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The study protocol was reviewed and approved by the Ethics Committee of Mianzhu Traditional Chinese Medicine Hospital before patient enrollment (Approval No. 2024.0; protocol version 1.0, version date: May 9, 2024). The ethics review was conducted through an expedited review procedure, and the study was approved for implementation. All patients or their authorized legal guardians provided written informed consent before participating in the study. The study was conducted in accordance with the Declaration of Helsinki and relevant institutional requirements for clinical research involving human participants.
Patient selection and baseline characteristics
A total of 96 patients diagnosed with Shi Bi who were enrolled and completed their full treatment regimen at Mianzhu Traditional Chinese Medicine Hospital between October 2024 and September 2025 were randomly assigned to a control group (n = 48) and an observation group (n = 48) using a random-number table. The detailed processes of patient screening, enrollment, randomization, follow-up, and the definition of analysis populations (including the specific attrition of cases for immune parameter evaluation) are delineated in the CONSORT flow diagram (Supplementary Figure 1). Allocation concealment was meticulously maintained utilizing sequentially numbered, opaque, sealed envelopes. Given the distinctive taste, aroma, and appearance of the TCM decoctions, a double-blind design was not feasible. Consequently, this study was conducted as an open-label trial with blinded outcome assessment; specifically, the radiologists evaluating tumor responses and the laboratory technicians analyzing blood samples were strictly blinded to patient group allocations. The final follow-up and data cutoff date for this study was December 31, 2025. It is confirmed that all 96 enrolled patients completed the full 3-month treatment regimen and underwent required clinical and laboratory evaluations prior to the initiation of data analysis. The inclusion criteria were defined as follows: 1) Meeting the Western medical diagnostic criteria for esophageal cancer as outlined in the Guidelines for Standardized Diagnosis and Treatment of Esophageal Cancer11, with histologically confirmed esophageal squamous cell carcinoma (ESCC) of advanced stage (stage IV), and documented disease progression during or after prior first-line platinum-based chemotherapy (assuring a homogeneous second-line treatment setting); 2) Meeting the TCM diagnostic criteria for phlegm-dampness and blood stasis syndrome according to Clinical TCM Oncology12. Primary symptoms included dysphagia, dry mouth and throat, eructation, and thoracic pain. Secondary symptoms included dull complexion, anorexia, emaciation, fatigue, constipation, dark purple tongue with petechiae, thick white greasy coating, and a slippery, rough pulse; 3) Karnofsky Performance Status (KPS) score ≥ 60, with an estimated survival of ≥ 3 months. The exclusion criteria included: patients with concomitant solid malignancies, autoimmune diseases, contraindications to the study medications, or inability to cooperate with long-term follow-up. Baseline characteristics between the two groups showed no statistical difference (P > 0.05), indicating comparability (Table 1).
Treatment protocol
Both groups received camrelizumab therapy. On day 1, 200 mg of camrelizumab was administered via intravenous infusion. The control group additionally received oral anlotinib (10 mg, once daily) for 14 consecutive days, followed by 7 days of rest. One treatment cycle lasted 21 days, and a total of 3 cycles were administered. For the observation group, the identical standard regimen of camrelizumab and anlotinib was implemented alongside TCM interventions. Concurrently, the observation group was administered modified San'ao and Erchen Decoctions. The basal formula consisted of Ephedra (10 g), Bitter Apricot Seed (15 g), Licorice (15 g), Poria cocos (50 g), Tangerine Peel (30 g), and Pinellia Ternata (10 g). For patients with blood-streaked sputum, Panax Notoginseng (15 g) was added. For those with severe chest pain, headaches, or limb soreness, Chuanxiong (30 g) was incorporated. To ensure batch-to-batch consistency and clinical reproducibility, all raw herbal materials were sourced from a certified Good Manufacturing Practice (GMP) supplier and authenticated by the Pharmacy Department of Mianzhu Traditional Chinese Medicine Hospital. Quality control was strictly executed in accordance with the Chinese Pharmacopeia (2020 Edition), which mandates macroscopic identification alongside high-performance liquid chromatography (HPLC) quantification of primary active components for each herb.
For the standardized preparation process, the prescribed herbal mixture was initially soaked in 1000 mL of distilled water for 30 min, followed by two sequential decoctions: 45 min for the first and 30 min for the second. The combined filtrates were subsequently concentrated to a standardized volume of 400 mL per daily dose. The final liquid was vacuum-sealed in sterile pouches and stored at 4 °C. Patients were instructed to warm the decoction before oral administration and to take approximately 133 mL three times daily after meals. One fresh daily dose was prepared from one herbal package for each day of treatment. Given that San’ao Decoction contains Ephedra, which contains ephedrine alkaloids with potential sympathetic excitatory effects, patients’ cardiovascular parameters—including blood pressure, resting heart rate, and electrocardiogram (ECG) tracings—were strictly monitored at baseline and before each treatment cycle. This surveillance was established to identify potential herb-drug interactions or exacerbated cardiovascular toxicities, particularly in patients concurrently receiving anlotinib.
Outcome measures
Primary and secondary endpoints. The primary efficacy endpoint was the objective response rate (ORR). Secondary endpoints included the disease control rate (DCR), Traditional Chinese Medicine (TCM) syndrome scores, serum tumor marker levels, immune function parameters, and safety outcomes. Tumor response was assessed after three treatment cycles according to the immune-modified Response Evaluation Criteria in Solid Tumors (RECIST) to account for atypical immune-related response patterns. Complete response (CR) was defined as the disappearance of all target lesions for more than 4 weeks. Partial response (PR) was defined as a decrease of at least 30% in the sum of target lesion diameters, maintained for more than 4 weeks. Progressive disease (PD) was defined as an increase of at least 30% in lesion size or the appearance of new lesions. Stable disease (SD) was defined as a response that did not meet either PR or PD criteria.
ORR = (CR + PR) / Total × 100% (1)
DCR = (CR + PR + SD) / Total × 100% (2)
TCM syndrome scores. TCM syndrome scores were assessed according to the Guiding Principles for Clinical Research of New Chinese Medicines. To ensure diagnostic consistency and reduce subjective bias, all TCM syndrome evaluations were independently performed by two senior TCM physicians who had received unified pre-trial training on the diagnostic criteria. Any disagreement in symptom grading or tongue/pulse differentiation was resolved through discussion or consultation with a third chief physician. Symptoms were graded as none, mild, moderate, or severe and assigned scores of 0, 2, 4, and 6 points, respectively. Tongue and pulse manifestations were scored as 1 point if present and 0 points if absent.
Serum tumor-marker assessment. Peripheral venous blood samples were collected at baseline and after three treatment cycles. For serum tumor-marker testing, 3 mL of venous blood was collected into serum-separation tubes after an overnight fast. Samples were allowed to clot at room temperature for 30 min and then centrifuged at 1,500 min for 10 min at 4 °C. The serum was separated and stored at −80 °C until analysis. Serum SCC-Ag, CA199, and CEA levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions. All samples were tested in duplicate, and assays were repeated when the intra-assay coefficient of variation exceeded 15%.
Immune-function assessment. Peripheral blood samples for immune function analysis were collected into EDTA anticoagulant tubes at baseline and after 3 treatment cycles and processed within 4 h of collection. CD3+, CD4+, and CD8+ T lymphocyte subsets were measured by flow cytometry using fluorochrome-conjugated monoclonal antibodies. Lymphocytes were first identified based on forward- and side-scatter characteristics, followed by the exclusion of debris and doublets. The percentages of CD3+, CD4+, and CD8+ cells were calculated within the gated lymphocyte population. Instrument calibration and fluorescence compensation were performed before each batch of analysis using standard quality-control beads. Because of insufficient blood volume or sample hemolysis during collection and processing, complete paired flow-cytometry data were successfully obtained from 40 patients in each group. Baseline demographic and clinical characteristics of this immune-function subset were comparable between the two groups (P > 0.05), supporting the reliability of the comparative immune analysis.
Safety assessment. Adverse events were recorded throughout the treatment period and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Safety monitoring included routine blood tests, liver and renal function tests, gastrointestinal symptoms, blood pressure, resting heart rate, and electrocardiographic findings. Particular attention was paid to possible hepatic, hematologic, gastrointestinal, and cardiovascular toxicities because the regimen included camrelizumab, anlotinib, and an Ephedra-containing decoction16.
Biosafety and waste disposal. All blood samples, used tubes, sharps, disposable consumables, and residual biological materials were handled as clinical waste. Residual herbal decoction materials and packaging were discarded in accordance with institutional medical-waste and biosafety procedures. All waste was collected, labeled, transported, and disposed of by trained personnel in accordance with the regulations of Mianzhu Traditional Chinese Medicine Hospital.
Statistical analysis
Statistical analyses were performed using SPSS 26.0 software. The sample size was determined based on the primary efficacy endpoint (ORR). Assuming an estimated ORR of 30% in the control group and an anticipated improvement to 60% in the observation group, a minimum of 39 patients per arm was required to achieve 80% power at a two-sided significance level of α = 0.05. To accommodate a potential dropout rate of approximately 20%, the sample size was expanded to 48 patients per group. Efficacy and safety analyses were conducted on the full analysis set (FAS), comprising all randomized patients who received at least one dose of their assigned treatment. Given that all 96 enrolled participants completed the prescribed treatment cycles and evaluations without loss to follow-up, no statistical imputation methods for missing data were required. Continuous variables were expressed as mean ± standard deviation and analyzed using independent samples t-tests. Categorical variables were expressed as percentages and analyzed using Chi-square (χ2 ) tests. A P-value < 0.05 was considered statistically significant.