating intestinal metabolites. 
EBioMedicine. 2023 May;91:104533. 
 
II. Research Objectives and Significance 
(1) By selecting suitable enrolled patients during the clinical diagnosis 
and treatment process and completing the treatment according to the 
study plan, we will evaluate the moderating effect of neoadjuvant 
chemoradiotherapy 
combined 
with 
PD-1 
monoclonal 
antibody 
(tislelizumab) and Probio-M9 treatment on the efficacy of pMMR/MSS 
locally advanced mid-low rectal cancer . 
(2) To explore the feasibility of organ-sparing surgery (non-TME surgery) 
or a wait-and-see strategy for patients who have achieved clinical 
complete remission or are expected to achieve pathological complete 
remission after the above treatments, and the impact on patients' 
functional and quality of life. This will provide new options for the 
treatment of locally advanced rectal cancer. 
  

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III. Research Content 
This study aims to include patients with locally advanced mid-to-
low rectal cancer and evaluate the moderating effects of neoadjuvant 
chemoradiotherapy 
combined 
with 
PD-1 
monoclonal 
antibody 
(tislelizumab) and the probiotic Probio-M9 as a special food intervention 
on the efficacy of locally advanced rectal cancer . Given that Probio-M9 
is already manufactured and marketed as a common food/dietary 
supplement by a relevant company ( Production License No.: 
SC10633070200513, Standard: Q/JYH0006S), this study will explore its 
synergistic value in standardized clinical treatment regimens. 
 
IV. Research Design 
1. Research Types and Overall Design 
This study was a prospective, randomized, controlled clinical 
trial . All subjects who met the inclusion criteria were randomly assigned 
to the experimental or control group in a 1 :1 ratio after completing the 
assessment during the screening period. 
2. Randomized controlled trial method 
A simple randomization method (without stratification) is used, 
where a random assignment system generates random sequences, and 
patients are divided into groups at a 1 :1 ratio: 
• Experimental group (Group A) : Received neoadjuvant 

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chemoradiotherapy + PD-1 monoclonal antibody (tislelizumab) + 
Probio-M9 ( this product is a marketed ready-to-eat probiotic food, 
classified as a general food/dietary supplement, taken orally once 
daily, 2g each time ) ; 
• Control 
group 
(Group 
B) 
: 
Received 
neoadjuvant 
chemoradiotherapy + PD-1 monoclonal antibody (tislelizumab) + 
placebo (oral, once daily). 
3. Sample size and calculation basis 
Currently, there are no relevant clinical studies. The studies available 
on clinical tirals are shown in the following figure: 
 
This project employs a 1 :1 randomized controlled trial (dr.:control) 
allocation, with an expected complete remission rate of 30% in the 
control group and 66% in the experimental group, and a dropout rate of 
8%. The final required sample size is 50 cases, with 25 cases in the 
experimental group and 25 cases in the control group. 
 
ClinicalTricals.gov. ID
研究发起中心
研究开始时间
预计入组
人数
疾病
治疗方案
研究设计
NCT05122546
美国希望之城医
疗中心
2021/11/1
30
转移性
肾细胞
nivolumab + cabozantinib S-
malate ± CBM588
RCT研究
NCT06865521
四川华西医院
2025/2/21
22
晚期结
直肠癌
anti-PD-1 monoclonal antibody +
TKI treatment/anti-PD-1
monoclonal antbody ±
Chemotherapy ± Bevacizumab
treatment + Akkermansia
probiotics
单臂队列
研究
NCT05094167
江西省肿瘤医院
2021/10/19
46
非小细
胞肺癌
Carilizumab+Platirum ±
Lactobacillus Bifidobacterim
V9(Kex02)
RCT研究

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4. Test subjects 
The target population of this study is patients with pathologically 
confirmed locally advanced mid-to-low rectal cancer who meet the 
indications for neoadjuvant chemoradiotherapy, are expected to receive 
neoadjuvant therapy based on chemoradiotherapy combined with PD-1 
monoclonal antibody therapy, and are deemed to have surgical or organ-
sparing potential after evaluation by a multidisciplinary team (MDT). The 
study focuses on evaluating changes in gut microbiota, efficacy, and 
safety in these patients after receiving adjuvant therapy with the probiotic 
Probio-M9. 
5 Intervention measures 
• Experimental 
group 
(Group 
A) 
: 
Neoadjuvant 
chemoradiotherapy + PD-1 monoclonal antibody (tislelizumab) + 
Probio-M9 (oral, once daily); 
• Control group (Group B) : Neoadjuvant chemoradiotherapy + 
PD-1 monoclonal antibody (tislelizumab) + placebo (oral, once 
daily). 

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Experimental group: Neoadjuvant chemoradiotherapy (50 Gy/25 
fractions, capecitabine 850-1000 mg/m², BID, PO, D1-D5, QW) was 
administered from week 1 to week 5. After radiotherapy, the patient rested 
for two weeks and then received two cycles of Capeox treatment (q3w; 
Day 1: Oxaliplatin, 130 mg/m² , IV.gtt; Day 1-Day 14: Capecitabine, 850-
1000 mg/m² , BID, PO). Tislelizumab (200 mg, IV.gtt, Q3W) was 
administered on week 2, week 5, and week 8, respectively. Probio-M9 (2 
g, QD) was administered once daily starting from week 1 . 
Control group: Neoadjuvant chemoradiotherapy (50 Gy/25 fractions, 
capecitabine 850-1000 mg/m² , BID, PO, D1-D5, QW) was administered 
from week 1 to week 5. After radiotherapy, a two-week rest period was 
followed by two cycles of Capex therapy (q3w; Day 1: Oxaliplatin, 130 
mg/m² , IV.gtt; Day 1-Day 14: Capecitabine, 850-1000 mg/m² , BID, PO). 
Tislelizumab (200 mg, IV.gtt, Q3w) was administered on week 2, week 5, 
and week 8, respectively. A placebo (2 g, QD) was administered once 
daily starting from week 1. 
6. Primary and Secondary Observation Indicators 
Primary endpoint : 
1) Major complete response rate (including clinical complete response 

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rate cCR and pathological complete response rate pCR ) 
 
Secondary endpoint : 
Table 1. AJCC 8th Edition Tumor Regression Criteria 
 
Table 2 MSKCC Tumor Regression Criteria 
 
1) Validity 
Pathological response rate (MPR); objective response rate (ORR, 
based on RECIST 1.1 criteria); disease-free survival (DFS); overall 
survival (OS); organ preservation rate (OPR); neoadjuvant therapy 
score for rectal cancer (NAR); quality of life score (QoL). 
2) Security 
3.1 Adverse events (AEs), including type, incidence, grade 
(judged according to NCI-CTCAE V5.0 criteria), severity, and 
relevance to the investigational drug; 

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3.2 Abnormal values of laboratory indicators, type, incidence, 
and grade (judged according to NCI-CTCAE V5.0 standards); vital 
signs, including blood pressure, pulse, respiratory rate, body 
temperature, and PS scores of ECG and ECOG. 
3) Changes in fecal intestinal flora (lactobacters, bifidobacteria, etc.). 
7. Safety and effectiveness evaluation 
• Safety assessment : Collect data on adverse events (AEs), serious 
adverse events (SAEs), abnormal laboratory indicators, and 
changes in vital signs; 
• Efficacy evaluation : The efficacy was comprehensively assessed 
by imaging, endoscopy, and surgical pathology. All pathological 
results were independently interpreted by two experienced rectal 
cancer pathologists, back to back. If the results differed 
significantly, a third expert was invited to review them. The 
pathological evaluation adopted the AJCC 8th edition of the 
diagnostic criteria for rectal cancer: tumor size, histological 
classification, depth of invasion, lymph node retrieval and number 
of metastases, whether the surgical margin was positive, tumor 
regression grade TRG, ypTNM staging, etc. 
8. Statistical Analysis Methods 
This study primarily employs intention-to-treat (ITT) analysis, with 
per-protocol (PP) used for sensitivity analysis. 

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Primary endpoint: To compare changes in gut microbiota abundance 
between the experimental and control groups. Initially, an independent 
samples t-test or analysis of covariance (ANCOVA, adjusted baseline) 
was used to select an appropriate parametric or non-parametric method 
based on the distribution. 
Secondary 
endpoints 
included 
radiological 
response 
rate, 
pathological response rate, organ preservation rate, and disease-free 
survival rate. Response rates were compared using the χ² test or Fisher's 
exact test; survival data were estimated using the Kaplan-Meier method 
with parallel log-rank tests, and multivariate analysis was performed 
using a Cox proportional hazards model when necessary. 
Safety endpoint: Descriptive statistical methods were used to 
calculate the frequency and percentage of adverse events and to compare 
differences between groups. 
Significance level: All tests were two-tailed, with a significance 
level of α = 0.05. 
V. Technical Approach 
 
VI. Selection/Exclusion Criteria and Withdrawal/Termination Criteria for 

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Trial Subjects 
Inclusion criteria:  
1. Sign a written informed consent form and voluntarily participate in 
this study; 
2. Age 18-75 years old, gender not limited; 
3. Histopathologically confirmed rectal adenocarcinoma; 
4. Clinical stage II-III as assessed by MRI (according to AJCC 8th 
edition); 
5. The lower edge of the tumor is ≤10cm from the anal verge; 
6. Surgically resectable; 
7. Able to swallow pills normally; 
8. ECOG PS 0-1; 
9. Has not previously received any anti-tumor treatment for rectal 
cancer, including radiotherapy, chemotherapy, surgery, etc.; 
10. Surgical treatment is planned after neoadjuvant therapy is 
completed; 
11. No contraindications to surgery; 
12. Major organ functions are normal, including: 
a) Complete blood count (no blood transfusions or blood products 
administered within 14 days prior to the first treatment, and no G-CSF or 
other hematopoietic stimulating factors used for correction): 
• Neutrophil count ≥1.5×10⁹ / L 

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澳濂澳
澳
• Platelet count ≥100× 10⁹ /L 
• Hemoglobin ≥ 90 g/L 
b) Blood biochemistry tests: 
Total bilirubin ≤ 1.5 × ULN 
 ALT ≤ 2.5×ULN, AST ≤ 2.5×ULN, 
• Serum creatinine ≤ 1.5 × ULN, or creatinine clearance ≥ 50 mL/min 
(Cocheroft-Gault formula) 
c) Coagulation function: 
• International Normalized Ratio (INR) ≤ 1.5 × ULN 
• Activated partial thromboplastin time (APTT) ≤ 1.5 × ULN 
Female subjects of childbearing age must undergo a serum 
pregnancy test within 72 hours prior to the start of administration of the 
study drug, and the result must be negative; 
Exclusion criteria:  
1. History of allergy to monoclonal antibodies, any component of PD-1 
monoclonal antibodies, capecitabine, or oxaliplatin; 
2. Have previously received or are currently receiving any of the 
following treatments: 
a) Any surgical, radiotherapy, chemotherapy, targeted therapy, 
immunotherapy, etc. treatment for tumors; 
b) The patient has been using immunosuppressive drugs or systemic 
hormones for immunosuppression (dose > 10 mg/day prednisone or 

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equivalent) within 2 weeks prior to the first use of the study drug; in the 
absence of active autoimmune disease, inhaled or topical steroids and 
corticosteroids at doses > 10 mg/day prednisone or equivalent may be 
used as replacements. 
c) Received a live attenuated vaccine within 4 weeks prior to the first use 
of the investigational drug; 
d) Underwent major surgery or suffered severe trauma within 4 weeks 
prior to the first use of the investigational drug; 
3. Patients with any active autoimmune disease or a history of 
autoimmune disease, including but not limited to: interstitial pneumonia, 
enteritis, hepatitis, hypophysitis, vasculitis, nephritis, hyperthyroidism, 
hypothyroidism (can be considered after hormone replacement therapy); 
patients with psoriasis or childhood asthma/allergy that has been 
completely remitted and requires no intervention in adulthood can be 
considered, 
but 
patients 
requiring 
medical 
intervention 
with 
bronchodilators should not be included; 
4. History of immunodeficiency, including HIV test positive, or other 
acquired or congenital immunodeficiency diseases, or history of organ 
transplantation or allogeneic bone marrow transplantation; 
5. The presence of uncontrolled cardiac clinical symptoms or diseases, 
including but not limited to: (1) NYHA class II or above heart failure, (2) 
unstable angina, (3) myocardial infarction within 1 year, (4) clinically 

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significant supraventricular or ventricular arrhythmias that are not 
controlled by clinical intervention or are still poorly controlled after 
clinical intervention. 
6. Severe infection (CTCAE > 2) that occurred within 4 weeks prior to 
the first use of the study drug, such as severe pneumonia, bacteremia, or 
infectious complications requiring hospitalization; baseline chest imaging 
showing active lung inflammation; symptoms and signs of infection 
within 14 days prior to the first use of the study drug; or patients 
requiring oral or intravenous antibiotic treatment, excluding those who 
used prophylactic antibiotics. 
7. Those who have been found to have active pulmonary tuberculosis 
infection through medical history or CT examination, or who have a 
history of active pulmonary tuberculosis infection within 1 year prior to 
enrollment, or who have a history of active pulmonary tuberculosis 
infection more than 1 year ago but have not received regular treatment; 
8. Possesses active hepatitis B (HBV DNA ≥ 2000 IU/mL or 104 
copies/mL) or hepatitis C (positive hepatitis C antibody, and HCV RNA 
above the detection limit of the analytical method); 
9. Patients who have been diagnosed with other malignancies within 5 
years prior to their first use of the investigational drug may be considered 
for enrollment unless they have a low risk of metastasis or death (5-year 
survival rate >90%), such as well-treated basal cell carcinoma of the skin, 

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squamous cell carcinoma of the skin, or cervical carcinoma in situ. 
10. Pregnant or breastfeeding women; 
11. The researchers determined that there were other factors that might 
force the study to be terminated midway, such as other serious illnesses 
(including mental illnesses) requiring combined treatment, alcoholism, 
substance abuse, family or social factors that may affect the safety or 
compliance of the participants. 
Exit Criteria 
1. The following withdrawals from the study were due to patient non-
compliance and reasons not related to the treatment itself. 
2. Subjects were found to be ineligible for inclusion/exclusion criteria 
after enrollment; 
3. Serious violation of test protocol requirements; 
4. The simultaneous use of other treatments affected the evaluation; 
5. Poor patient compliance; 
6. Patients who require emergency surgical resection due to intestinal 
obstruction, intestinal perforation, intestinal bleeding, or other reasons 
after enrollment in the study; 
7. Patients who, after being enrolled in the study, experience severe 
allergic reactions or intolerance to chemotherapy, radiotherapy, or 
immunotherapy; 
8. Patients who become pregnant during the trial period or at least 3 

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months after the last dose after enrollment in the study ; 
9. Patients who request to withdraw from the study cohort for various 
reasons after being enrolled; 
10. Patients who have other non-tumor conditions that prevent them 
from continuing this treatment regimen; 
11. Patients who, after being enrolled in the study , took other 
immunosuppressants and other probiotics for various reasons ; 
1.2 . Patients who are unable to complete the research plan after being 
selected for the study for various reasons (if a patient withdraws from the 
trial, the reason and time of withdrawal should be recorded in the CRF 
form). 
VII. 
Contingency 
Plan 
for 
Handling 
Common 
Adverse 
Reactions/Serious Adverse Reactions 
Adverse event classification is detailed in Appendix 1. 
Contingency Plan 
1. Adverse Event Handling 
⚫ All subjects were required to be closely monitored for adverse events 
(AEs) and serious adverse events (SAEs) during the trial. 
⚫ For immune-related adverse reactions (such as immune colitis, 
hepatitis, thyroid dysfunction, etc.), they should be graded and 
managed in accordance with the Society for Immunotherapy of 
Cancer (SITC) guidelines for the management of adverse reactions to 

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immunotherapy; for immune-related adverse reactions of grade ≥3, 
PD-1 
monoclonal 
antibody 
should 
be 
suspended 
and 
immunosuppressive therapy such as glucocorticoids should be given. 
⚫ The toxicities associated with radiotherapy and chemotherapy (such 
as bone marrow suppression, intestinal reactions, and skin damage) 
should be assessed according to the CTCAE v5.0 standard, and the 
dosage should be adjusted or treatment delayed if necessary. 
⚫ For probiotic-related adverse reactions (such as bloating, diarrhea, 
infection), discontinue probiotic use according to the severity and 
provide symptomatic and supportive treatment. 
2. Contingency Plan for Trial Termination/Withdrawal 
⚫ If a patient experiences intolerable adverse reactions, significant 
disease progression, or if the investigator assesses that the risks of 
continuing the trial outweigh the benefits, the trial treatment should 
be terminated immediately. 
⚫ When a patient requests to withdraw from the trial, their wishes 
should be respected, and efforts should be made to complete the final 
efficacy and safety assessment , i.e., the primary and secondary 
endpoints . 
⚫ The reasons for exiting or terminating must be recorded in detail and 
included in the security analysis set. 
3. Intervention interruption or alternative solutions 

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⚫ If intervention measures are interrupted temporarily due to force 
majeure (such as an epidemic or insufficient drug supply), the 
intervention measures can be resumed according to the patient's 
specific condition , and a risk assessment record should be made. 
⚫ If necessary, surgery or other standard treatment procedures can be 
initiated earlier after MDT assessment, and intention-to-treat (ITT) 
analysis can be included in the outcome analysis. 
4. Abnormalities in drug/probiotic supply and management 
⚫ If the investigational drug or probiotic has quality problems or supply 
disruptions, the intervention should be immediately suspended and 
reported to the ethics committee and the sponsor, while a temporary 
alternative supply plan should be developed. 
5. Safety Emergency Handling 
⚫ In the event of a life-threatening serious adverse event, standard first 
aid measures should be administered immediately, and the incident 
should be reported to the ethics committee and the national adverse 
drug reaction monitoring system. 
⚫ Emergency unblinding may be performed if necessary to allow for 
targeted medical intervention (if the design is double-blind). 
VIII. Quality Control 
(1) Surgical standardization: The surgery shall be performed by an 
experienced surgeon, and all key steps of the surgery shall be documented 

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and traceable. Surgeons performing open surgery shall have at least 30 
cases of open surgery experience. Surgeons performing laparoscopic 
surgery shall have at least 30 cases of open surgery and 30 cases of 
laparoscopic surgery experience respectively. 
(2) Enrolled patients shall undergo the required examinations, 
treatments, and evaluations, and these shall be recorded in the CRF form. 
All forms must be completed completely and signed and dated by the 
person completing them. The study supervisor shall collect the enrollment 
case information and CRF forms from each center each month, urge each 
center to complete the CRF forms, and review and sign them. If the forms 
are not completed, the reason must be stated. 
(3) Clinical data and follow-up quality control: Follow-up is carried 
out by experienced professional follow-up personnel; clinical data is 
improved by gastrointestinal surgeons. 
Nine Data Statistical Methods 
Public health statisticians and principal investigators developed and 
documented a statistical analysis plan based on the research protocol. 
Statistical analyses were then conducted according to the plan. Efficacy 
analyses employed both intention-to-treat (ITT) principles and evaluable 
case analysis methods. The analysis was performed using computer 
software (IBM SPSS 25.0 or Prism 7.0). For descriptive statistics, rates 
were used for categorical data, and means and 95% confidence intervals 

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were used for continuous data. For comparisons between two groups, t-
tests were used for continuous data, and χ² tests were used for categorical 
data. Survival analyses employed Kaplan-Meier and Log Rank methods, 
and prognostic analyses used a Cox proportional hazards model. At all 
stages, probability p-values were used for statistical inference, and a 
statistical significance threshold of 0.05 was used. 

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Appendix 1: CTCAE-based grading of immune-related adverse 
events caused by immune checkpoint inhibitor therapy and timing of 
discontinuation, and the Clinical Practice Guidelines of the Society 
for Cancer Immunotherapy (SITC) regarding adverse reactions. 
I. General Principles of the Clinical Practice Guidelines on Adverse 
Reactions from the Society for Cancer Immunotherapy (SITC): 
Patients should be encouraged to use contraception while receiving 
immunotherapy. Fertility issues should be discussed before treatment. 
Before initiating ICI treatment, the following tests should be 
performed: complete blood count (CBC) and differential, comprehensive 
metabolic assessment (CMP), thyroid-stimulating hormone (TSH), and 
free thyroxine (fT4). Urinalysis should be considered to assess underlying 
kidney disease. 
Baseline electrocardiogram (EKG) should be performed in patients 
at high risk of myocarditis (e.g., those with coexisting heart disease, 
diabetes, or those using combined anti-PD-(L)1 and anti-CTLA-4 ICI 
therapy). Baseline troponin measurement can also provide information 
for assessing potential future cardiotoxicity. 
When starting hormone therapy, patients should be specifically 
asked about potential toxic reactions, including hyperglycemia, mood 
disorders, insomnia, gastritis, weight gain, and opportunistic infections 
(such as Pneumocystis carinii pneumonia). 

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During ICI treatment, complete blood cell count, CMP, TSH, and 
fT4 should be monitored intermittently. 
Patients should consult with specialists for diagnosis and treatment 
advice in the following situations: when the toxicity reaction is grade ≥3; 
when any level of toxicity reaction is unresponsive to hormone therapy; 
when hospitalization or treatment with less toxic drugs is required due to 
toxicity reaction, such as the occurrence of neurotoxic or rheumatic 
toxicity reactions (e.g., arthritis that does not affect daily life, mild pain 
with erythema or joint swelling). 
The diagnosis of all irAEs requires exclusion of other possible 
causes (such as diarrhea/colitis associated with Clostridium difficile), and 
treatment for irAEs should be initiated when clinically deemed 
appropriate. 
For patients with life-threatening autoimmune diseases, ICI 
treatment should only be initiated after a thorough discussion of the risks 
and benefits of ICI therapy between the patient and healthcare provider, 
and after consideration of alternative therapies. The risks of autoimmune 
disease flare-ups should be weighed against the benefits of ICI therapy, 
especially in cancer patients with a high expected incidence of adverse 
reactions. 
Patients with a pre-existing non-life-threatening autoimmune disease 
should be consulted about the possibility of its onset or worsening before 

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receiving ICI treatment. These patients should be closely monitored for 
any exacerbation of their existing autoimmune disease while receiving 
ICIs, and consideration should be given to having appropriate 
professionals conduct concurrent monitoring and treatment. 
Unless there are special circumstances, patients with irAEs grade 1 
should continue ICI treatment under close monitoring for symptom 
worsening. 
Unless there are special circumstances, patients who develop grade 2 
irAEs should temporarily discontinue ICI treatment and be given 
hormone therapy according to the toxicity. 
The decision to discontinue ICI treatment in patients can be complex. 
Factors that may lead clinicians to decide against ICI therapy include 
serious or life-threatening irreversible adverse events (irAEs), the need 
for long-term use or the use of multiple immunosuppressants, long-term 
use of ICI therapy, and/or patients who have achieved complete remission 
or demonstrated significant clinical benefit. 
Unless there are special circumstances, patients who have 
experienced irAEs grade 2 may resume ICI treatment only when their 
symptoms and signs have subsided or their symptoms are controlled with 
≤10 mg of prednisone/day (or equivalent dose). 

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Whether patients who have experienced grade 3 or 4 irAEs should 
undergo ICI treatment again requires a risk assessment based on the 
expected benefits of the treatment and the potential toxicity risks. 
Patients with any level of myositis, myocarditis, or neurological 
symptoms (such as myasthenia gravis): 1. Should consult a specialist. 2. 
Should undergo a series of diagnostic tests, as overlapping symptoms 
may occur, putting the patient at higher risk. 3. Should undergo a series of 
laboratory tests, including: erythrocyte sedimentation rate (ESR), C-
reactive protein (CRP), creatine kinase (CK), antibody testing 
(acetylcholine, specific kinase (MusK), striational), aldolase, troponin, 
EKG, nerve conduction studies, and electromyography (EMG). 4. In 
addition to receiving classic irAE treatment (e.g., hormones), frequent 
lung evaluations are necessary. 
 
 
 
 
 
 
 
 
 

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CTCAE-based grading of immune-related adverse events and timing of 
discontinuation induced by immune checkpoint inhibitor therapy 
1. Skin diseases 
 1 ) Maculopapular rash/dermatitis 
g
rade 
describe 
1 Macules/papules covering less than 10% of the body surface area, 
with or without symptoms (such as itching, burning, or tightness). 
2 Macules/papules cover 10%-30% of the body surface area, with or 
without symptoms (such as itching, burning, tightness); mild 
limitation of daily living abilities. 
3 Macules/papules covering more than 30% of the body surface 
area, with or without related symptoms; limiting the ability to 
perform daily activities. 
4 Grade 4 maculopapular rash/dermatitis is not included in CTCAE. 
Timing for discontinuing medication: Level 3: Discontinue 
medication 
  
 2 ) Skin itching 
grade 
describe 
1 
Mild or localized; localized areas require intervention. 
2 
Intense or widespread; intermittent attacks; skin changes caused 
by scratching (e.g., edema, papules, abrasions, lichenification, 
exudation/scabs); oral medication intervention; mild limitation of 
daily living abilities. 
3 
Intense or widespread; persistent; limiting self-care ability or 

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sleep restriction; requiring oral corticosteroids or 
immunosuppressive therapy. 
4 
Level 4 is not included in CTCAE 
Timing for discontinuing medication: None 
2. Gastrointestinal diseases 
 1 ) Colitis 
grade 
describe 
1 
Asymptomatic; clinical or diagnostic observation only; no 
intervention measures. 
[Grade 1 diarrhea frequency ≤ 4 times/day] 
2 
Abdominal pain; mucus or blood in stool 
[Grade 2 diarrhea frequency: 4–6 times/day] 
3 or 4 
Severe abdominal pain; changes in bowel habits; need for 
medical intervention; presence of peritoneal signs. 
[Grade 3 diarrhea frequency ≥ 7 times/day] 
Level 4: Life-threatening consequences; urgent intervention 
required. 
Timing of medication discontinuation: Level 2 medication suspension 
  
 2 ) Hepatitis 
grade 
describe 
1 
ULN < AST, ALT < 3 × ULN; ULN < total cholesterol < 1.5
2 
3×ULN < AST, ALT ≤ 5×ULN; 1.5×ULN < Total cholestero
3 and 4 
AST, ALT > 5 × ULN; Total cholesterol > 3 × ULN 
Timing of drug discontinuation: If AST/ALT increases by ≥50% for ≥1 week, 

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discontinued. 
 
3. Endocrine 
 1 ) Hypophysitis 
grade 
describe 
1 
Asymptomatic or mild symptoms; requiring only clinical or
2 
Moderate symptoms; requiring minimal, localized, or no
independently. 
3 
Severe symptoms or, although medically significant, no
extension of existing hospitalization; completely contraindi
4 
It could have life-threatening consequences; urgent interven
Timing of discontinuation: If there is a grade ≥2 irAE, discontinue ICI until sympto
started. 
  
 2 ) Hypothyroidism 
grade 
describe 
1 
Asymptomatic; requires only clinical or diagnostic ob
2 
Symptoms present; requires thyroid hormone replacem
3 
Severe symptoms; requiring hospitalization; limiting a
4 
It could have life-threatening consequences; urgent int
Timing of discontinuation: If there is a grade 3 or higher irAE, ICI should be suspen
better. 
  
 3 ) Hyperthyroidism 
grade 
describe 

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1 
Asymptomatic; requires only clinical or diagnostic observation; no 
intervention is needed. 
2 
Symptoms present; requires thyroid hormone suppression therapy; 
mildly affects daily living abilities. 
3 
Severe symptoms; requiring hospitalization; limiting ability to care 
for oneself 
4 
It could have life-threatening consequences; urgent intervention is 
required. 
Timing of drug discontinuation: ≥ Grade 3 irAE 
  
 4 ) Type 1 diabetes (hyperglycemia) 
grade 
describe 
1 
ULN < fasting blood glucose < 8.9 mmol/L 
2 
8.9 < fasting blood glucose < 13.9 mmol/L 
3 
13.9 < fasting blood glucose < 27.8 mmol/L; hospitalizatio
4 
Fasting blood glucose > 27.8 mmol/L; life-threatening con
Timing of medication discontinuation: ICI should be discontinued in type 1 dia
ketoacidosis. 
Type 1 diabetes without diabetic ketoacidosis, grade ≥3, ICI should be discontinued. 
 
4. Lungs 
 1 ) Pneumonia 
grade 
describe 
1 
Asymptomatic; only clinical or diagnostic observation requi
2 
Symptoms present; mildly affecting the ability to care for on

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3 
Severe symptoms; limited ability to care for oneself; require
4 
Life-threatening respiratory damage; requiring emergency in
Timing for discontinuation: Level 1: Consider suspending ICI; Level 2: Susp
discontinue ICI. 
 
 
 2 ) Pulmonary sarcoidosis 
grade 
describe 
1 
Undefined in CTCAE 
≥2 levels 
 
To date, there have been no studies on the side effects of using chec
to treat sarcoidosis. 
Timing for discontinuation: Consider suspending ICI for Level 1 cases; suspend ICI
above. 
 
5. Rheumatism 
 1 ) Inflammatory arthritis 
grade 
describe 
1 
Mild pain accompanied by inflammatory symptoms (morning 
stiffness), skin redness, and joint swelling (which may include 
soft tissue swelling, joint effusion, or synovitis). 
2 
Moderate pain associated with inflammatory symptoms, skin 
erythema, joint swelling, and limited ability to perform daily 
activities using tools. 
3 
Severe pain associated with inflammatory symptoms, skin 
erythema, joint swelling, irreversible joint damage, disability, 
and limited ability to perform daily activities of self-care. 

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Timing for discontinuing medication: None 
 
6. Infusion reaction 
grade 
describe 
1 
Mild, transient reaction; no intervention required. 
2 
Treatment or infusion interruption is required, but the patient 
responds rapidly to symptomatic treatment (such as 
antihistamines, 
nonsteroidal 
anti-inflammatory 
drugs, 
anesthetics, 
and 
intravenous 
infusions); 
prophylactic 
medication: ≤24 hours. 
3 
Symptoms that persist for a long time (e.g., no rapid response 
to symptomatic medications or discontinuation of intravenous 
fluids), or symptoms that recur after initial improvement, 
require hospitalization to prevent sequelae. 
4 
Life-threatening, emergency intervention 
Timing of drug discontinuation: Permanent discontinuation for grade 3/4. 
 
7. Cardiovascular diseases 
grade 
describe 
1 
Abnormalities in cardiac function-related biomarkers, including 
electrocardiogram (ECG). 
2 
Abnormal screening test results, accompanied by mild 
symptoms 
3 
Symptoms appear even with moderate abnormality detection or 
mild activity. 

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4 
Moderate to severe decompensation requires intravenous 
medication or interventional treatment and is life-threatening. 
Timing of drug discontinuation: Level 4: Permanent discontinuation of 
medication 
 
8. Blood diseases 
 1 ) Anemia 
grade 
describe 
1 
Hgb < lower limit of normal -10.0 g/dL, lower limit of normal -
6.2 mmol/L; lower limit of normal -100 g/L 
2 
Hgb＜10-8g/dl; 6.2-4.9mmol/l; 100-80g/l 
3 
Hgb < 8.0 g/dL; 4.9 mmol/L; 80 g/L, requires blood transfusion. 
4 
Life-threatening, emergency intervention 
Timing of drug discontinuation: Level 4 - permanent discontinuation of 
medication 
  
 2 ) Decreased platelet count 
grade 
describe 
1 
Lower limit of normal value -75000mm³ ; <Lower limit of normal 
75.0x10 e9 /l 
2 
< 75000-50000mm 3 ; < 75.0-50.0x10 e9 /l 
3 
<50000-25000mm 3 ; <50.0-25.0x10 e9 /l 
4 
<25000mm³ ; <25.0x10⁹ / l 
Timing of drug discontinuation: Significant clinical symptoms, steroid-r

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thrombocytopenia 
 
9. Kidney disease 
 1 ) Nephritis 
grade 
describe 
1 
An increase in creatinine levels >0.3 mg/dL, or creatinine 
levels 1.5–2.0 times higher than baseline. 
2 
Creatinine 2-3 times higher than baseline 
3 
A creatinine level more than 3 times the baseline or >4.0 
mg/dL requires hospitalization. 
4 
Life-threatening, requires dialysis 
Timing of drug discontinuation: Level 4: Permanent discontinuation of 
medication 
 
10. Nervous system 
 1 ) Encephalopathy/leukoencephalopathy/reversible posterior 
leukoencephalopathy syndrome 
grade 
describe 
1 
Mild symptoms 
2 
Moderate symptoms; limited ability to perform daily 
activities using tools. 
3 
Severe symptoms; limited ability to perform daily living 
activities and care for oneself. 
4 
Life-threatening, requiring emergency intervention. 

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Timing of drug discontinuation: Permanent discontinuation for grade 3/4. 
 
 2 ) Peripheral motor and sensory neuropathy 
grade 
describe 
1 
Refer to the level definitions of each disease 
2 
 
3 
 
4 
 
Timing of drug discontinuation: Permanent discontinuation for grade 3/4. 
11. Nervous system 
 1 ) Encephalopathy / leukoencephalopathy / reversible posterior 
leukoencephalopathy syndrome 
1 2. Ophthalmology 
 1 ) Uveitis 
grade 
describe 
1 
Asymptomatic, discovered only through clinical examination 
2 
Anterior uveitis requires drug intervention. 
3 
Posterior uveitis or panuveitis 
 
4 
Blindness in the affected eye (equal to or worse than 20/200) 
Timing for discontinuing medication: Permanent discontinuation for levels 3 
and 4. 
 

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 2 ) Episcleritis 
grade 
describe 
1 
Asymptomatic, discovered only through clinical examination 
2 
Symptoms include moderate visual impairment (equivalent to or 
better than 20/40), limiting daily living activities with the use of 
tools. 
3 
Symptoms include significant visual impairment (worse than 
20/40), limiting the ability to perform daily living activities and 
self-care. 
4 
Blindness in the affected eye ( equal to or worse than 20/200) 
Timing for discontinuing medication: Permanent discontinuation for levels 3 
and 4. 
 
 3 ) Blepharitis 
grade 
describe 
CTCAE 
does 
not 
define 
a 
classification. 
 
Timing for discontinuing medication: None