Effect of PRM1201 Combined With Adjuvant Chemotherapy on Preventing Recurrence and Metastasis of Stage III Colon Cancer: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Background Chemotherapy is the standard adjuvant treatment for colon cancer. Chinese herbal formula PRM1201 improves the efficacy of chemotherapy when used in combination with Cetuximab or Bevacizumab in patients with metastatic colorectal cancer. This study aims to explore the benefits of treatment with chemotherapy plus PRM1201 in the postoperative adjuvant setting. Methods In this parallel-group study, patients who had undergone curative resection for stage III colon cancer were randomly assigned to receive adjuvant chemotherapy (FOLFOX q2w for 6 months, or CapeOx q3w for 6 months) plus PRM1201 (chemo+PRM1201 group) or adjuvant chemotherapy plus placebo (chemo+placebo group). The primary endpoint was disease-free survival (DFS), and the secondary endpoints were quality of life (QOL) and toxicity. Results A total of 370 patients were randomly assigned to chemotherapy plus PRM1201 group (n = 184) and chemotherapy plus placebo group (n = 186). Up to October 30, 2019, 96 events of recurrence, metastasis, or death had been reported, of which 38 events were in the group of chemotherapy plus PRM1201 and 58 events in the chemo+placebo group. The 3-year DFS rate was 77.1 and 68.6% in the chemo+PRM1201 and chemo+placebo group, respectively (hazard ratio [HR], 0.63; 95% CI, 0.42 to 0.94). The QOL of patients in the chemo+PRM1201 group were significantly improved in terms of global quality of life, physical functioning, role functioning, emotional functioning, fatigue, and appetite loss. The incidence of grade 3 or 4 treatment-related adverse event (TRAEs) were similar between the two arms. Conclusions Chemotherapy in combination with PRM1201 improved the adjuvant treatment of colon cancer. PRM1201 can be recommended as an effective option in clinical practice. Clinical Trial Registration Chinese Clinical Trials Registry, identifier ChiCTR-IOR-16007719.


INTRODUCTION
Colon cancer (CRC) is a common type of gastrointestinal tumor which has a high rate of fatality, ranking as the second leading cause of cancer-related death worldwide (1). After receiving standard treatments, such as surgery, and chemotherapy, recurrence and metastasis are still the most important factors related to the survival of patients (2). The International Duration Evaluation of Adjuvant Therapy (IDEA) study showed that more than 25% of patients with stage III colon cancer had recurrence or metastasis within 3 years after surgery even with standard adjuvant chemotherapy (3). Additionally, the 5-year survival rate for metastatic colon cancer is only 10% (4).
In addition to surgery and chemotherapeutic drugs, most Chinese patients seek help from traditional Chinese medicine during or after standard first-line therapy. As an important component of medicine in China nowadays, Chinese herbal medicine (CHM) plays an important role in cancer treatment and is covered by most Chinese health insurance. A large number of clinical trials have shown that CHM has its own advantages in coping with malignant tumors. The effects of CHM in alleviating symptoms, improving quality of life (QOL), and reducing side-effects and adverse events caused by chemotherapeutic drugs have been confirmed (5)(6)(7). However, few studies have focused on the effects of CHM treatment on survival outcomes in stage III colon cancer, and thus, its use remains controversial.
PRM1201 is a Chinese herbal formula composed of seven herbs. A network pharmacology study showed that the therapeutic targets of the active components of PRM1201 are closely related to colorectal cancer (8). Both in vitro and in vivo experiments showed that this formula can inhibit the invasion and metastasis of colon cancer (9,10). A randomized, controlled, and double-blinded clinical trial confirmed that PRM1201 combined with chemotherapy and Cetuximab or Bevacizumab can prolong the progression free survival of metastatic colorectal cancer (11). To determine whether PRM1201 can benefit patients with colon cancer in an earlier stage, we conducted this prospective interventional study in patients with stage III colon cancer. The study protocol was in compliance with the Declaration of Helsinki and the "Good Clinical Practice" guidelines, and approved by the institutional research ethics committee of all medical centers. The study was registered in the Chinese Clinical Trial Registry (No.: ChiCTR-IOR-16007719). All patients provided signed informed consent before enrollment in the study.

Inclusion and Exclusion Criteria
The inclusion criteria were as follows: (1) pathologically confirmed colon cancer and had undergone curative resection within 8 weeks; (2) stage III in TNM pathological staging according to the American Joint Committee on Cancer; (3) men or women aged from 18 to 75 years; (4) Karnofsky's performance scoring (KPS) ≥70; (5) no strict heart, liver, kidney, or hematopoietic system disease or other factor affecting drug evaluation; and (6) volunteers who had given informed consent.
Patients with any of the following conditions were excluded: (1) mental disorder, pregnancy, or lactation; (2) participant in other clinical trial; (3) any adverse reaction to composition of PRM1201; and (4) incomplete information.

Randomization and Blinding
Patients were randomly assigned to receive either chemotherapy plus PRM1201 (chemo+PRM1201) or chemotherapy plus placebo (chemo+placebo) at a ratio of 1:1. The randomization list was generated by SAS 8.2 and randomization was performed centrally by a statistician with no clinical involvement in the trial. PRM1201 and placebo were in granule form and identical in appearance, weight, color, smell, and taste. All physicians, researchers, and patients were blinded to treatment allocation.  Table 1). The details of total ion chromatogram of PRM1201 are shown in Supplementary  Figure 1. PRM1201 was standardized to 12.0 g per package and the color, shape, weight, smell, taste, and packaging of the placebo were similar to PRM1201. The intervention period lasted the entire chemotherapy cycles, 6 months in total. In addition, quality control of PRM1201 were evaluated by profiling of major phytochemical components at three time points (January 1 2016, May 7 2016, and October 12 2016). As shown in Supplementary  Figure 2, both the overall cluster patterns and the percentages of individual major components indicated a good stability of PRM1201 used in the trial period.

Intervention
All eligible patients were scheduled to receive standard adjuvant chemotherapy. The mFOLFOX6 or CapeOx regimen was recommended by the physician according to the National Comprehensive Cancer Network (NCCN) guidelines. Chemotherapy treatment was started within 8 weeks after surgery. Interruption happened when disease recurrence or metastasis was observed for any other reasons causing chemotherapy termination.

Assessments
The primary endpoint was disease-free survival (DFS). This was calculated from the time of the intervention until any signs or symptoms of first recurrence or metastasis, or death from any cause be recorded. Patients were assessed before randomization. The baseline assessment involved a history and physical examination, complete blood count (CBC), liver and kidney function tests, carcinoembryonic antigen level measurement, chest computed tomography, and abdominal ultrasonography or computed tomography. Follow-up assessment consisted of periodic history and physical examination, adverse events, CBC, laboratory tests, and imaging studies. Colonoscopy was also performed yearly to assess recurrence and metastasis. Only imaging, cytologic analysis, or biopsy was accepted as evidence of recurrence or metastasis. The diagnosis of relapse could not be made on the basis of elevated carcinoembryonic antigen level. The Common Terminology Criteria of the National Cancer Institute (NCI-CTC, version 4.0) was used to grade treatmentemergent adverse events.
QOL scores and adverse events of patients were observed as the secondary endpoints. The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30) was used to assess QOL (12). The QLQ-C30 includes 30 items to evaluate functional scales (physical, role, cognitive, emotional, and social functioning), global health status, symptom scales (fatigue, nausea and vomiting, and pain), and six single symptoms (dyspnea, insomnia, loss of appetite, constipation, diarrhea, and financial impact). The score of each item ranges from 1 (not at all) to 4 (very much), and raw scores were transformed to a 0-to-100 scale linearly. Higher scores on the functional scales and global QOL indicate a better QOL, whereas lower scores on the symptom scales indicate fewer complaints. When the score changed by over 10, this was seen as a significant variation (13).

Statistical Analysis
PASS version 15.0.5 (NCSS LLC., USA) was used for the sample size calculation. The 3-year disease-free survival (DFS) rate of stage III colon cancer patients after surgery in control group was estimated to be 68% (14,15). Chemotherapy plus PRM1201 increased DFS to 79% in the adjuvant treatment of colon cancer and using an a value of 5% and power (1-b) of 80%, we calculated that a sample size of 327 was required. In consideration of a 10% loss rate in follow-up, there were a total of 364 cases needed in this study.
SPSS version 21.0 (IBM Corp., Armonk, USA) was used for the statistical analysis. DFS was compared using a two-sided log-rank test. Hazard ratios with 95% confidence intervals were calculated using the Cox proportional hazards model. The Kaplan-Meier method was used to draw survival curves. Standard parametric or non-parametric tests were used for continuous factors and c2 tests were used for categorical factors. Continuous variables were presented as means (SD) and medians (interquartile range [IQR]). A value of P < 0.05 was considered significant.

Patients' Characteristics
Between January 2016 and June 2016, a total of 420 patients were screened from seven medical centers and 370 patients were included in the study. Patients were randomly assigned to receive chemotherapy plus PRM1201 (184 patients) or chemotherapy plus placebo (186 patients) ( Figure 1). All patients received at least one cycle of treatment. The patients' demographic and clinical characteristics were well matched between these two arms ( Table 1). In both groups, the time between surgery and the start of chemotherapy were within 8 weeks.

Disease-Free Survival
At the time of analysis (median follow-up, 29.60 months), 38 (20.4%) patients in the group given chemotherapy plus PRM1201 had relapsed or died, as compared with 58 (31.5%) in the control group ( Table 2). The 1-year, 2-year, and 3-year DFS rates were 91.2, 84.5, and 77.1% in the chemo+PRM1201 arm, and they were 89.7, 79.2, and 68.6% in the chemo+Placebo arm respectively, with a P value of 0.024. This result indicated that the DFS in the group given chemotherapy plus PRM1201 was significantly higher than in the control group. The hazard ratio (HR) for recurrence in the group given chemotherapy plus PRM1201, as compared with the control group was 0.63 (95% Confidence Interval [CI], 0.42 to 0.94), corresponding to a 37% reduction in the risk of recurrence (Figure 2).

Quality of Life
EORTC QLQ-C30 scores were used to assess the QOL of patients. Patients were evaluated using the questionnaire for the baseline QOL assessment, and there was no significant difference in baseline scores in all domains and items between these two groups (Supplementary Figure 3). The most common symptoms were fatigue (mean 31  PRM1201 than in the chemotherapy plus placebo group (P <0.05) ( Figure 3A). For symptom scales, the improvement ratios in fatigue (50.1 vs. 13.2%), and appetite loss (32.6 vs. 15.8%) were significantly higher in the group given chemotherapy plus PRM1201 than in the control group (P < 0.05) ( Figure 3B).

Adverse Events
There was no serious adverse event that occurred in either group. Neutropenia (108 of 370 patients), diarrhea (43 of 370 patients), and paresthesia (42 of 370 patients) were the most grade 3 or 4 treatment-related adverse events (TRAEs) in all patients with adjuvant treatment. Grade 3 or 4 neutropenia was less common in patients with chemotherapy plus PRM1201 than those with chemotherapy plus placebo (P = 0.292). The proportions of patients with chemotherapy related diarrhea (P = 0.545), vomiting (P = 0.371), and nausea (P = 0.317) were also lower in the PRM1201 arm than in the control arm. While the incidence of other grade 3 or 4 TRAEs between both arms was similar ( Table 4).

DISCUSSION
Resection and chemotherapy are the most effective therapies in clinical management for stage III colon cancer patients (16)(17)(18). Oxaliplatin (19) and capecitabine (20) are the most commonly used chemotherapeutic drugs, and FOLFOX and CapeOx regimens are the first-line chemotherapy for stage III colon cancer according to the NCCN guidelines. A high-quality meta-analysis showed that the 5-year DFS rates of stage II and III colorectal cancer patients receiving assisted chemotherapy were 79.3 and 63.6%, respectively (21). The MOSAIC study, which had a 9.5-year follow-up period, showed that the 5-year DFS rate of stage III CRC patients in the FOLFOX group was 66.4% (22). There is a consensus that postoperative recurrence and metastasis mainly occur in the first 2 years after surgery and that improving the 2-year DFS should be the main focus of preventing disease progression. In our study, the 1-year, 2-year, and 3-year rates of DFS were 89.7, 79.2, and 68.6% in patients receiving chemotherapy plus placebo, similar to 69.3% and 60.68% to 69.15% reported by the other two clinical trials in China (14,15).
In China, CHM combined with chemotherapy is commonly used in clinical practice. A number of clinical studies have   confirmed that CHM can reduce the toxicity of chemotherapeutic drugs, but the effects of CHM on survival outcomes remain undefined. In this study, our primary outcome was DFS. It was shown that the 1-year, 2-year, and 3-year rates of DFS were 91.2, 84.5, and 77.1% in the group given chemotherapy plus PRM1201. It suggested that the herbal medicine PRM1201 can reduce recurrence and metastasis and can prolong the DFS of patients with stage III colon cancer.
Although chemotherapy can significantly prolong DFS and OS, it causes a lot of negative effects, such as neutropenia, dehydration (23), and peripheral sensory neuropathy (24). It was reported that 12.4% of oxaliplatin patients suffered grade 3 peripheral neurotoxicity (25), and 20-50% of them had to reduce their dosage or even stop taking treatment because of intolerable drug related toxicity (26), leading to worse outcomes (21). According to Baek Y's research, lack of appetite, insomnia, and  fatigue are the most frequently observed symptoms in patients receiving chemotherapy in South Korea (27), and all of these symptoms can significantly reduce the QOL of patients (28). Many clinical studies have demonstrated a reduction in the QLQ of colorectal cancer patients in western countries (29)(30)(31). In our study, all of these adverse effects and the reduction of QLQ-C30 scores were observed in patients receiving chemotherapy plus placebo. Therefore, CHM combinational therapy has a distinct advantage in the treatment of cancers. It is generally acknowledged in China, and in Asia as a whole, that CHM can promote the quality of life, alleviate the adverse effects of chemotherapy, improve the effects of chemotherapy, and relieve negative clinical symptoms of cancer patients. The PRM1201 can significantly reverse the decline in QLQ, especially in terms of improved scores in the global functioning, physical functioning, role functioning, emotional functioning, fatigue, and appetite loss. We also observed the decreases in these adverse effects and QLQ-C30 scores in patients who received the placebo. At the same time, the effects of reducing the incidence of grade 3 or 4 neutropenia of PRM1201 were observed. However, the adverse effects mentioned above are not the main outcome of this study. The evaluation of this part has certain limitations, and further observations and improved efficacy evaluation are needed. During the trial period, there were no major side effects of RPM1201 other than few reports of increased frequency of diarrhea. While the diarrhea can produce tolerance in short time after persistent receiving PRM1201 and there were no differences between two groups in diarrhea according to QOL score or TRAEs.
The clinical efficacy of CHM in preventing recurrence and metastasis of colon cancer is attributed to its multiple pharmacological activities, such as inhibiting angiogenesis, reducing chemotherapeutic drug resistance, regulating immunity, and improving the tumor microenvironment. In our preliminary study, we detected serum VEGF, MMP-2, and MMP-9 in patients with colon cancer. It was found that PRM1201 can inhibit the expression of angiogenetic factors, indicating that the effect of RPM1201 on anti-tumor metastasis might be related to the inhibition of angiogenesis. In combination with basic experimental studies, the mechanism of anti-metastasis of PRM1201 is associated with the regulation of Wnt/b-catenin signaling pathway and HIPK2-p53 signaling pathways (9,10). Through clinical observations, it was found that the clinical efficacy of PRM1201 might be associated with the regulation of intestinal microbiota. Further rigorously designed study is needed to confirm this assumption.
This study was a perspective, randomized, double-blinded, placebo-controlled clinical trial. We focused on the effect of PRM1201 in preventing the recurrence and metastasis of stage III colon cancer treated with chemotherapy after surgery. We confirmed that PRM1201 can prolong the DFS of patients with stage III colon cancer, and improve the QOL of patients. However, the anti-tumor mechanism of the THM formula is not fully understood, and further studies are warranted.

CONCLUSIONS
This clinical trial demonstrated that oral PRM1201 significantly improve the DFS of patients with stage III colon cancer with adjuvant chemotherapy after surgery. It showed that PRM1201 has great potential to increase survival outcome, prevent recurrence and metastasis, and improve the QOL of patients. PRM1201 can be recommended as an effective option in clinical practice.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by IRB of Shuguang Hospital affiliated with Shanghai University of TCM. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
QL was involved in the conception and design of the study. RJ, NL, GC, HX, PZ, HML, JH, XY, HG, WY and QL were involved in provision of study materials or patients. RJ, YuZ, LHZ, YiZ, CW, XS, YF, HJL, HL, ZH, YL, YaZ and GH were involved in acquisition of data. RJ, QJ, ZL and WY were involved in data analysis and interpretation. RJ drafted the manuscript and QL, WY and NL were involved in the critical revision of the manuscript. All authors contributed to the article and approved the submitted version.