Clinical study of Xianjun formula combined with standard western therapy in metastatic colorectal cancer

Abstract

BACKGROUND

Colorectal cancer (CRC) represents a common malignancy for which treatment choices remain constrained with a poor prognosis in advanced stages. Immune checkpoint inhibitors (ICIs) are highly effective in microsatellite instability-high or mismatch repair-deficient CRC, but this subgroup is small. Most patients with metastatic CRC (mCRC) exhibit microsatellite-stable (MSS) or proficient mismatch repair (pMMR) status and derive minimal benefit from ICI monotherapy, largely due to an immunologically “cold” tumor microenvironment. Although combination strategies with anti-angiogenic agents, histone deacetylase inhibitors, or gut microbiota modulation have been explored, effective options remain limited for patients who have progressed after third-line or later therapies. Our preliminary studies suggest that Xianjun formula can modulate the tumor immune microenvironment and synergize with immunotherapy, offering a potential novel strategy for late-line treatment.

AIM

To evaluate overall survival (OS) in patients with MSS mCRC receiving Xianjun formula combined with standard Western therapy in the third-line or later setting, and to explore progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR).

METHODS

This multicenter, prospective, single-arm exploratory study will enroll 44 patients with MSS/pMMR mCRC who have progressed after at least three lines of therapy. Patients will receive oral Xianjun formula granules in combination with investigator-selected standard Western treatments until disease progression, unacceptable toxicity, or withdrawal. OS serves as the primary endpoint; secondary endpoints include PFS, ORR, DCR, and treatment-related adverse events graded according to CTCAE v6.0. Patients will be followed every 3 weeks, with radiologic evaluations every 2 months. Survival will be analyzed using the Kaplan-Meier method. This single-arm study is designed to generate hypotheses and inform the design of future randomized controlled trials.

RESULTS

As an exploratory study, this trial aims to preliminarily evaluate survival trends in late-line MSS mCRC patients treated with Xianjun formula combined with standard Western therapy. Improvements in OS and PFS are anticipated, along with acceptable safety profiles, providing a foundation for subsequent investigations employing larger cohorts or randomized controlled methodologies.

CONCLUSION

This prospective exploratory study is designed to provide preliminary evidence on the efficacy and safety of Xianjun formula combined with standard Western therapy in patients with heavily pretreated MSS mCRC. By targeting the immunosuppressive tumor microenvironment, this combination strategy may offer a feasible and potentially beneficial treatment option for patients who lack effective standard therapies in the late-line setting. The results of this study are expected to support further large-scale or randomized controlled trials evaluating integrative therapeutic approaches in MSS mCRC.

Key Words: Xianjun formula; Metastatic colorectal cancer; Microsatellite stable; Late-line therapy; Overall survival; Integrative medicine

Core Tip: Most metastatic colorectal cancer (mCRC) patients are microsatellite stable (MSS) and derive limited benefit from immune checkpoint inhibitor-based therapies, especially in late-line settings. This prospective, multicenter, single-arm clinical trial explores the feasibility and survival benefit of integrating Xianjun formula, a traditional Chinese medicine compound, with standard Western therapies in refractory MSS mCRC. By targeting the tumor immune microenvironment, this study aims to provide preliminary clinical evidence supporting an integrative therapeutic strategy for patients lacking effective treatment options.

INTRODUCTION

Colorectal cancer (CRC) remains among the most common digestive tract cancers globally, posing substantial clinical challenges in advanced stages due to limited therapeutic options and unfavorable prognosis. Previous clinical studies have demonstrated that immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 elicit significant and durable antitumor responses in CRC patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR), and these agents have become standard treatment options for this specific population[1]. However, MSI-H/dMMR tumors represent only a minority of CRC cases. Most patients with metastatic CRC (mCRC) harbor microsatellite-stable (MSS) or proficient mismatch repair (pMMR) tumors, which generally show limited responsiveness to ICI monotherapy, indicating substantial intrinsic resistance to immunotherapy in this population[2].

Mechanistic studies have revealed that MSS mCRC is typically characterized by a non-inflamed tumor immune microenvironment, with insufficient infiltration of effector immune cells-particularly CD8⁺ T lymphocytes and activation of multiple immunosuppressive signaling pathways, thereby restricting the antitumor efficacy of immune checkpoint blockade[3].

In recent years, combination strategies have been explored to overcome immunotherapy resistance in MSS mCRC. The REGONIVO trial first reported enhanced antitumor activity of regorafenib combined with nivolumab compared with ICIs monotherapy, providing key clinical evidence supporting anti-angiogenic therapy combined with immunotherapy[1]. A randomized phase II trial further demonstrated that a combination of PD-1 inhibition, histone deacetylase inhibition, and anti-VEGF therapy significantly improved progression-free survival (PFS) and objective response rate (ORR) in patients with MSS/pMMR mCRC[2]. In the third-line setting, studies have shown that fruquintinib combined with PD-1 inhibitors exhibits antitumor activity with manageable safety profiles, achieving improved overall survival (OS) and PFS compared with historical controls[4]. Moreover, emerging evidence suggests that incorporating gut microbiota modulation-such as fecal microbiota transplantation-into immunotherapy and anti-angiogenic regimens may further enhance immune responses and improve survival outcomes in selected MSS mCRC patients[5,6].

Despite these advances, effective and evidence-based treatment options remain scarce for patients who progress beyond third-line therapy. Against this background, increasing attention has been directed toward the potential role of traditional Chinese medicine in regulating the tumor immune microenvironment. Our preliminary studies indicate that Xianjun formula, when combined with ICIs, significantly suppresses CRC cell proliferation and exhibits synergistic antitumor effects.

MATERIALS AND METHODS

Study design and endpoints

We designed this exploratory clinical study as a multicenter, prospective, open-label, single-arm trial to be conducted at Xiyuan Hospital, China Academy of Chinese Medical Sciences, and the Traditional Chinese Medicine Hospital of Xinjiang Uygur Autonomous Region. Patients with MSS mCRC who are scheduled to receive or have received third-line or later systemic therapy within the past month will be enrolled. Under real-world treatment conditions, all eligible patients will receive investigator-selected standard Western therapy combined with Xianjun formula granules until the occurrence of disease progression, intolerable adverse effects, or study discontinuation. Figure 1 displays the study’s participant flow diagram.

Figure 1 Study flow chart. OS: Overall survival; PFS: Progression-free survival; ORR: Objective response rate; DCR: Disease control rate.

The primary endpoint of our study is to evaluate OS in patients with MSS mCRC receiving Xianjun formula in combination with standard Western therapy during the third-line or later treatment phase, whilst exploring potential survival benefits.

The secondary outcome measures include: (1) PFS; (2) Incidence and safety profile of treatment-related adverse events (AEs); and (3) Exploratory analyses of survival outcomes associated with Xianjun formula combined with different standard western treatment regimens.

Eligibility criteria

Western medical diagnosis will follow the Chinese Society of Clinical Oncology Guidelines for the Diagnosis and Treatment of Colorectal Cancer (2025 edition). Traditional Chinese medicine diagnosis will be based on the National Standard of the People’s Republic of China, Clinical Terminology of Traditional Chinese Medicine-Part 2: Syndromes.

Inclusion criteria: (1) Age 18-80 years, regardless of sex; (2) Histologically confirmed CRC, stage IV, with MSS status by genetic testing or pMMR by immunohistochemistry; (3) Patients who are scheduled to receive, or have received within the past month, third-line or later systemic therapy; (4) Diagnosis of spleen-kidney yang deficiency syndrome according to traditional Chinese medicine criteria; (5) Eastern Cooperative Oncology Group (ECOG) performance status 0-2; (6) Estimated life expectancy ≥ 3 months; (7) At least one measurable lesion per RECIST v1.1; (8) Adequate cardiac, pulmonary, hepatic, and renal function; (9) Normal coagulation function without active bleeding or thrombotic disorders; (10) Voluntary participation with written informed consent; (11) Expected good compliance with treatment and follow-up; and (12) Deemed by investigators likely to benefit from the study treatment.

Exclusion criteria: (1) Symptomatic brain metastases; (2) Concurrent uncontrolled malignant tumors other than CRC; (3) dMMR or MSI-H status; (4) Uncontrolled hypertension (systolic blood pressure ≥ 140 mmHg or DBP ≥ 90 mmHg despite medication); (5) Significant cardiac disease (New York Heart Association class ≥ II heart failure; unstable angina; myocardial infarction within 1 year; clinically significant arrhythmias requiring intervention); (6) Conditions affecting oral drug absorption (e.g., inability to swallow, intestinal obstruction); (7) High bleeding risk, including active peptic ulcer disease, recent melena or hematemesis, or hemoptysis within 1 month; (8) Thrombotic disorders or requirement for long-term anticoagulation or antiplatelet therapy; (9) Major surgery, open biopsy, or significant trauma within 28 days prior to enrollment; (10) Concurrent participation in another clinical trial; and (11) Any other conditions deemed inappropriate by investigators.

Discontinuation and study termination

Enrolled individuals have the right to withdraw from the study at any time for any reason. The investigator may also discontinue a participant if continued participation is considered inappropriate. Reasons for discontinuation include: (1) Disease progression confirmed by RECIST v1.1 criteria; (2) Occurrence of unacceptable toxicity or safety concerns; (3) Withdrawal of informed consent by the participant; (4) Loss to follow-up or inability to continue treatment or assessments; (5) Major protocol violations affecting study integrity; (6) Enrollment in another clinical trial that prohibits concurrent participation; and (7) Death or other conditions judged by the investigator to make further participation unsuitable. The investigator may terminate the entire study early if significant safety concerns or major protocol issues arise.

Interventions

Western medicine interventions: In accordance with the Chinese Society of Clinical Oncology Guidelines for the Diagnosis and Treatment of Colorectal Cancer (2025 edition), the treatment regimen shall be determined by the investigator. The specific protocols are as follows: (1) Fruquintinib: 5 mg, orally, once daily, on days 1-21, repeated every 28 days; (2) Regorafenib: 160 mg, orally, once daily, days 1-21, repeated every 28 days; or dose titration may be employed during the first cycle: 80 mg/day in week 1, 120 mg/day in week 2, 160 mg/day in week 3; (3) Trifluridine and tipiracil hydrochloride: 35 mg/m² (single maximum dose 80 mg), orally, twice daily, days 1-5 and days 8-12, repeated every 28 days; (4) Trifluridine and tipiracil hydrochloride + bevacizumab: Trifluridine and tipiracil hydrochloride: 35 mg/m² (maximum single dose 80 mg), orally twice daily on days 1-5 and 8-12, repeated every 28 days; bevacizumab 5 mg/kg, intravenous infusion on day 1, repeated every 14 days. Alternatively: Trifluridine and tipiracil hydrochloride 35 mg/m² (maximum single dose 80 mg), orally, twice daily, on days 1-5, repeated every 14 days; bevacizumab 5 mg/kg intravenous infusion on day 1, repeated every 14 days; and (5) Chidamide + bevacizumab + sintilimab: Chidamide 30 mg orally twice weekly; bevacizumab 7.5 mg/kg intravenous infusion every 3 weeks; sintilimab 200 mg intravenous infusion, every 3 weeks.

Traditional Chinese medicine intervention: All enrolled patients will receive Xianjun formula granules in addition to standard Western treatment. The granules are administered as a fixed prescription: One sachet twice daily. Traditional Chinese Medicine theory posits that patients with advanced CRC, particularly those who have undergone multiple lines of systemic therapy, often exhibit spleen-kidney yang deficiency. Symptoms include fatigue, anorexia, aversion to cold, and generalized weakness. Xianjun formula, composed of ingredients such as Rhubarb and Epimedium (fried), aims to warm and tonify spleen-kidney yang. Preclinical studies by our group indicate that this formula can also modulate the tumor immune microenvironment and enhance antitumor immune responses. Therefore, spleen-kidney yang deficiency syndrome was adopted as an inclusion criterion to ensure consistency between syndrome differentiation and herbal intervention.

Dose and regimen adjustment guidelines

Dose adjustment: According to CTCAE v6.0 criteria, grade 1-2 adverse reactions may be managed with routine symptomatic treatment and concomitant medication recorded. For grade 3 adverse reactions persisting after symptomatic management, initiate the first dose reduction to 80% of the standard dose. Subsequent dose reductions shall be to 50% of the standard dose.

Regimen adjustment: Upon a third occurrence of grade 3 adverse reactions, the treatment regimen shall be changed. For patients switching regimens due to poor tolerability, if disease progression has not been assessed per RECIST v1.1 criteria, PFS shall be temporarily excluded from calculation. Their PFS shall be calculated based on the time to disease progression assessed per RECIST v1.1 criteria. If disease progression is assessed, the investigator shall determine the change of treatment regimen.

Modification or discontinuation of western therapy: Should multiple regimen changes prove intolerable, conventional western medical treatment shall be discontinued. Subsequently, the investigator can alter the treatment protocol based on the individual patient’s clinical requirements and preferences.

Follow-up treatment

Each treatment cycle lasts 3 weeks. Safety assessments will be conducted every cycle, and radiologic evaluations will be performed every 2 months. Treatment may be continued beyond initial radiographic progression if atypical responses are suspected and clinical benefit is observed.

Supervision and quality assurance

An interdisciplinary team comprising oncology, clinical methodology, and biostatistics experts has rigorously peer-reviewed and refined this protocol. Before the trial starts, every investigator is required to undergo compulsory training on standard operating procedures, which include eligibility criteria, intervention protocols, case report form completion, RECIST v1.1 application, and data governance. Throughout the study, dedicated personnel will handle data entry, management, and monitoring.

Traditional Chinese medicine quality control

“Xianjun Formula Granules” are manufactured by Beijing Kangrentang Pharmaceutical Co., Ltd.

Ethical considerations

The study has been approved by the Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (No. 2025XLA172-2).

Sample size estimation

The sample size calculation was based on preliminary data from a previous study by our group evaluating a traditional Chinese medicine-based integrative strategy for mCRC in the third-line setting. In that study, patients receiving Chinese medicine combined with targeted therapy demonstrated a median OS of approximately 13.8 months, compared with 7.9 months in patients receiving Western therapy alone[6].

Assuming a two-sided α of 0.05 and power (1-β) of 80%, the required sample size was estimated using PASS version 15.0.5. The calculation indicated that 40 patients would be required to detect a survival difference of this magnitude. Considering a potential dropout rate of approximately 10%, the final planned sample size was set at 44 patients.

This sample size is considered sufficient for an exploratory clinical study aimed at evaluating survival trends and informing the design of future randomized controlled trials. Although the treatment regimen and herbal formulation differed from the present study, the patient population and treatment setting were comparable, making these data a reasonable reference for exploratory sample size estimation.

Statistical analysis

Statistical analyses will be performed using JMP version 14.0 or SPSS version 24.0. Continuous variables will be assessed for normality using the Kolmogorov-Smirnov test and presented as mean ± SD or median (range), as appropriate. Categorical variables will be summarized as n (%).

OS and PFS will be analyzed using the Kaplan-Meier method, with median survival times and 95%CIs reported. Given the anticipated heterogeneity in background Western treatments, exploratory subgroup analyses based on treatment regimen categories and baseline clinical characteristics will be conducted when sample size permits. Multivariable Cox proportional hazards regression will be used to explore potential prognostic factors associated with survival. Missing data will be addressed using multiple imputation, with sensitivity analyses (e.g., complete-case analysis) performed to assess robustness. All statistical tests will be two-sided, with a significance level of P < 0.05. Given the single-arm design, all analyses are considered exploratory. Additionally, exploratory analyses will be performed to evaluate changes in traditional Chinese medicine syndrome patterns and their potential association with survival outcomes.

Primary outcome

OS: Time from enrollment to death from any cause or last follow-up.

Secondary outcomes

(1) PFS; (2) ORR; (3) Disease control rate; (4) Incidence and severity of AEs graded by CTCAE v6.0; and (5) Exploratory analysis of traditional Chinese medicine syndrome evolution and its correlation with clinical outcomes (e.g., PFS, OS).

Safety assessments

AEs, including type, incidence rate, grading (assessed according to CTCAE v6.0 criteria), severity, duration, and relationship to the study drug. Laboratory abnormalities, including type, incidence rate, grading (assessed according to CTCAE v6.0 criteria), duration, etc. Vital signs, including blood pressure, pulse rate, respiratory rate, electrocardiogram, and ECOG Performance Status score. All investigators have received standardized training on CTCAE v6.0 criteria prior to study initiation, and the case report forms have been updated accordingly.

RESULTS

This study is designed as an exploratory clinical trial to assess the clinical feasibility and survival outcomes of Xianjun Formula combined with standard Western therapy in patients with heavily pretreated MSS mCRC. The primary analysis will focus on OS, with secondary evaluations of PFS, tumor response, disease control, and treatment-related AEs. Data collection will be conducted prospectively, and interim descriptive analyses will summarize patient characteristics, treatment exposure, and safety profiles. The results of this study are expected to clarify survival trends and tolerability of this integrative treatment strategy in the late-line setting, providing preliminary evidence to support further confirmatory studies.

DISCUSSION

Patients with MSS mCRC, particularly those in the third-line or later setting, face significant therapeutic challenges due to limited responsiveness to ICI-based therapies and the lack of effective standard options. Although combination strategies incorporating anti-angiogenic agents, epigenetic modulators, or microbiota interventions have shown activity in selected populations[2], durable benefit remains inconsistent. This multicenter, prospective, single-arm exploratory study was therefore designed to investigate an integrative strategy combining Xianjun formula with standard western therapies, with OS as the primary endpoint to capture clinically meaningful benefit in the late-line setting.

Preclinical and preliminary clinical evidence suggests that Xianjun formula may modulate the tumor immune microenvironment and enhance antitumor immune responses, providing a biological rationale for its integration with Western treatments. While the single-arm design and treatment heterogeneity limit definitive efficacy comparisons, this study aims to generate real-world evidence on feasibility, safety, and survival trends, thereby informing the design of future larger-scale or randomized controlled trials.

Several methodological limitations should be acknowledged. First, the single-arm design without a concurrent control group introduces potential selection bias and limits causal inference; results should therefore be interpreted as hypothesis-generating. To minimize bias, standardized eligibility criteria, centralized radiologic evaluation per RECIST v1.1, and prospective data collection will be implemented. Exploratory comparisons with historical controls and propensity score-based adjustments may be considered post hoc. The findings from this study are expected to provide critical preliminary evidence to support the design of future randomized controlled trials evaluating Xianjun formula-based integrative therapy in MSS mCRC.

CONCLUSION

This multicenter, prospective, single-arm exploratory study is designed to evaluate the feasibility, safety, and potential survival benefit of Xianjun formula combined with standard Western therapy for individuals with extensively pre-treated MSS mCRC. By targeting the immunosuppressive tumor microenvironment through an integrative treatment approach, this study addresses a critical therapeutic gap in the late-line setting. The findings are expected to provide preliminary clinical evidence supporting further investigation of Xianjun formula-based combination strategies and to lay the groundwork for larger, randomized controlled studies in MSS mCRC.