The effect of mistletoe extract on tumor response in neoadjuvant chemoradiotherapy for rectal cancer


 Background

Mistletoe extract, which is usually used as a complementary agent for cancer patients, provides an anticancer effect on various malignancies. The present study aimed to evaluate the effect of mistletoe extract (Abnoba Viscum Q®) on tumor responses in neoadjuvant chemoradiotherapy for locally advanced rectal cancer.
Methods

The rectal cancer patients who underwent neoadjuvant chemoradiotherapy were analyzed from Jan 2018 to Jul 2020. In the mistletoe group (MG), the patients were administered Abnoba Viscum Q® subcutaneously during chemoradiotherapy, and it was maintained just before surgery. Patients' demographics, clinical outcomes, and histopathological outcomes were compared between chemoradiation with the MG and nonmistletoe group (NMG).
Results

A total of 52 patients were included. There were MG of 15 patients and NMG of 37 patients. Baseline demographics were statistically similar between the two groups except the CA19-9 and tumor location levels from anal verge. There was no difference in the clinical stage for both groups. We also observed better tumor response in MG in terms of TRG, T stage, and overall TNM stage. Tumor response was significantly better in MG comparing NMG in terms of pathologic complete response rate (53.3% vs 21.6%, p = 0.044), good responder of tumor regression grade (66.7% vs 32.4%, p = 0.024), T downstaging (86.7% vs 43.2%, p = 0.004), and overall downstaging (86.7% vs 56.8%, p = 0.040). The toxicities during NCRT in both groups were minimal.
Conclusion

MG treated with chemoradiation combined with mistletoe extract showed better outcomes than NMG in terms of tumor responses. This diversity in treatment may elevate the method to hope for better oncologic outcomes. Prospective and randomized studies with long-term follow-up are warranted to confirm and extend this study.


Introduction
Colorectal cancer is one of the most common solid tumors with the third highest incidence and the secondhighest mortality rate among malignancies worldwide [1]. The standard treatment for patients with locally advanced rectal cancer is neoadjuvant chemoradiotherapy (NCRT) before radical surgery to reduce locoregional recurrence and toxicity of chemoradiation [2,3]. Tumor down-staging and pCR after NCRT for rectal cancer are associated with good survival outcomes compared to non-response to NCRT [4][5][6][7][8].
Although NCRT has been associated with reducing the locoregional recurrence of rectal cancer, locoregional recurrence causes poor oncologic outcomes to the patient. Therefore, it is essential to enhance tumor response to achieve good oncological results in patients with rectal cancer treated with NCRT. More toxic chemoagents and biologics have been tried to add to conventional NCRT. Oxaliplatin or target agents have been added to NCRT for patients with advanced rectal cancer. Unfortunately, they increase toxicities without better tumor response [9][10][11][12]. We need a new regimen with little toxicity and good compliance for rectal cancer patients to raise the complete remission rate.
Mistletoe (scienti c name: Viscum album) is a semi-parasitic plant in the order Santalales. The extracts from European mistletoe are widely used in complementary and alternative agents to treat patients with various malignancies, including colorectum, lung, oral, pancreas, and breast cancers. Their components, including viscotoxin and lectin, exhibit antitumor effect based on direct cytotoxicity and the increase of the cell mediated immunity [13,14]. Mistletoe extracts showed safety and improved quality of life during chemotherapy in oncologic patients [15][16][17]. Although their antitumor effect and safety have been reported, so far, there is no clinical study regarding tumor regression after NCRT combined with mistletoe extract in rectal cancer. In the present study, we investigated the effect of mistletoe extract on tumor response in NCRT for patients with locally advanced rectal cancer.

Patients selection
A single-center retrospective observational study included the consecutive 52 patients with clinical stagerectal adenocarcinoma treated with long-course NCRT from January 2018 to July 2020 (for this period, mistletoe extract began to be used during NCRT) in Gil Medical Center: a tertiary-referral hospital. The patients with incomplete NCRT were excluded. The conditional searching of patient information was performed by the Clinical Research Data Warehouse (CRDW) system, and detailed data were collected from electronic medical records in Gil Medical Center.
The institutional review board approved the study through the ethics committee of our hospital (approval no. GFIRB2020-304).

Neoadjuvant chemoradiotherapy
All patients treated with long-course NCRT based on the National Comprehensive Cancer Network guidelines. Radiation that delivered 50.4 Gy in 28 fractions over ve weeks in the pelvis was given to 53 patients. Dosing schedules for concurrent chemotherapy were capecitabine 825 mg/m2 per oral twice daily ve days per week during pelvic radiotherapy, or 5-uorouracil 400 mg/m2 intravenous bolus with leucovorin 20 mg/m2 intravenous bolus for four days during week 1 and 5 of NCRT.

Mistletoe extract
Abnoba Viscum Q® (ABNOBA GmbH, Pforzheim, Germany) was used for mistletoe extract. There was no criterion for selecting the patients to be treated with Abnoba Viscum Q®. The patients were administered it subcutaneously: rstly 0.02 mg/ample 3 times a week for 3 weeks, followed by 0.2 mg/ample 3 times a week for 3 weeks, and then 2 mg/ample 3 times a week for 3 weeks, and nally 20 mg/ample 3 times a week; the nal 20mg dose was maintained just before surgery. The patients administered Abnoba Viscum Q® by themselves after learning how to inject Abnoba from the medical staff, followed up in an outpatient clinic every 3 weeks, receiving the amples. The administration of Abnoba Viscum Q® was transiently halted if there were any local or systemic adverse events, such as severe urticarial, local swelling over 5 cm, or high fever over 38 ℃.

Assessment parameters and statistical analysis
Patients demographics, clinical outcomes, and histopathological outcomes were compared between NCRT with mistletoe group (MG) and nonmistletoe group (NMG). Clinical TNM stage and mesorectal fascia (MRF) involvement were assessed by computed tomography and magnetic resonance image. Abdominoperineal CT and rectal MRI were performed twice before and after NCRT to evaluate the tumor and lymph node status. Tumor distance from anal verge (AV) was measured by magnetic resonance image. Tumor regression grade (TRG) was graded on a scale of 0 (complete response: no viable cancer cells) to 3 (poor or no response) according to American Joint Committee on Cancer (AJCC) classi cation [18]. Response to treatment were graded as good (AJCC grades 0 and 1) or poor responder (AJCC grades 2 and 3) 'T, N, and overall TNM downstaging' were de ned as downstaging between baseline imaging results and pathologic stage after curative resection. Continuous variables were analyzed by Mann-Whitney test, and categorical variables were analyzed Chi-square test or Fischer's exact test. The signi cant differences of each variable between two groups were de ned as p value less than 0.05. All statistical analyses were performed with IBM SPSS Statistics 23 for Windows (IBM SPSS Inc., Chicago, IL, USA).

Baseline patient characteristics
A total of 52 patients were included. There were MG of 15 patients and NMG of 37 patients. The median age was 68 (range: 38-86) years, and the sex ratio was 35 men: 17 women. Baseline demographics were statistically similar between the two groups except CA19-9 and tumor distance from AV. CA 19 − 9 was higher in NMG (p = 0.01), and tumor distance from AV was longer in NMG (p = 0.008). MRF involvement presented 10 (66.7%) in MG, and 19 (51.4%) in NMG (p = 0.314). All of the patients in MG were clinically stage , while NMG showed 3 (8.1%) patients in stage and 34 (91.9%) patients in stage . There was no statistical difference in the clinical stage for both groups (p = 0.548). The details of baseline patient demographics are demonstrated in Table 1. Perioperative outcomes All patients received a total of 50.4 Gy of pelvic radiation. The median interval to surgery is 9 (range: 5.9-14.1) weeks after the completion of NCRT. The interval time in MG and NMG was 8,6 and 8.7 weeks, respectively, with no statistical difference (p = 0.808). All patients were treated with oral capecitabine in concurrent chemotherapy except one patient (5-uorouracil/leucovorin). Surgery contained 11 low anterior resections, 34 ultra-low anterior resections, 2 abdominoperineal resections, 3 Hartmann's operations, and 2 transanal excisions. There was no statistical difference in perioperative clinical outcomes between both groups (Table 2).  Table 3. According to Common Terminology Criteria for Adverse Events ver 3.0 (CTCAE), toxicity was strati ed from grade 1 to grade 4. The most common toxicity for both groups was proctitis. There were 9 (60.0%) patients with grade 1 proctitis in the MG, and 10 (27.0%) patients with grade 1 proctitis in the NMG. Grade 2 neutropenia and grade 3 anemia were observed in the NMG, while no patients suffered from those adverse events in the MG. Pruritus with or without skin rash on the injection site was occurred in 3 (20.0%) patients in the MG, whereas there was no pruritus in the NMG (p = 0.021). All other adverse events, including nausea, vomiting, diarrhea, constipation, oral mucositis, and peripheral neuropathy, were grade 1, and there were no statistical differences between both groups.

Evaluation of tumor responses
Estimations of pCR, downstaging, and TRG were assessed in histopathologic reports after curative surgery. Table 4 shows the histopathological outcomes after curative resection. There were no statistical differences in tumor size, histologic grade, ypN stage, distal resection margin, circumferential resection margin involvement, harvested lymph nodes, and perineural invasion between both groups. However, ypT stage presented the trend toward higher stage in NMG than MG (p = 0.005). Lymphovascular invasion was more frequently detected in NMG than MG (32.4% vs 13.3%, p = 0.04)  including T-stage, lymphovascular invasion, TNM stage, TRG, and pCR. These results proposed that mistletoe extract may lead to more improved oncologic outcomes.
Rectal cancer, which is approximately one-third out of colorectal cancer, has relatively higher locoregional recurrence rates than colon cancer because mid-to-lower rectum has no serosa to protect tumor invasion through the muscle layer, and it is technically more demanding to obtain enough safety margin [19]. In addition, it is technically challenging to achieve su cient resection margin to prevent locoregional recurrence due to the close proximity to pelvic structures and organs.
Chemoradiotherapy for rectal cancer usually is needed locoregional therapy due to the relatively high risk of locoregional recurrence compared to colon cancer. Preoperative irradiation is more effective because tumor oxygenation is better with preoperative therapy than postoperative therapy [20]. NCRT improved locoregional control and associated with reduced toxicity compared to postoperative chemoradiotherapy [2,21]. NCRT using 5-FU or capecitabine is usually used as standard therapy for locally advanced rectal cancer before radical surgery [22,23]. NCRT of stage II or III of rectal cancer before surgery improves locoregional control and reduces the toxicity of chemoradiation [2,24]. Despite these treatment efforts, the 5-year incidence of locoregional recurrence rates of rectal cancer is reported to range from 6 to 10.7% [2,8,25,26]. Tumor downstaging and pCR after NCRT facilitate increased overall survival and disease-free survival, including locoregional therapy [4][5][6][7][8].
Therefore, it is crucial to get more tumor downstaging and pCR to achieve good oncological results in patients with rectal cancer treated with NCRT.
In terms of the period from the completion of NCRT to surgery, curative surgery is recommended within 5 ~ 12 weeks. Longer intervals (more than the classical 6 ~ 8 weeks) from completion of NCRT increase the rate of pCR by 6% in rectal cancer [27]. The interval over 8 weeks is associated with increased odds of pCR compared with an interval of 6 to 8weeks [28]. The median interval to surgery in current study was 8.6 weeks in MG and 8.7 weeks in NMG.
Mistletoe is a parasitic plant that parasite on trees. It was rst introduced as a cancer treatment by Rudolf Steiner [29]. Cancerostatic protein components from Viscum album were identi ed later as the cytotoxic viscotoxins and mistletoe lectins [30]. The mistletoe extract induces apoptosis and cytotoxicity to cancer [31]. It also stimulates immunocompetent cells to promote immunomodulation by mistletoe lectin as betagalactoside-speci c lectin [32]. The most important pharmacological components of mistletoe extract are lectins and viscotoxins, which exhibited antitumoral and immunomodulating effects. Mistletoe extract had been considered as a proven old medicine for various cancer patients. However, so far mistletoe therapy to patients with cancer has been controversial in terms of quality of life and oncologic outcomes in the literature [33][34][35][36].
Abnoba Viscum Q® showed the radioprotective effect, and it was similar to the effect of amifostine that was approved as a clinical radioprotectant [37]. Viscotoxin in mistletoe extract had potential antioxidant activity [38]. Mistletoe extract combined with chemotherapy and/or radiotherapy was used for the stage I-III colorectal cancer, showing reduced adverse reaction after adjuvant therapy and better oncologic outcomes in terms of disease-free survival [39]. We adopted Abnoba Viscum Q®, which was expected as the dual effects, radioprotectivity and tumor regression, in the patients with locally advanced rectal cancer.
Tumor regression after conventional NCRT appears in the majority of patients, and approximately 12-38% of patients can achieve pathologic complete response (pCR) [40]. To enhance pathologic tumor response and improve locoregional control, more toxic chemoagents and biologics, including oxaliplatin or target agents, have been tried to add to conventional NCRT to increase pCR and improve oncologic outcomes including reduced recurrence in patients with rectal cancer. However, oxaliplatin as a radiation sensitizer did not improve clinical outcomes, including pathologic complete response, and patients' survival, while it increased chemotoxicity [9,10,[41][42][43]. Clinical studies including target agents such as cetuximab, panitumumab, and bevacizumab in NCRT for advanced rectal cancer did not increase pCR, but instead increased signi cant toxicities [11,12,44,45] [9,10,27,[40][41][42]. We expect good oncological results in the future by using additional mistletoe extract for NCRT in rectal cancer with these pathological results.
Patients receiving NCRT with more toxic chemoagent, including oxaliplatin or bevacizumab, experienced increased rates of grade 3 or 4 toxicity [9-11, 43, 45]. On the other hand, mistletoe extract with chemotherapeutical regimes for solid tumors can improve in health related quality of life [15,47]. Mistletoe extract reduced the rate of gastrointestinal adverse effects such as diarrhea in gastric cancer during adjuvant chemotherapy [15]. In the present study, all adverse events, including gastrointestinal symptoms, oral mucositis, and peripheral neuropathy, were grade I. Only NMG had grade II neutropenia, and grade III anemia; on the other hand, pruritus was observed in MG. There were no statistical differences between MG and NMG. It shows that mistletoe extract is safe and less toxic agents for the treatment of rectal cancer patients at NCRT. Mistletoe extracts help reduce chemotherapy-induced toxicity and enhance treatment tolerability.
Our study had some limitations. First, the number of patients who received NCRT may not be enough to analyze the effect of mistletoe extract for locally advanced rectal cancer. We had practical constraints of patients' nancial strain because the mistletoe extract does not be reimbursed under the national insurance system.
Second, this is a retrospective nonrandomized study. Third, downstaging assessment of rectal tumor after NCRT has unavoidable limitations in its accuracy because the evaluation of preoperative tumor staging was based on image studies. Fourth, it is not fully known how mistletoe extract works to tumor regression.
Nevertheless, the strength of this study is signi cant in that it is the rst study to achieve good results using mistletoe extract together during NCRT for locally advanced rectal cancer.

Conclusions
Neoadjuvant chemoradiation with Abnoba Viscum Q® showed better tumor response for rectal cancer than conventional chemoradiation. Further large populated prospective randomized studies with long-term follow-up will be required to con rm the effectiveness of mistletoe extract in chemoradiation for patients with locally advanced rectal cancer.